Article Type : Research Article
Authors : Abofila MTM, Absheenah ANA, Azab AE, Moussa EA, Imam UM, Ng MH, Ramasamy R, Chen HC and Ganabadi S
Keywords : Osteoarthritis; Stifle joint; Rabbit; Histopathology; Allogenic stem cells; Stem cells therapy
Background: Osteoarthritis (OA) is the most common form of
arthritis and cause physical disability. The ability of autologous mesenchymal
stem cells to regenerate lost articular cartilage in OA been clearly confirmed.
Objectives: The aim of this study was to estimate the
allogeneic stem cells as treatment for OA by histopathological evidence.
Materials and Methods: Twenty-four (24) males New Zealand white rabbits were
used in this study. They were divided into four groups (n=6); Rabbit stem
cell-treated group (RSTG), Sodium Hyaluronate-treated group (SHTG), Media stem
cell-treated group (MSTG) and Normal saline-treated group (NSTG). OA was
induced by a single intra-articular injection of monosodium iodoacetate (MIA)
2.5 mg/0.3 ml normal saline (NS). After 4 weeks of OA induction the (RSTG) was
given a single intra articular injection of rabbit bone marrow-derived
mesenchymal stem cells (BM-MSCs) at a density of 1.5X106 cells / 0.3 ml media,
while the (SHTG) was treated with four injections of 0.3 ml 0.1% sodium
hyaluronate at weekly intervals starting 4 weeks post OA induction. Lastly,
both the (MSTG) & (NSTG) received an injection of the same volume of medium
without cells & normal saline as respectively. Rabbits were euthanized by
intravenous injection of sodium phenobarbital (Dolethal) 100mg/kg at 20 weeks
post-OA induction then histopathology images were assessed.
Results: The results showed that there were significant
differences among all groups in histopathological scoring of the stifle joints
evaluation at week 20. The RSTG showed the best histopathological scoring
followed by SHTG, which are restricted to pain relief, delayed progression of
the disease, and improving general mobility while the MSTG and NSTG showed the
worst scores. Conclusion: In conclusion, a single intra-articular injection of
allogeneic stem cells could promote the regeneration of damaged articular
cartilage in OA as evidenced by improved histopathological outcomes.
Osteoarthritis (OA) is
a degenerative disease of the joint characterized by the degradation of
articular cartilage with loss of matrix and also cyst and osteophyte formation
[1]. Osteoarthritis is the most common form of arthritis, and mostly results in
physical disability. OA affects major weight bearing joints leading to pain,
physical incapacity and reduced quality of life [2]. Other researchers conform
to the view that the disease is primarily degenerative in nature and the
inflammatory changes are secondary [3]. The chondrocytes of articular cartilage
play an important role in the early stages of the disease development. OA were
classified into primary and secondary. Primary degenerative joint disease
refers to those cases, which have no apparent predisposing factor and commonly
occur in older humans or animals but the secondary degenerative joint disease
refers to those cases that have an apparent predisposing factor. OA is
multifactorial in origin and normally does not have a single cause or factor that
could be used to explain why OA does not behave in the same way over the world
[4]. Both groups of researchers; found that the occurrence and clinical
presentation of degenerative joint disease vary between the developed and
developing countries due to geo-ethnic differences in lifestyle and many other
factors such as nutritional, genetic, gender, cultural and occupational [5,6].
The previous authors added that, poor health and nutritional awareness are
other factors that might affect both the occurrence and clinical presentation
of OA. OA affects a large number of humans and animals at different ages; it
commonly affects horses, dogs, and cats. At least, 80% of joint problems are
classified as degenerative joint disease [7]. Wise et al. suggested an association
between deteriorated measures of mental health and OA pain and risk of pain
flares [8]. General mental health is a modifiable component of health and maybe
a new avenue for preventing outbreaks of OA pain. There are several drug
classes available for OA management in both humans and animals. Most of these
drugs are limited to pain control, symptom alleviation, delayed progression of
the disease, and improving general mobility and exercise tolerance as well as
eliminating the risk factors [9]. OA drug treatments include non-steroids such
as diclofenac, ibuprofen, naproxen, and ketoprofen, as well as corticosteroids,
narcotic as morphine and hyaluronic acid [10]. The efficiency of these
treatments is still controversial because unfavorable gastrointestinal
complications have been reported [11]. Pharmacological treatment options for OA
are still very limited, making the search for more options worthwhile [12].
