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UNIVERSITY OF AGRICULTURAL
SCIENCIES AND VETERINARY MEDICINE
CLUJ-NAPOCA
FACULTY OF VETERINARY MEDICINE
DOCTORAL SCHOOL
SYNOPSIS OF PhD. THESIS
HETEROLOGOUS TRANSPLANTATION OF
MOUSE MEZENCHYMAL STEM CELLS
AS ALTERNATIVE THERAPY IN ANIMAL
MODEL OF OSTEOPOROSIS
SCIENTIFIC COORDINATOR
PROF. IOAN ŞTEFAN GROZA, PhD, DVM PhD STUDENT
ILEA IOANA CRISTINA
CLUJ-NAPOCA
2013
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RESEARCH PURPOSES
Osteoporosis is a metabolic disorder characterized by decreased bone
mineral content and bone mineral density, the bone becoming more porous
(Jagelaviciene E. and Kubilius R., 2006). The main accepted causes that lead to
osteoporosis are: estrogen deficiency, aging, genetic disorders, nutritional
deficiencies and physical inactivity. The diagnosis and prevention of
osteoporosis are difficult to provide due to lack of visible symptoms in early
stages, being considered a “silent epidemic disease” (Cooper C. și col., 2011).
Due to multifactorial pathogenesis, the pharmacological treatments based
on biophosphates ( Black et al., 2006), hormones and bone anabolic agents are
not able to efficiently control this disease, therefore it becomes necessary to
further evolve with new therapeutic agents that are under investigation (Mulder
et al. 2006). Currently, one of the most innovative, promising, but in the same
time challenging additional treatment is the mesenchymal stem cells (MSCs)
therapy (Teitelbaum S.L., 2010).
In order to introduce cellular therapy in current practice it is essential to
offer accessible animal models for experimental studies. Currently, there are
characterizations of different animal models including primates, dogs, rabbits,
rats, mice etc. (Turner S.A., 2001). Nonetheless, there are many requirements
and limitations due to the ethical problems and diagnosis issues for some species,
especially in small laboratory animals.
Considering these aspects, the main objectives of this thesis conducted
over four years in the Department of Veterinary Reproduction, Obstetrics and
Gynecology - Cluj-Napoca, were:
to characterize a murine animal model with severe osteoporosis open
to all research laboratories, by assessing two methods of inducing experimental
osteoporosis: ovariectomy and ovariectomy associated with heparin;
to describe and quantify the shape and structure changes of the
femoral shaft on high-resolution radiographic images by analyzing various
morphometric variables and fractal analysis;
to assess different methods for in vitro isolation and propagation of
murine MSCs derived from bone marrow and inguinal adipose tissue,
morphological and functional characterization, as well as highlighting the
multipotent character by phenotypic characterization;
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to assess the engraftment after systemic heterologous MSCs
transplantation and the MSCs therapeutic potential in murine animal models
with severe osteoporosis.
MATERIALS AND METHODS
The research was conducted during the period October 2009 - June 2013
in the Department of Veterinary Reproduction, Obstetrics and Gynecology,
Faculty of Veterinary Medicine Cluj-Napoca, in collaboration with the
specialized dental imaging office "SC. Digital Dental expert".
In order to obtain mouse models of severe osteoporosis a number of 36
CD1 mice were used. The experimental groups were formed by 24 females that
underwent ovariectomy and the control group (n = 12) underwent sham-
operation. Subsequently, ovariectomized females were randomized into two
groups: group OVX (n = 12) and OVX-H group (n = 12). The OVX-H group
received heparin (1U/gmc) for 64 days.
The screening of metabolic changes was performed before and after
surgery by monitoring the body mass and blood biochemical parameters (Ca + +,
PO4, and serum ALP with UV-VIS spectrophotometer Screen Master Touch).
In vivo quantification of femoral radiographic changes was performed at
days 46 and 85 by assessment of morphometric parameters: EAD (extracortical
average diameter, mm), MAD (medullary average diameter, mm) and MCI
(mean cortical index) and by fractal analysis (fractal dimension, FD). The results
were statistically analyzed.
At day 85 of the study the histological examination was provided.
