Keywords stem cells - osteoporosis - bone - BMP 2 - osterix - TRAP - PKH-26
Introduction
Osteoporosis is a skeletal disease characterized by a total reduction in bone density
due to the body’s inability to manage the mineral content of the bones and damage
to bone architecture, leading to reduced bone strength.[1 ] Its complications such as osteoporotic fracture were associated with significant
morbidity as well as treatment costs.[2 ] Females are known to be more susceptible to osteoporosis than males, a situation
resulting from the menopause, which induces hormonal imbalances in them, including
decreased levels of estrogen which, in turn, reduce bone density.[3 ]
Osteoporosis constitutes a complication of prosthodontics-related dental treatment
caused by significant bone resorption, affecting retention and stabilization of dentures.
One novel, effective, and practical alternative for missing tooth replacement involves
dental implants, as demonstrated by the prevalence of their use in the United States,
having increased significantly from 0.7% in 2000 to 5.7% in 2016.[4 ]
One prerequisite for successful implants is the occurrence of osseointegration, which
represents a clearly defined structural and functional relationship between the bone
and the surface of the implant. It is necessary to observe the thickness of the jawbone
in postmenopausal females before implementing prosthodontic treatment to prevent implant
placement failure, thereby ensuring effective osseointegration.[5 ]
Ovariectomized rats are the most commonly used animal subjects in pathophysiological
studies of osteoporosis, since they can illustrate the effect of estrogen deficiency
on the decline of trabecular bone mass in humans. Osteoporosis will first attack the
axial bone (mandible) and, subsequently, limb bones. It can be used as a diagnostic
marker of osteoporotic abnormalities systemically.[6 ]
Stem cells constitute as yet undifferentiated immature cells which are capable of
differentiating and self-renewal, depending on the potential of the tissues in which
they are produced. Given the presence of a widespread defect, the number of stem cells
in the body is insufficient to rapidly regenerate tissue. Therefore, a strategy for
administering stem cells from outside the body is required.[7 ]
An ideal source for tissue engineering must satisfy several conditions, including
ease of access, adequate cell counts, and a high-degree of immunocompatibility. An
important source that has been the object of investigation for some time is that of
human umbilical cord mesenchymal stem cells (hUCMSCs), the inducted form of which
can assume the role of osteoprogenitors and promote bone formation. Previous research
has confirmed that the application of hUCMSCs promotes high-levels of osteoblast differentiation
in an osteoporotic model, a finding that is supported by increased numbers of osteoblasts
and expression of transforming growth factor-β1(TGF-β1) and Runt-related transcription
factor 2 (Runx2),[8 ] and the other research has confirmed that increased expression of ALP, type I collagen
and osteocalcin, and a larger trabecular bone area in osteoporotic rats induced by
hUCMSCs is the proof of increased osteoblastogenesis.[9 ]
Greater bone regeneration is characterized by an increase in bone formation markers
such as bone morphogenetic protein-2 (BMP-2), Osterix (Osx) expression, and decrease
in bone resorption markers like tartrate-resistant acid phosphatase (TRAP) expression.
BMP-2 is a protein superfamily of transforming growth factor beta (TGF-β) active in
osteogenesis, whereas Osx is a specific transcription factor of osteoblasts which
activates gene receptors during differentiation of preosteoblasts into mature osteoblasts.[10 ]
[11 ] TRAP is highly expressed in chondroclasts and able to degrade phosphoproteins such
as osteopontin (OPN) from skeletons. A hUCMSCs-induced reduction in osteoclasts was
evidenced by lower levels of TRAP expression.[12 ] This research aims to demonstrate the ability of hUCMSCs to promote osteoporotic
mandibular bone regeneration through BMP-2, Osx, and TRAP expression.
Materials and Methods
Sample Preparation
Ethical commission of health research of Universitas Airlangga Faculty of Dental Medicine
has approved this study (Number: 043/HRECC.FODM/II/2019 and Number: 042/HRECC.FODM/II/2019).
This research represented a true experimental study. Samples were collected from 25
three-month-old, female Wistar strain Rattus norvegicus Albinus 180 to 200 g in weight. Samples were randomly classified into five groups: a normal
group with sham surgery (N ), an ovariectomy group whose members were injected with gelatin solvent for 4 weeks
(G4) or 8 weeks (G8), and an ovariectomy group whose members were injected with hUCMSCs
and gelatin for 4 weeks (SC4) or 8 weeks (SC8). RSUD Dr. Soetomo Surabaya (Dr. Soetomo
Academic Medical Center Hospital) and the Ethical Commission of the Faculty of Veterinary
Medicine, Universitas Airlangga, approved the health research ethics for the research
reported here.
Ovariectomy Preparation
An ovariectomy involving the Khajuria procedure for was performed osteoporotic conditions,
and sham surgery on the control group members was conducted. The subjects underwent
these procedures on osteoporosis after 12 weeks.[13 ]
hUCMSCs Culture Preparation
Umbilical cords were retrieved from the healthy C-sections of newborn babies at RSUD
Dr. Soetomo, Surabaya, Indonesia. The cords were then cut into 1 cm lengths, while
the veins, arteries, and adventitia were removed to obtain Wharton’s jelly, which
was subsequently handled by Han’s method. The medium was changed once every 3 days.[7 ]
Injection of Osteoporotic hUCMSCs into Mice Subjects and the Gelatin Solvent Procedure
The left mandibular of the samples was penetrated through the skin under the molar
region into trabecular area with a perforator needle (Stabident; Miami, USA) under
anesthesia. The needle was then retrieved. Members of the G4 and G8 subject groups
were injected with 50 µL of gelatin solvent, while 400.000 cells in 50 µL of gelatin
solvent were injected into the SC4 and SC8 group subjects.