Recently, attention has been focused on agents that could stimulate the
endogenous production of cytokines that can arrest the disease and, in some
cases, help rebuild the cartilage in joints that have been damaged by the
disease [13].
Bajada et al. reported
that replacement of either lost or defective tissues can be achieved with the
assistance of regenerative medicine when current therapies are inadequate [14].
Regenerative medicine comprises the use of tissue engineering and stem cell
technology as the stem cells are suitable and effective biological agents that
can help damaged tissues to regenerate because of their ability to renew
themselves and differentiate into several types of body tissues such as bones,
heart, liver, muscles, etc. under the influence of growth factors but by a
process yet undefined and also their differentiation capacity depend on its
type either embryonic or non-embryonic stem cells.
Joanne et al., reported that autologous adult
stem cells are a much better potential source of cells than mature
chondrocytes, because of their better compatibility and less likelihood of provoking
an immune rejection [15]. Furthermore, mesenchymal stem cells (MSCs) have been
shown to treat degenerative joint disease, influence regeneration of articular
cartilage and slow the progression of the disease [16]. Transplantation of MSCs
to affected discs in stifle joints of rabbits showed proliferation and
differentiation into desired cells resulting in regeneration of affected joints
[17]. Thus, it is evident that previous studies on management of degenerative
joint disease using autologous stem cells have shown promising results. In this
study, we investigated the possibility of using allogeneic and xenogeneic stem
cells as therapy for OA to study healing of the joints and articular cartilage
following experimentally induced OA. Successful use of both allogeneic and
xenogeneic stem cells therapies to replace degraded articular cartilage will
provide an opportunity to reduce the cost, time and effort that are involved
currently in the treatment of OA in humans and large animals such as sport
horses, which are susceptible to joint disease. The main objective of this work
is to evaluate the usefulness of rabbit bone marrow derived mesenchymal stem
cell (allogeneic stem cell) therapies in comparison with sodium hyaluronate in
the replacement of degraded articular cartilage through histopathological
examination.
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.
Table 1: Histological grading
scale.
|
Grading
Scales Histological
OA feature |
Grade (0) |
Grade (1) |
Grade (2) |
Grade (3) |
|
Chondrocyte Loss |
Normal |
Mild |
Moderate |
Severe |
|
Chondrocyte Cloning & Hypertrophy |
Normal |
Mild |
Moderate |
Severe |
|
Chondrocyte Disorganization |
Normal |
Mild |
Moderate |
Severe |
|
Surface Irregularity of Articular
Cartilage |
Normal |
Mild |
Moderate |
Severe |
|
Fibrillation of Cartilage Surface |
Normal |
Mild |
Moderate |
Severe |
|
Safranin O Stain Reduction |
Normal |
Mild |
Moderate |
Severe |
|
Degeneration/Necrosis |
Normal |
Mild |
Moderate |
Severe |
|
Marginal Osteophyte Formation |
Normal |
Mild |
Moderate |
Severe |
|
Subchondral Changes |
Normal |
Mild |
Moderate |
Severe |
|
Total Histological OA Score |
0 – 27 |
|||
Ethical consideration
The Animal Care and Use
Committee (ACUC), Faculty of Veterinary Medicine, University Putra Malaysia
(UPM) approved the use of animals, on 9th April 2010 (Ref.
UPM/FRV/PS/3.2.1.551/ AUP-R94).
Statistical analysis
According to Duncan, [28], nonparametric
statistical methods were used to analyze the data from the study using SPSS
version 16 for the window software package. P value < 0.05 was considered
statistically significant. Data was expressed as mean ± standard deviation (SD)
of mean. Kruskal-Wallis and Mann Whitney (one tail) tests were conducted on
histopathological scoring among all groups.
Histopathological
evaluation was done on stifle joints after 20 weeks of OA induction (16 weeks
after application of different treatments). Right (normal) joint samples were
observed under 200µm powered objective and showed smooth articular cartilage
surface with the underneath layer of flattened chondrocytes in tangential zone.