Longitudinal sections of the femur were stained with Hematoxylin-Eosin staining
(HE).
Prior to assessing the therapeutic potential of mesenchymal stem cells in
induced osteoporosis, the bone marrow and inguinal adipose tissue derived
MSCs from inbred male mice were morphologically, functionally and
phenotypically characterized.
In vitro characterization of MSCs clonogenic and proliferative potential
was performed at different passages by calculating the number of fibroblasts
colony forming units (CFU-Fs) and the cell doubling number (NCD). MSCs
multipotency was assessed by differentiation into osteogenic, chondrogenic and
adipogenig lineages. Phenotypic characterization of differentiated cells was done
by RT-PCR method in order to highlight the specific markers: Osteopontin,
Osteocalcin, Runx2 and Osterix (for osteogenic induction), Aggrecan and Type
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II Collagen (for chondrogenic induction) and PPAR-ƴ2 and LPL (for adipogenic
induction).
Following morphological, functional and phenotypical characterization,
the bone marrow derived MSCs were used in cellular therapy.
For evaluation of MSCs therapeutic potential, a number of 36 female with
severe osteoporosis induced according to the protocol described above were
used. Both OVX (n = 18) and OVX-H (n = 18) groups were randomized as
follows: OVX - C (n = 12) and OVX-H-C (n=12) received undifferentiated
heterologous MSCs via intraperitoneal route (8.7 x 105cel in 100μl saline
solution) and the OVX-M (n = 6) and OVX-H-M (n = 6) groups received 100 μl
saline solution.
Before transplantation, the cell line was phenotypically characterized by
RT-PCR method in order to highlight the expression c-kit specific multipotency
marker. MSCs’ tracking after intraperitoneal inoculation was performed by BrdU
(5-bromo-2-deoxyuridine) labeling.
Homing capacity of BrdU labeled MSCs was assessed at days 7 and 14 in
OVX-C and OVX-H-C groups. Blood, spleen and bone marrow samples were
analyzed by flow cytometry using BD FACS Canto II device.
Osteoporosis screening was performed by X-ray examination at day 0
(before transplantation) and days 32 and 64 (after transplantation). The x-rays
were analyzed according to the protocol previously described.
At day 64 after MSCs transplantation, all subjects were killed. To
highlight the donors’ Y chromosome in the recipients, PCR method was used.
Histological examination was performed on longitudinal sections of
femora and lumbar vertebrae stained with Hematoxylin-Eosin (HE).
RESULTS AND DISCUSSIONS
The recovery rate after ovariectomy surgery performed by two dorso-
lateral incisions was 100% without further complications.
Periodic assessment of body weight showed a statistically significant
increase in OVX group at days 46 and 85 (36.40 ± 2.35 g, respectively 38.60 ±
2.16 g) compared to Control group (32.40 ± 1.51 [p <0.05], respectively 34.60 ±
2.51 [p <0.001]) and OVX-H group (31.71 ± 3.87 g [p <0.05], respectively 33.28
± 3.98 g [p <0.001]).
In all groups, the Ca + + and PO4 values were within physiological limits
of the species (Ca + +: 7.1-10.1 mg / dl, PO4: 5.7-9.2 mg / dl) throughout entire
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study. A significant decrease of serum ALP activity was recorded in OVX-H
group at days 46 and 85 (119.77 ± 11.01 U/l, respectively 112.29 ± 5.90 U/l, [p
<0.001]) compared to Control group (141.80 ± 14.71 U/l [p <0.05], respectively
141.53 ± 10.23 U/l [p <0.001]) and OVX group (134.90 ± 9.82 U/l [p <0.001],
respectively 133.65 ± 6.87 U/l [p <0.001]) as a consequence of decreased
osteoblasts number and activity induced by long term heparin administration (64
days).
Radiological examination showed at days 46 and 85 of the study
pathological changes of bone shape and structure in OVX and OVX-H groups
compared to Control group (fig. no. 1).
Fig. no. 1 – Radiographic morphological and structural
aspects in murine species (original)
At day 46, a slight decrease in opacity was obvious in the proximal and
distal epiphysis in both OVX (fig. no. 1B) and OVX-H (Figure no. 1C) groups
compared to Control group (fig. no. 1A). Shape changes were observed along
femoral shaft, where "throttled" appearance was detectable in both OVX and
OVX-H groups. All pathological changes were more pronounced in OVX-H
group.
