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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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