Chondrocytes were normally arranged in parallel rows in the transitional and
radial zones of the articular cartilage while the subchondral bone revealed
normal distribution of trabeculae composed of osteocytes and canaliculli
surrounding bone marrow filled with blood forming elements. Additionally,
intercellular matrix were deeply and uniformly stained with Safranin O fast
green stain in the non-calcified part (region extending from articular surface
to tidemark) and to a lesser extent in the calcified region (Figure 1).
Figure 1:
Right (normal) articular cartilages and subchondral bones of the femoral
condyle and tibial plateau observed under 200 µm powered objective. (A) H and E staining of the femoral condyle; (C)
H and E staining of the tibial plateau.
Both A and C revealed smooth articular cartilage surface with underneath
flattened chondrocytes of the tangential zone. Chondrocytes were distributed in
parallel rows in transitional and radial zones. There was no pathological
change detected in the subchondral bone. (B) Safranin O staining of the femoral
condyle; (D) Safranin O staining of the tibial plateau. Both B and D revealed
uniformly normal staining of the extracellular matrix.
The histopathological
observations for left OA stifle joint (articular cartilage and subchondral
changes) showed loss of chondrocyte cells, cloning and hypertrophy of
chondrocytes, chondrocyte disorganization, surface irregularity of articular cartilage,
and fibrillation of articular cartilage surface, reduction in intensity of
Safranin O stain, degeneration, necrosis and marginal osteophyte formation.
These observations have been described according to the different groups as
follows:
·
RSTG:
Left (OA) articular cartilages and subchondral bones of the femoral condoyle
and tibial plateau were observed under 200µm powered objective after staining
with H and E stain revealed smooth articular cartilage surfaces with mild
chondrocyte loss in the tangential zone. Chondrocytes were distributed in
parallel rows in transitional and radial zones but besides that there were no
pathological changes detected in the subchondral bone. Also, there was mild
loss of staining with Safranin O fast green stain of the intercellular matrix.
The total histopathological score for this group was 9.16 ± 0.56 derived from
the summation of 3.83 ± 0.28 (femoral condyle) and 5.33 ± 0.28 (tibial plateau)
(Figure 2, 3) (Tables 2 and 3).
Figure 2: Left (OA) articular cartilages and subchondral bones of the femoral condyle and tibial plateau of the RSTG observed under 200 µm powered objective. (A) H and E staining of the Femoral condyle and (C) H and E staining of the Tibial plateau; both A and C revealed smooth articular cartilage surfaces with mild chondrocyte loss (C.L) in the tangential zone. They also showed mild cellular loss (C.L) in transitional and radial zones with mild chondrocyte colonies (C.C), hypertrophy (H) and chondrocyte disorganization (D.O). Besides that, there was no pathological change detected in the subchondral bone. (B) Safranin O staining of the Femoral condyle; (D) Safranin O staining of the Tibial plateau; both B and D revealed mild loss of staining of the intercellular matrix.
Figure 3: Left (OA) articular cartilages and subchondral bones of the femoral condyle and tibial plateau of the SHTG observed under 200 µm powered objective. (A) H and E staining of the Femoral condyle; (C) H and E staining of the Tibial plateau; both A and C revealed moderate to severe fibrillation (F) and chondrocyte loss (C.L) in tangential zone. They also showed moderate cellular loss (C.L) in transitional and radial zones with moderate chondrocyte disorganization (D.O). Besides that, the subchondral bone showed mild subchondral fibrosis formation (F.F). (B) Safranin O staining of the Femoral condyle; (D) Safranin O staining of the Tibial plateau; both B and D revealed moderate to severe loss of staining of the intercellular matrix.