At day 85 of radiological examination, the shaft "throttled" appearance
was more severe in both OVX (fig. no. 1D) and OVX-H (fig. no. 1E) groups. In
addition, bone pathological structure changes were observed along the medullary
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cavity: the presence of radiopaque structures like soap bubbles (reinforcement
lines).
Quantifying shape changes by morphometric analysis at day 46 revealed a
significant increase of EAD (p <0.01) only in OVX-H group (1.608 ± 0.19 mm)
compared to Control group (1.404 ± 0.03). These results suggest faster bone
density and bone strength loss when estrogen deficiency was associated with
heparin administration.
At day 85 was recorded a significant increase of EAD in both
experimental groups (OVX: 1.639±0.15 mm [p <0.001], OVX-H: 1.782±0.10
mm [p <0.001]) compared to Control group (1.404 ± 0.03 mm). In addition a
significant increase was observed in OVX-H group compared to OVX group (p
<0.05), which suggests that compressive shaft deformation was more quick and
marked due to the associated effects of estrogen deficiency and heparin (Graphic
no. 1).
Graphic nr. 1. – EAD variation in Control, OVX and OVX-H groups
MAD significantly increased (p <0.001) in OVX-H group in both
examination periods (1.085 ± 0.10 mm and 1.185 ± 0.07 mm) compared to
Control (0.897 ± 0.06 mm) and OVX (0.946 ± 0.10 mm) groups. This finding
suggests early extending (46 days) of medullary cavity in heparinized females
and more severe lesions caused long term heparin administration; in OVX group
(1.117 ± 0.14 mm), this lesion was statistically confirmed (p <0.001) only at day
85 (graphic no. 2.).
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Graphic nr. 2. – MAD variatiom in Control, OVX and OVX-H groups
MCI assessment did not allowed characterization of pathological cortical
changes in in any of the examination stage (p> 0.05). The cortical changes were
hard to detect either due to the small size of the animals or to low sensitivity of
X-ray examination (graphic no. 3).
Graphic nr. 3. – MCI variation in Control, OVX and OVX-H groups
The structural medullary cavity changes quantified by fractal analysis
revealed a significant increase (p <0.001) of the FD only at day 85 in OVX-H
group (1.61 ± 0.04) compared Control (1.49 ± 0.04) and OVX (1.50 ± 0.05)
groups. This results suggest that bone texture changes were more pronounced
after association of ovariectomy with heparin administration (graphic no. 4.)
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Graphic nr. 4. – Variația FD la loturile Martor, OVX și OVX-H
Histological examination performed at day 85 confirmed the diagnosis of
osteoporosis in line with the results of radiological examination, morphometric
and fractal analysis.
In both OVX (fig. no. 2.B) and OVX-H (fig. no. 2.C) groups was revealed
reduced trabecular bone volume and compressive deformation of the femoral
head and growth plate (as Z letter) compared to Control group (fig. no. 2.A).
Irregular appearance of the cortex and the presence of trabeculae in the
middle third of the medullary cavity in OVX (fig. no. 2.E) and OVX-H (fig. no.
2.F) groups compared to control (fig. no. 2. D) was the histological confirmation
of the reinforcement lines detectable on X-rays. The trabeculation process is the
result of spongy bone proliferation along medullary cavity walls as a body's
defense mechanism. In OVX-H group the trabeculation process was more
advanced than in OVX group.
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Fig. no. 2. – Histological findings in Control, OVX and OVX-H groups
Detailed axamination of histological changes in both OVX and OVX-H
groups also revealed pathological changes of osteoporosis (fig. no. 3).
Fig. no. 3. – Osteoporosis specific histological findings
The bone marrow derived cell populations showed specific MSCs
morphological characteristics (fibroblast-like cells) at primary culture
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(P0) (fig. no. 4.) and after successive passages (P1, P2, P3, P4 [fig. no.
5.]).
