Keywords
bone marrow mesenchymal stem cells - hypoxic precondition - salivary gland defect
- SDF1-CXCR4 - Bcl-2 - α-amylase
Introduction
Salivary gland is one of the normal tissues frequently affected by the head and neck
radiation therapy. Irreversible salivary gland defect frequently occurs due to the
radiation exposure. The salivary gland damage results in the decrease of saliva production
and in a very severe condition called xerostomia.[1] Following irradiation-induced, irreversible hyposalivation often occur because the
stem cell sterilization of the primitive salivary glands.[2] It is required to apply an alternative approach to treat the severe damage of glands
and the left tissue. One of the alternative approaches for this purpose is the stem
cell therapy.
Some factors that affect the success of stem cell therapy include the following: stem
cells strongly attach and survive in the defect area and they can integrate with the
surrounding microenvironment.[3] However, considering the low amount of survived cells, which shows the low viability
of the transplanted stem cells, the effectiveness of stem cell therapy decreases.
The low rate of viability is probably due to the conventional method of culture, which
was done under normoxic condition with 21% O2. It is contrary to the in vitro environment, which is a hypoxic condition with 1 to
7% O2,[4] depending on the stem cell location and type. Therefore, it is assumed that the
same condition as the micro environment is necessary, to get a desirable result regarding
the viability of the transplanted stem cells in the injured tissue and is expected
to allow the stem cells proliferation and differentiation into the origin-like cells.[5] In addition to the appropriate microenvironment, there are others factors that also
play a role in the success of stem cell therapy, the factors that can induce stem
cells to migrate into the defect area. One of the mediators that plays an important
role in the migration process into the defect area is stromal derived-cell factor
1 (SDF1) through binding with CXCR4 receptor.[6]
The purpose of this study was to explain the mechanism of regeneration of salivary
gland defect due to ionizing radiations by BM-MSCs transplantation that has been given
hypoxic preconditioning with 1% of O2 concentration.
Materials and Methods
Ethical Approval
All animal studies were performed via a protocol approved by the Institutional Animal
Care and Use Committee of Faculty of Veterinary Medicine, Universitas Airlangga, and
complied with the National Research Council's guidelines (366-KE) through ethical
seminar.
Salivary Gland Damage in Animal Model
Damage to salivary glands in a healthy male Wistar rat was induced by single dose
of 15 Gy radiation in the ventral region of the neck. Rats used in this study were
3 to 4 months old and each with 250 to 300 g weight. Rats were kept in an individual
plastic cage in the laboratory for Experimental Animal of Institute of Tropical Disease,
Universitas Airlangga with adequate ventilation.
Treatment
This research was a true experimental posttest control group design. The bone marrow
mesenchymal stem cells (BM-MSCs) were isolated from the femur of Wistar male rats.
Stem cell culture was divided into two conditions: normoxic (O2: 21%) and hypoxic conditions for 48 hours of incubation in a low oxygen tension chamber
consisting of 95% N2, 5% CO2, and 1% O2. Forty male Wistar rats were divided into four groups: two groups of control and
two groups with damage, each with 10 replicates. They are as follows:
-
The negative control group (T0–): rats with normal salivary glands (not irradiated)
and without MSCs treatment.
-
The positive control group (T0+): rats with damaged salivary glands and without MSCs
treatment.
-
The treatment Group 1 (T1): rats with damaged salivary glands, given MSCs transplant
under normoxia 24-hour postradiation.
-
The treatment Group 2 (T2): rats with damaged salivary glands, given MSCs transplant
under hypoxia 24-hour postradiation.
The regeneration process of salivary gland damage was determined after 4 weeks post
BM-MSCs transplantation by studying the expression of several chemokines and proteins,
such as binding of SDF1-CXCR4, Bcl-2 on the tissue by immunohistochemistry methods
and the activity of enzyme α-amylase produced by acinar cells through enzyme-linked
immunosorbent assay (ELISA) activity as a marker of salivary gland regeneration process.
Statistical Analysis
Data were analyzed statistically with normality test and MANOVA test using Statistical
Package for the Social Sciences (SPSS) 17.0 (IBMTM, Chicago, Illinois, United States).