Table 2: Histopathology scoring
of left (OA) stifle joint (Femur) for different groups at week 20.
|
Observations |
RSTG |
SHTG |
MSTG |
NSTG |
|
Chondrocyte Loss
|
1 1 0 0 0 0 |
2 2 2 3 2 2 |
3 2 2 3 3 3 |
3 3 3 3 2 3 |
|
Average Pathology
Score |
0.33
|
2.17
|
2.67
|
2.83
|
|
Chondrocyte Cloning
and Hypertrophy |
1 0 2 0 1 1 |
3 2 2 2 2 3 |
2 3 3 3 3 3 |
2 3 2 2 3 3 |
|
Average Pathology
Score |
0.83
|
2.33
|
2.83
|
2.50
|
|
Chondrocyte
Disorganization
|
0 0 1 1 1 1 |
3 2 2 2 2 2 |
3 2 3 2 3 3 |
3 3 3 3 2 3 |
|
Average Pathology
Score |
0.67
|
2.17
|
2.67
|
2.83 |
|
Surface Irregularity
of Articular Cartilage |
0 0 2 1 0 0 |
2 2 2 3 2 1 |
3 3 2 2 2 3 |
3 3 3 2 3 2 |
|
Average Pathology
Score |
0.50
|
2.00
|
2.50
|
2.67
|
|
Fibrillation of
Cartilage Surface |
0 0 0 2 0 0 |
1 2 2 2 2 2 |
2 3 3 3 3 2 |
3 2 3 3 3 3 |
|
Average Pathology
Score |
0.33 |
1.83 |
2.67 |
2.83 |
|
Safranin O Stain
Reduction |
1 1 1 0 1 1 |
2 3 2 2 3 2 |
3 3 3 3 3 3 |
3 3 3 3 3 3 |
|
Average Pathology
Score |
0.83
|
2.33
|
3.00
|
3.00
|
|
Degenerative/Necrosis |
0 0 0 1 0 0 |
2 2 3 2 2 2 |
3 3 3 3 2 3 |
3 3 3 3 2 2 |
|
Average Pathology
Score |
0.17
|
2.17
|
2.83
|
2.67
|
|
Marginal Osteophyte
Formation |
0 0 0 0 0 0 |
3 3 2 2 2 2 |
2 2 3 3 3 3 |
3 2 2 3 3 2 |
|
Average Pathology
Score |
0.00
|
2.33
|
2.67
|
2.50
|
|
Subchondral Changes |
0 1 0 0 0 0 |
2 2 2 2 1 1 |
2 2 3 3 3 3 |
3 2 3 3 3 3 |
|
Average Pathology
Score |
0.17
|
1.67
|
2.67
|
2.83 |
|
Total Averages Pathology Scores ± SD |
3.83± 0.28 |
19.00± 0.22 |
24.51±
0.13 |
24.66± 0.16 |
|
0: No Change, 1: Mild, 2: Moderate, 3: Severe |
||||
Table 3: Histopathology scoring
of left (OA) stifle joint (Tibia) for different groups at week 20.
|
Observations |
RSTG |
SHTG |
MSTG |
NSTG |
|
Chondrocyte Loss
|
1 1 0 0 0 0 |
2 2 2 3 2 3 |
2 3 3 3 3 3 |
3 3 3 3 3 3 |
|
Average Pathology
Score |
0.33
|
2.33
|
2.83 |
3.00 |
|
Chondrocyte Cloning
and Hypertrophy |
1 0 2 0 2 1 |
3 2 2 3 2 3 |
3 3 3 3 3 3 |
3 3 3 3 3 2 |
|
Average Pathology
Score |
1.00
|
2.50
|
3.00
|
2.83
|
|
Chondrocyte
Disorganization
|
0 0 1 1 2 1 |
3 3 2 2 2 2 |
2 3 3 3 3 3 |
3 3 3 3 3 3 |
|
Average Pathology
Score |
0.83
|
2.33
|
2.83
|
3.00 |
|
Surface Irregularity
of Articular Cartilage |
0 1 2 1 0 0 |
2 2 2 3 2 2 |
3 3 2 2 3 3 |
3 3 3 2 3 3 |
|
Average Pathology
Score |
0.67
|
2.17
|
2.67
|
2.83
|
|
Fibrillation of
Cartilage Surface |
1 0 1 2 0 0 |
2 2 2 2 2 3 |
3 3 3 3 3 2 |
3 3 3 3 3 3 |
|
Average Pathology
Score |
0.67 |
2.17 |
2.83 |
3.00 |
|
Safranin O Stain
Reduction |
1 1 1 0 2 1 |
2 3 2 2 3 3 |
3 3 3 3 3 3 |
3 3 3 3 3 3 |
|
Average Pathology
Score |
1.00
|
2.50
|
3.00
|
3.00
|
|
Degenerative/Necrosis |
0 0 1 1 0 0 |
2 2 3 2 3 2 |
3 3 3 3 2 3 |
3 3 3 3 3 2 |
|
Average Pathology
Score |
0.33
|
2.33
|
2.83
|
2.83
|
|
Marginal Osteophyte
Formation |
0 0 0 0 0 1 |
3 3 2 2 3 3 |
3 2 3 3 3 3 |
2 3 3 3 3 3 |
|
Average Pathology
Score |
0.17 |
2.67
|
2.83
|
2.67 |
|
Subchondral Changes |
0 1 0 0 0 1 |
2 2 2 2 1 2 |
3 3 3 3 3 2 |
3 3 3 3 3 3 |
|
Average Pathology
Score |
0.33 |
1.83 |
2.83
|
3.00 |
|
Total Averages Pathology Scores ± SD |
5.33± 0.28 |
20.83± 0.23 |
25.65±
0.09 |
26.32± 0.11 |
|