Fig. no. 4. – MSCs morphology
at P0 (original)
Fig. no. 5. – MSCs morphology
at P4 (original)
Assessment of clonogenic potential (NCD) and CFU-Fs (70% fibroblast-
like colonies) revealed the presence of cells with MSCs functional properties at
P4. Cell population doubling time was approximately 2-3 days.
Adipose tissue derived cells (ASCs) harvested by enzymatic dissociation
method yielded morphologically different cell populations (round-, polygonal-,
enlarge-, flattened- shape cells) than the specific ASCs. Heterogenous cell
populations were found in the primary culture (A0, fig. no. 6) and during all
three successive passages (A0, A1 and A3). At A3 spontaneous osteogenic
differentiation was also noticed (fig. no. 7.)
Fig. no. 6. – ASCs morphology
at A0 (original)
Fig. no. 7. – ASCs morphology
at A3 (original)
The heterogeneous cell population, asymmetric division, extremely high
proliferation rate and occurrence of spontaneous osteogenic and adipogenic
differentiation at passage A3 suggested that the cell pupulation was composed
by adipose progenitor cells (APCs), not ASCs.
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The enzymatic cleavage associated with mechanical dissociation for
isolation of adipose tissue derived cells yielded a cell population with specific
ASCs morphology at passage A'0 (fig. no. 8.). Cell populations heterogeneity at
the end of the successive passages (A'1, A'2 and A'3 [fig. no. 9.]), increased
proliferation rate (NCD) and CFU-Fs results (20% fibroblast-like colonies)
suggested their exclusion from multipotency evaluation.
Fig. no. 8. – ASCs morphology
at A’0 (original)
Fig. no. 9. – ASCs morphology
at A’3 (original)
Multipotent capacity of bone marrow derived MSCs was highlighted by
the three lineage-specific differentiation (osteogenic, adipogenic and
condrogenic) and their phenotypic characterization by RT-PCR method (fig. nr.
10.).
Fig. no. 10. – The electrophoretic profile of the amplificated specific7
cellular markers (original):
A – Osteogenic differentiation, B – Condrogenic differentiation,
C- Adipogenic differentiation
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Analysis of genetic information transmitted by the mRNA of
undifferentiated bone marrow derived MSCs confirmed the expression of c-kit
marker, by RT-PCR method (fig. no. 11). Afterwards the cells were used for
evaluation of the therapeutic potential in osteoporosis.
Fig. no. 11. – The electrophoretic
profile of the G3PDH și c-kit genes
(original)
The 214 bp band corresponds to
G3PDH house-keeping gene.
The 1090 bp band corresponds to
c-kit gene, confirming the MSCs
multipotency.
Homing capacity of inoculated BrdU-labeled cells into bone marrow was
demonstrated after transplantation. At day 7, a percentage of 0.5% BrdU positive
cells were found at day 7 in OVX-C group and 0.6% in OVX-H-C group. At day
14, the percentage of BrdU positive cells was 0.2% in both groups.
In OVX-C group, the radiological exam showed improvement of the
osteoporotic lesions at days 32 and 64 (fig. no. 12). In contrast, in OVX-M group
(fig. no. 13) the shaft throttled aspect was more pronunced and the medullary
cavity had increased opacity.
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Fig. no. 12. – Radiographic changes in OVX-C group
after MSCs transplantation (original)
Fig. no. 13. – Radiographic changes in OVX-M group after
saline solution administration (original)
In OVX-H-C group (fig. no. 14.), the osteoporotic lesions were detectable
at day 32, a slight improvement being observed at day 64, especially along the
medullary cavity.
In OVX-H-M group (fig.nr. 15), at day 32 a more pronounced
compressive deformation was found. In addition, at day 64, a percentage of 33%
of animals suffered complications (diaphyseal fractures), suggesting the
continous development osteoporosis.
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Fig. no. 14. – Radiographic changes in OVX-H-C group after
MSCs transplantation (original)
Fig. no. 15. – Radiographic changes in OVX-H-M group after
saline solution administration (original)
Morphometric analysis was performed in order to assess the visible
changes on the X-rays.