Immunohistochemical Methods for Observation of SDF1-CXCR4 and Bcl-2
Immunohistochemistry (IHC) was performed to determine the expression of SDF1-CXCR4
and Bcl-2.[7] First, salivary glands were transversely incised from paraffin blocks. Monoclonal
antibodies, namely anti-SDF1-CXCR4 and anti-Bcl-2, were used in this technique. Samples were observed under a light microscope at a
magnification of 200× to check SDF1-CXCR4 and Bcl-2 expression. The expression of
each variable was described by the number of cells with brown discoloration due to
diaminobenzidine (DAB-chromogen) in each incision.[8]
Histological Observation of Salivary Glands
Light microscopy examination was performed to study salivary gland histology and regenerate
acinar cells. After that, samples were prepared for histology in the following steps:
submandibular gland of rats was fixed in 10% formalin buffer, followed by dehydration
using a series of alcohol, from 70, 80, 90, to 96% (absolute). Gland tissues of rats
were cleared in a xylene solution. The tissues were infiltrated with an embedding
agent, the liquid paraffin. Microtome was set with a distance at 4 to 6 µ for sectioning,
and the sections were placed on a slide. The embedding process must be reversed to
get the paraffin wax out of the tissue and allow water soluble dyes to penetrate the
sections. Therefore, the slides are “deparaffinized” by washing them through xylenes
to alcohols to water before any staining can be done. The routine H&E staining was
performed. After that, Canada balsam was used to mount the stained section and covered
with a coverslip. Submandibular gland and acinar cell regenerations are observed and
identified based on the histological measures of that of the normal tissue.
Results
The IHC examination results showed that in the hypoxic group the expression of binding
SDF1-CXCR4 and Bcl-2, and α-amylase enzyme increased significantly than in the normoxia group. The mean
of SDF-1 and CXCR4 expression significantly expressed in hypoxic preconditioning group
than in the normoxic group. A significant difference (p < 0.05) between normoxia and hypoxia group with SDF1-CXCR4 binding also showed in
MANOVA test ([Table 1]). It can be seen from comparison of CXCR4 (brown chromogen) expression between treatments
that hypoxic group in acute conditions showed mainly CXCR4 expression (that was stronger
than the expression in the normoxia group ([Fig. 1]). It also can be seen from the green color of the microscopic image of salivary
gland tissue that occupies most of the ductal basal membrane. The distribution of
BM-MSCs, as depicted by labeled PKH 26, shows that green coloration is stronger in
hypoxic groups than in the normoxic group ([Fig. 2]). The mean of Bcl-2 expression was significantly expressed in hypoxic preconditioning
group than in the normoxic group; the MANOVA test results indicated a significant
difference (p < 0.05; [Table 2]). It can be seen from a comparison of the expression of Bcl-2 (brown chromogen)
between treatments that groups with acute hypoxia showed stronger Bcl-2 expression compared with the acute normoxia group ([Fig. 3]). Furthermore, there were significant differences in MANOVA test (p < 0.05) of α-amylase expression between the treatment groups. The acute hypoxia group
showed a significant increase in α-amylase enzyme activity compared with that in the
acute normoxia group ([Table 3]).
Fig. 1 Comparison of CXCR4 (brown chromogen) expression between treatments. In the slide
it appears that hypoxic group in acute conditions showed mainly CXCR4 expression (A) that was stronger than that in the normoxic group (B). Immunohistochemical staining, 400× magnification; Nikon H600L microscope; 300-megapixel
camera DS Fi2.
Fig. 2 Microscopic images of BM-MSCs labeled using PKH 26. The green light in the image
shows the distribution of labeled BM-MSCs. Hypoxic condition (A), normoxic condition (B).
Fig. 3 Comparison of the expression of Bcl-2 (brown chromogen) between treatments. In the
slide, it appears that groups with acute hypoxia (A) showed stronger Bcl-2 expression compared with those in the acute normoxic group
(B). Immunohistochemical staining, 400× magnification; Nikon H600L microscope; 300-megapixel
camera DS Fi2.
Table 1
Average value and standard deviation (SD) expression of SDF1-CXCR4
|
Groups
|
SDF1-CXCR4
|
p-Value
|
|
Mean
|
SD
|
|
Abbreviation: SD, standard deviation.
|
|
Negative control group (T0–)
|
1.640
|
0.555
|
0.000
|
|
Positive control group (T0+)
|
1.160
|
0.219
|
|
Treatment group 1 (T1)
|
2.920
|
0.540
|
|
Treatment group 2 (T2)
|
3.720
|
1.825
|
Table 2
Average values and standard deviations of expression of Bcl-2
|
Groups
|
Bcl-2
|
p-Value
|
|
Mean
|
SD
|
|
Abbreviation: SD, standard deviation.
Note: p < 0.05 is significant.
|
|
Negative control group (T0–)
|
23.600
|
5.176
|
0.000
|
|
Positive control group (T0+)
|
4.000
|
1.870
|
|
Treatment group 1 (T1)
|
5.400
|
1.140
|
|
Treatment group 2 (T2)
|
11.800
|
1.303
|
Table 3
Average values and standard deviations of α-amylase enzyme expression
|
Groups
|
α-amylase
|
p-Value
|
|
Mean
|
SD
|
|
Abbreviation: SD, standard deviation.