0: No Change, 1: Mild, 2: Moderate, 3: Severe. |
||||
Figure
4: Left (OA) articular cartilages and subchondral bones of the femoral
condyle and tibial plateau of the MSTG observed under 200 µm powered objective.
(A) H and E staining of the Femoral condyle; (C) H and E staining of the Tibial
plateau; both A and C revealed very severe fibrillation (F). They also showed
severe to complete cellular loss in tangential, transitional and radial zones.
Besides that, the subchondral bone showed severe fibrosis and cyst formation
(C.F). (B) Safranin O staining of the Femoral condyle; (D) Safranin O staining
of the Tibial plateau; both B and D revealed severe to complete loss of
staining of the intercellular matrix.
·
SHTG:
Left (OA) articular cartilages and subchondral bones of the femoral condyle and
tibial plateau were observed under 200µm powered objective after staining with
H and E stain revealed moderate to severe fibrillation and chondrocyte loss in
tangential zone. Also, there were moderate to severe cellular loss in
tangential and radial zones with moderate to severe chondrocyte colonies,
hypertrophy, necrosis and disorganization. Besides that, subchondral structures
showed some changes including replacement of bone marrow elements with fibrous
tissue, with accompanying cyst formation in some joints. Also, it revealed
moderate to severe loss of staining with Safranin O fast green stain of the
intercellular matrix. The total histopathological score for this group was
39.83 ± 0.45 derived from the summation of 19.00 ± 0.22 (femoral condyle) and
20.83 ± 0.23 (tibial plateau) (Figure 4).
·
MSTG:
Left (OA) articular cartilages and subchondral bones of the femoral condyle and
tibial plateau were observed under 200µm powered objective after staining with
H and E stain revealed severe to very severe fibrillation and chondrocyte loss
in tangential zone. Also, it showed moderate cellular loss in tangential and
radial zones with severe to very severe chondrocyte colonies, hypertrophy,
necrosis and disorganization. Besides that, the subchondral bone showed mild
subchondral fibrosis with small subchondral cyst formation and marginal
osteophyte formation. Also, there was severe to very severe loss of staining
with Safranin O stain of the intercellular matrix. The total histopathological
score for this group was 50.16 ± 0.22 derived from the summation of 24.51 ±
0.13 (femoral condyle) and 25.65 ± 0.09 (tibial plateau) (Figure 5,6).
Figure 5: Picture of marginal
osteophyte formation on the femoral condyle (A) and tibial plateau (B) of the
MSTG observed under 100 µm powered objective.
·
NSTG:
Left (OA) articular cartilages and subchondral bones of the femoral condyle and
tibial plateau were observed under 200µm powered objective after staining with
H and E stain revealed severe to very severe fibrillation and chondrocyte loss
in tangential zone. Also, it showed moderate cellular loss in tangential and
radial zones with severe to very severe chondrocyte colonies, hypertrophy,
necrosis and disorganization. Besides that, subchondral structures showed some
changes including replacement of bone marrow elements with fibrous tissue.