In OVX-M group (graphic no. 5.) no significant difference (p> 0.05) was
found between EAD measured at days 0, 32 and 64. These results suggest the
maintenance of the throttled shaft in untreated subjects. A significant decrease of
MAD values was found at day 32 (0.8720 ± 0.14 mm, p <0.001) compared to
day 0 (0.9932 ± 0.06 mm). The reduction of medullary cavity size suggests an
intense trabeculation process in the internal wall. At day 64, the significant
increase of MAD (0.9946 ± 0.05 mm, p <0.05) compared to day 32 (0.8720 ±
0.14 mm) suggests pronounced compressive deformation associated with bone
strength decrease. Also, MCI significantly increased at day 32 (0.3455 ± 0.07, p
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<0.001) compared to day 0 (0.2512 ± 0.03) and a significantly decreased at day
64 (0.2222 ± 0.05, p <0.01). These continuous cortical changes are related to the
progress of characteristic osteoporotic lesions.
In OVX-C group (graphic no. 6) a significant decrease of EAD was found
at day 64 (1.287 ± 0.09 mm, p <0.05) compared to day 0 (1.343 ± 0.04 mm). The
reduction of extracortical diameter size could be related to increased bone
strength. The significant decrease of MAD at days 32 (0.8630 ± 0.08 mm, p
<0.001) and 64 (0.8852 ± 0.10 mm, p <0.001) compared to day 0 (1.062 ± 0.03
mm), associated with significant increase of MCI at days 32 (0.3424 ± 0.05, p
<0.001) and 64 (0.3139 ± 0.04, p <0.001) compared to day 0 (0.2088 ± 0.03)
indicate reduction of medullary cavity size as a consequence of the osteoporotic
lesions improvement.
Graphic no. 5. – Morphometric parameters
in OVX-M group
Graphic no. 6. – Morphometric parameters
in OVX-C group
In OVX-H-M group (graphic no. 7.), EAD significantly increased at day
32 (1.501 ± 0.05201 mm, p <0.001) compared to day 0 (1.320 ± 0.14 mm)
suggesting that the shaft throttled aspect was pronounced. MAD significantly
increased at days 32 (1.127 ± 0.02 mm, p <0.001) and 64 (1.059 ± 0.10 mm)
compared to day 0 (0.9556 ± 0.14 mm) and MCI significantly decreased at day
64 (0.2353 ± 0.06, p <0.05) compared to day 0 (0.2791 ± 0.04). These results
suggest that reduced cortical thickness is associated with compressive
deformation and delimitation loss between cortex and medullary cavity due to the
trabeculation process.
In OVX-H-C group (graphic no. 8.), EAD significantly increased at day
32 (1.741 ± 0.08 mm, p <0.001) compared to day 0 (1.629 ± 0.14 mm). At day
64 (1.723 ± 0.07 mm) was comparable with day 32. MAD followed the same
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trend at day 32. MCI significantly decreased at day 32 (0.2497 ± 0.06, p <0.05)
compared to day 0 (0.2961 ± 0.04) and increased at day 64 (0.2906 ± 0.03)
following the same pattern.
Graphic no. 7. – Morphometric parameters
in OVX-H-M group
Graphic no. 8. – Morphometric parameters
in OVX-H-C group
These findings indicate that although compressive deformation increased
in the first month, after 2 months was achieved a stoppage of osteoporosis
development. In addition was found a slight improvement of lesion as supported
by the results of EAD, MAD and MCI.
The fractal analysis showed no significant structural changes in OVX-M
group at days 32 and 64 compared to day 0. These results suggest maintenance of
osteoporotic lesion (graphic no. 9).
In OVX-C group, FD significantly decreased at days 32 (1.495 ± 0.03, p
<0.001) and 64 (1.551 ± 0.07, p <0.001) compared to day 0 (1.660 ± 0.003),
indicating reduce tissue complexity (improvement of character osteoporotic
levcxcvnmsions) (graphic no. 9.).
In OVX-H-M group, FD significantly increased at days 32 (1.662 ± 0.008,
p <0.001) and 64 (1.653 ± 0.03 p <0.001) compared to day 0 (1.540 ± 0.04).
These results suggest increased complexity of the analyzed structures related to
newly formed reinforcement lines (graphic no. 10).