Note: p < 0.05 is significant.
|
|
Negative control group (T0–)
|
289,259.000
|
18,645.313
|
0.000
|
|
Positive control group (T0+)
|
95,330.400
|
31,503.973
|
|
Treatment group 1 (T1)
|
152,088.200
|
6,434.510
|
|
Treatment group 2 (T2)
|
186,118.400
|
5,971.156
|
Discussion
Stem cell transplantation is one of the promising therapies that is predicted to restore
the function of the salivary gland damage by regenerating acinar cells. Stem cell
therapy can be successful in addition to the need for adaptive stem cells and when
transplanted stem cells are attached and integrated with their niche. Stem cell niches
are needed to increase and manage the viability of stem cells transplanted in the
damaged area. They have the same microenvironment as the physiological micro-conditions
of the original cell and can support stem cells to proliferate and differentiate into
the original cell.[4] However, there are problems that still exist in cell-based therapies, namely the
delivery cell process or cell migration to the area of injury or what is commonly
called homing. It is suspected that several proteins and chemokines play a role in
the migration process into the damaged area, such as stromal derived-cell factor 1
(SDF1) through binding with CXCR4 receptor.[1]
In the previous study, effect of hypoxic preconditions on the expression of CXCR4
and SDF-1 was studied using IHC. The results showed that 1% hypoxic preconditioning
treatment significantly increased CXCR4 and SDF-1 expression, which play an important
role in increasing the ability of MSCs to migrate to the damaged area and induce endogenous
stem cells to proliferate and differentiate into the desired cells.[9] Thus, it is expected that there will be a process of improving the microenvironment
of the resident stem cells, which will eventually lead to the regeneration process.
In this study, transplantation of adaptive MSCs was administered by direct injection
into salivary glands of Wistar rats damaged by exposure to 15 Gy of single dose ionizing
radiations.
The results of this study indicate that BM-MSCs cells that have been given hypoxic
preconditioning have better therapeutic ability than those under normoxic conditions
so that they can induce cell repair processes. It can be seen from the green color
of the microscopic image of salivary gland tissue that occupies most of the ductal
basal membrane. This shows the migration of BM-MSCs in the basal membrane of the ducts
and acinar cells which are heavily damaged by radiation exposure. The distribution
of PKH 26-labeled BM-MSCs shows that green coloration is stronger in hypoxic group
than in the normoxic group. The IHC examination results in the hypoxic group showed
that the expression of binding SDF1-CXCR4 and Bcl-2 increased significantly than in the normoxic group.
A recent study states that the activation of SDF-1-CXCR4 bonds in tissues plays a
role in the transduction of various signals that can regulate several biological functions,
such as cell survival, proliferation, chemotaxis, and cell differentiation, which
is in line with result of this study. One of the main functions of SDF1-CXCR4 bond
is regulating the trafficking of BM-MSCs cells in during homing in the injured area.[10]
Furthermore, the MANOVA test results showed that there were significant differences
in the expression of α-amylase between the treatment groups. The hypoxia group showed
a significant increase in the activity of α-amylase enzyme compared with that in the
acute normoxic group after transplantation. The results of this study indicate the
regeneration of the damaged salivary glands.[11]
All these have shown the influential effect of low O2 concentration on MSCs biology and raised serious concern over its therapeutic efficiency
and biosafety.[12] Environmental stress to the in vitro cultured MSCs can be caused by higher O2 concentration. Moreover, in recent years after many transplantation studies, early
senescence, longer population doubling time, DNA damage,[13] and poor engraftment have presented clear evidence regarding the negative influence
of ambient O2 concentration on MSCs.[14] A large number of number of studies suggest that hypoxia activates several transcription
factors in the nucleus, such as HIF-1α and NFκβ, and these factors interact with paracrine
factors such as MEK and PI3K/Akt.[15] All these interactions increase the secretion of several growth factors, such as
stromal-derived factor 1 (SDF-1), hepatocyte growth factor (HGF), and vascular endothelial
growth factor (VEGF) with increased expression of each receptor such as CXCR4, increased
secretion of some anti apoptotic proteins, such as Bcl-2 and Bcl-xL as a survival
factor.[16]
Conclusion
This study concluded that (1) BM-MSCs transplantation with hypoxic preconditioning
of 1% O2 increases the expression of SDF1-CXCR4 and Bcl-2 that contribute to cell migration
and cell survival. (2) BM-MSCs can improve regeneration process of the damaged salivary
glands by increasing the activity of the α-amylase as a marker of regeneration process
in the salivary gland tissue.