There was accompanying cyst formation in some joints and marginal osteophyte
formation. Additionally, it showed severe to very severe loss of staining with
Safranin O stain of the intercellular matrix. The total histopathological score
for this group was 50.98 ± 0.27 derived from the summation of 24.66 ± 0.16 (femoral
condyle) and 26.32 ± 0.11 (tibial plateau) (Figures 7 and 8; Tables 2 and 3).
Overall, there were significant differences at P < 0.05 among different
treated groups for the average histopathological observations (Figure 7).
Figure 6: Left (OA) articular
cartilages and subchondral bones of the femoral condyle and tibial plateau of
the NSTG observed under 200 µm powered objective. (A) H and E staining of the
Femoral condyle; (C) H and E staining of the Tibial plateau; both A and C
revealed very severe fibrillation (F) and erosion (E). They also showed severe
to complete cellular loss in tangential, transitional and radial zones. Besides
that, the subchondral bone showed severe fibrosis and cyst formation (C.F). (B)
Safranin O staining of the Femoral condyle; (D) Safranin O staining of the
Tibial plateau; both B and D revealed severe to complete loss of staining of
the intercellular matrix.
Figure
7: Picture of marginal osteophyte formation on the femoral condyle (A) and
tibial plateau (B) of the NSTG observed under 100 µm powered objective.
The present work on the
histopathological observations in stifle joints indicated that there were
significant differences among the different groups after 20 weeks of OA
induction (16 weeks after the start of treatments). The right normal joints
revealed no detectable histopathological changes in the joint structures
(articular cartilage and subchondral changes), and there were no significant
differences among the different groups. Histopathological changes in the left
OA-induced stifle joint were detected and were significantly differences among
different groups after 20 weeks of OA induction. In the present work, the
allogeneic stem cell-treated rabbits (RSTG) showed regeneration ofarticular
cartilage in the left OA induced stifle joint and the histological appearance
showed normal to mild changes in joint structures. That might be due to
allogeneic stem cell secretions which activate the residual stem cell and help
rebuild cartilage in affected articular cartilage. This is in agreement with
previous works in which it was proposed that MSCs are able to adhere to
articular cartilage surfaces in the absence of other substrates in human and
rabbit [29,30]; in other previous studies, also consistent with the present
work, MSCs cultured with TGF-?3 and IGF-1 have been successfully used to repair
articular cartilage defects in-vitro [31,32] and in-vivo [33-35]. Other
previous studies using histological observation found that the knee joint
treated with autologous MSCs clearly demonstrated the repair of articular cartilage,
meniscal tissue regeneration and retardation of the progressive destruction
normally seen in those models of OA [16,36-38]. In yet another recent study, it
was suggested that allogeneic MSC-based cartilage repair over generations are
feasible and may also validate the use of immature porcine models as clinically
relevant to test the feasibility of synovial MSCs-based therapies in chondral
lesions [39]. Also, a recent study conducted in the rabbit model of OA treated
with allogeneic MSCs revealed lesser grade of cartilage degeneration, formation
of osteophyte and subchondral sclerosis than the control group. The quality of
cartilage was significantly better in the cell-treated group compared to
controls. One possible reason for this may be the genetic similarity of
laboratory rabbits. However, large groups and longer periods of the study may
provide additional support for the use of this therapeutic approach as a new
way of engineering cartilage [40]. All the previous studies mentioned earlier
are consistent with the present work. However, a subsequent equine study
performed in equine model developed carpal osteochondral fragment, the results
of which showed no response to treatment with bone marrow-derived cells
histologically. It seems that the outcome of this technique can be greatly
enhanced by the timely administration of MSCs into the joint space [41].
Several studies using cell-labeling techniques have shown that, after
intra-articular injection, MSCs preferentially localize to structures of articular
soft tissue with little or no adhesion of the injected cells to the cartilage
surface [16,30, 42,43] and little or no retention in cartilage defects
[30,42-46]. In light of these results, the beneficial effects of
intra-articular administered MSCs on articular cartilage are probably mediated
primarily by effects on other joint tissues or by soluble factors or by cells
attraction to chemokines [47,48], like subchondral mesenchymal progenitor
cells, which are sensitive to these chemotactic signals. In addition, OA
articular chondrocytes secrete morphogenetic factors that stimulate
chondrogenic differentiation of MSCs with the phenotypic characteristics of
joint, as opposed to populations of endochondral chondrocytes [49]. Previous
studies mentioned earlier are in contrasts to the current study perhaps because
some of these studies were on aged animal models or because of the varied
effects of MSC therapy on different cartilage defects. In the present study,
sodium hyaluronate (SHTG) showed reduced OA progression compared with control,
and revealed moderate to severe histopathological changes in the joint
structures likely as a result of sodium hyaluronate ability to decline OA
progression in affected OA stifle joint structure. It is agree with recently studies
illustrated by Kim et al., [25] which explored the effect of growth hormone in
OA therapy compared to the effect of hyaluronic acid in rabbit MIA model of OA.