In OVX-H-C group, statistical difference was found between FD at 32
(1.675 ± 0.01, p> 0.05) and 64 (1.675 ± 0.006, p> 0.05) compared to day 0
(1.611 ± 0.01), which shows that lesions were comparable after transplantation
(graphic no. 10).
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Graphic no. 9. – Fractal dimension in
OVX-M and OVX-C groups
Graphic no. 10. – Fractal dimension in
OVX-H-M and OVX-H-C groups
The morphometric and fractal analysis results of the femoral shaft were
confirmed by histological examination (fig. no. 16).
Fig. no. 16. – Histological findings at day 64 after transplantation:
A – Healthy control, B – OVX-M group, C – OVX-C group,
D – OVX-H-M group, E – OVX-H-C group
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The histological examination of the lumbar vertebrae revealed severe
osteoporotic lesions in both OVX-M (fig. no. 17.) and OVX-H-M (fig. no. 19.)
groups. The predominant lesions were increased number of resorption lacunae
with active osteoclasts, decreased trabecular volume, increased trabecular
spacing and large areas of necrosis in the growth plate.
Fig. nr. 17. – Characteristic lesions of severe osteoporosis in OVX-M group (original)
Fig. nr. 18. – Histological findings after transplantation in OVX-C group (original)
In OVX-C (fig. no. 18.) and OVX-H-C (fig. no. 20.) groups, bone
regenerative processes were demonstrated by increased number of new formed
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blood vessels, increased number of inactive and active osteoblasts on trabeculae
surface, reduced number and areas of necrosis in the growth plate and slight
increase of trabecular volume. The regenerative aspects and activate osteoblasts
were more increased in OVX-C group.
Fig. no. 19. – Characteristic lesions of severe osteoporosis in OVX-H-M group (original)
Fig. no. 20. – Histological findings after transplantation in OVX-H-C group (original)
Identification of male MSCs engraftment in the females’ bone tissue by
PCR method (highlighting the Sry gene of the Y chromosome) demonstrated that
regenerative processes in the groups subjected to MSCs transplantation were
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determined by their presence, confirming the regenerative potential in mouse
animal models with severe osteoporosis (fig. no. 21.).
Fig. no. 21. – The electrophoretic profile of the 722 bp fragment corresponding
Sry gene (sequence 256-978) on chromosome Y (original)
CONCLUSIONS AND RECOMMENDATIONS
high-resolution X-ray examination revealed the characteristic changes of
osteoporosis at day 85 after starting the induction;
morphometric analysis allowed quantification of bone shape changes
specific to osteoporosis at days 46 and 85 of examination;
fractal analysis revealed the characteristic bone structure changes of
osteoporosis only at day 85 of examination;
the association of estrogen deficiency with heparin administration leads
to a more advanced stage of osteoporosis;
isolation and cultivation of bone marrow derived cells yielded to cell
populations with specific characters MSCs;
the multipotent capacity of bone marrow derived MSCs confirmed the
possibility of their use in regenerative therapy;
BrdU positive cell engraftment after transplantation has been
demonstrated in the bone marrow of the recipients;
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radiological examination associated with morphometric and fractal
analysis enabled quantification and screening of osteoporosis evolution after
transplantation;
the histological examination confirmed the existence of regenerative
processes in the femoral shaft and lumbar vertebrae of the groups subjected to
cellular therapy;
male derived MSCs engraftment in female bone at 64 days after
transplantation confirmed the regenerative potential of MSCs in osteoporosis.
Recommendations
we recommend the use of murine species as experimental model for
osteoporosis; to obtain an advanced stage of disease, we propose ovariectomy
associated with heparin administration;
for cellular therapy studies, we recommend the use of inbred mice in
order to reduce the risk of GVHD disease (Graft-versus-host disease);
we recommend the use of radiological examination associated with
morphometric measurements and fractal analysis for the screening of
osteoporosis after transplantation ;
to standardize and introduce a therapeutic protocol in the current
medical practice, we recommend a expended experiment on a larger number of
animals;
in severe cases of osteoporosis, we recommend to increase the number
of transplanted MSCs, to repeat inoculations at different time intervals and
surveillance of treated animals at least 4-6 months.
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