Another work was Similar to present research, which concluded that the HA
therapy was used in the treatment of high quality cases with OA and there was
no cure [50]. While disagree with research published by Mihara et al., [51]
that tested the effect of intra-articular injections of high molecular weight
sodium hyaluronate (HA) and NSAID on affected joints in a model of OA-induced
rabbit with partial meniscectomy, and showed that in the HA group, the damaged
cartilage area decreased and cartilage degeneration was ameliorated. Disagree
may be due to different way of OA induction, or could be due the type of
hyaluronate that used in each research. Also, another work in a rabbit model of
OA induced by ACLT concluded by Amiel et al., [52] that was un likely with
current work which indicated that the repeat courses of HA can reduce the
degree of articular degeneration observed over a 26-week follow-up period
compared with no treatment, this contrast might be due to the different
repeated courses of HA lead to different result. In the current study, the left
stifle joint induced with OA from the MSTG or NSTG showed increased severity of
OA progression and revealed severe to very severe microscopic appearance in
joint structures. It is likely that both treated groups did not respond to the
treatments, which may have resulted in the progression of OA with severe
consequences. These findings are in accordance with previous studies in which
severe histopathological lesion of OA were observed in the stifle joint
injected with basal medium only [37,39,40]. In this aspect, another study
illustrated that normal saline showed more severity in histopathological
lesions compared to other groups [25,51,52]. Generally, there is limited
information on the use of xenogeneic stem cells approach for OA therapy. This
limitation in data may be due to the fact that greater antigenic differences
exist between different species than within the same species, and so the immune
response to xenografts is much stronger than allograft. Thus, induction of
tolerance is essential to the success of clinical xenotransplantation. The
ability to induce tolerance across highly disparate xenogeneic barriers remains
poorly studied. The genetic incompatibility between species can also affect the
induction of xenograft tolerance by mixed chimerism. While we are still far
from achieving tolerance in clinical xenotransplantation, recent studies using
a transgenic mouse model have demonstrated the principle that mixed
hematopoietic chimerism can induce mouse and human T cell tolerance to
xenografts from pig. The induction of tolerance through mixed chimerism depends
on the successful engraftment of donor hematopoietic cells in the hosts and the
ability of the chimeric cells to eliminate the development and/or decline the
xenoreactive T cell function [53].
Overall, the histopathology results for
articular cartilage and subchondral bone from the current study indicate that
the allogeneic treatments have the best therapeutic effect on OA, followed by
xenogeneic therapy which showed significant improvement in the histopathology
of the articular cartilage and subchondral. Sodium hyaluronate on the other
hand delayed the progression of OA, while the media of stem cells and normal
saline showed the most severe histopathological changes in the articular
cartilage and subchondral. These appear to mirror findings from previous
researches [37,39,40].
Overall, the current study evaluated the
usefulness of rabbit BM-MSCs (allogeneic stem cells) therapies in comparison
with sodium hyaluronate in the replacement of degraded articular cartilage
through histopathological scores for articular cartilage and subchondral bone
of the stifle joint were evaluated, which indicated that the treatment with
rabbit BM-MSCs was the most effective therapy, followed by Sodium hyaluronate
therapy. Both media without cells and normal saline treatments produced the
most severe histopathological changes and proved that these agents had no
remedial effect on degenerative joint disease (OA).
The researchers really appreciate the effort and
assistance offered by University Putra Malaysia, University Kebangsaan Malaysia
and Al-Zawia University-Libya. We thank Dr. Rajash Ramasamy and Dr. Angel Ng
for their assistance, advice and encouragement in conducted this research.