Procedure for Injection of hUCMSCs into the Mandibular Osteoporotic Subjects Using
Labeled Cells
Osteoporotic subjects were prepared for injection with PKH-labeled 26 hUCMSCs before
being allocated to one of two groups, two subjects to the 4-week group, and two to
the 8-week group. Four and eight weeks postinjection of hUCMSCs with PKH 26 labeling,
the subjects were terminated in the same manner as those which had not been labeled.
Termination of Experimental Subjects and Microscopic Analysis of Testing Samples
Following the elapse of the testing period, termination of the research subjects was
undertaken. Immunohistochemical staining using a monoclonal mouse BMP-2 (Abcam, United
Kingdom), Osx (Santa Cruz Biotechnology, Inc, Europe), and TRAP (Bioss Antibodies,
Netherlands) antibodies was conducted. A light microscope (Nikon H600L, Tokyo, Japan)
fitted with a DS-Fi2 300 MegaPixel digital camera and a Nikon Image System photo editor
program was employed for the purposes of microscopic observation. The results were
measured using the Remmele immunohistochemistry index scale to count the total number
of osteoblast cells (both surface and mesenchymal) from five different fields of view
at 400x magnification. Fresh bone was cut with a LEICA cryocut in a dark room at a
temperature of between − 40°C and − 60°C. PKH-26 contains fluorescent proteins which
were then observed by means of a fluorescent microscope (OLYMPUS FSX-100).[14 ] The fresh bone to be cut must be wrapped in aluminum foil and conditioned in a dark
room.
Statistical Analysis
All the data was shown in five different experimental groups. Statistical analysis
using the SPSS version 15.0 (SPSS, Inc.; Chicago, IL, USA), ANOVA and Tukey HSD tests
was performed with the statistically significant p < 0.05 being considered.
Results
Isolation and Culture of Human Umbilical Cord Mesenchymal Stem Cells
The isolation and culture of hUCMSCs have yet to be published. The experiment result
verified that the cells were hUCMSCs by evaluating the surface marker of isolated
cells, i.e., CD45–, CD73 +, CD90 +, and CD 105 +.
Labeling PKH 26
To identify the effect of hUCMSCs on the osteoporosis model, an evaluation is required
to ensure that MSC inside the mandibular bone originates from exogenous hUCMSCs. At
the next stage of the process, hUCMSCs labeling of each subject in the SC4 and SC8
groups using PKH26 was completed. Postinjection observation of the mandibles of the
members of the group SC4 4 weeks later detected the presence of strongly fluorescent,
green, labeled cells. The microscopic view of PKH26 expression can be seen in [Fig. 1 ].
Fig. 1 Microscopic image of human umbilical cord mesenchymal stem cells (hUCMSCs) with PKH26
labeling.
The Expression of BMP-2, Osx, and TRAP
The microscopic expression of BMP-2 is shown in [Fig. 2 a ]. The number of BMP-2 expressions in each group is contained in [Fig. 3 ]. The BMP-2 normality test with Kolmogorov–Smirnov is normal (p = 0.950) and the homogeneity test with Levene statistic is homogenous (p = 0.434). The one-way ANOVA showed significantly difference between groups (p = 0.000). The Tukey HSD test showed BMP-2 expression had significantly increased
in the SC4 and SC8 groups compared with the other groups. The lowest BMP-2 expression
was observed in the G4 and G8 groups (SC4 = 0.041; SC8 = 0.004 to G4) (SC8 = 0.019
to G8).
Fig. 2 Microscopic image of bone morphogenetic protein-2 (BMP-2) (a ), Osterix (Osx) (b ) and tartrate-resistant acid phosphatase (TRAP) (c ) expression. Arrows show the expression in an immunoreactive osteogenic cell.
Fig. 3 Graph of mean value and standard deviation (SD) in the bone morphogenetic protein-2
(BMP-2) expression of each group.
The expression of Osx is shown in [Fig. 2 (b) ]. The number of Osx expressions in each group is presented in [Fig. 4 ]. The Osx normality test with Kolmogorov–Smirnov is normal (p = 0.868) and the homogeneity test with Levene statistic is homogenous (p = 0.138). The one-way ANOVA showed significantly difference between groups (p = 0.000). The Tukey HSD test showed Osx expression had significantly increased in
the SC4 and SC8 groups compared with the other groups. The lowest Osx expression was
observed in the G4 and G8 groups (SC4 = 0.010 to G4) (SC4 = 0.002; SC8 = 0.030 to
G8).
Fig. 4 Graph of mean value and standard deviation (SD) in the Osterix (Osx) expression of
each group.
The expression of TRAP is shown in [Fig. 2 (c) ]. The number of TRAP expressions in each group in contained in [Fig. 5 ]. The TRAP normality test with Kolmogorov–Smirnov is normal (p = 0.922) and the homogeneity test with Levene statistic is homogenous (p = 0.782). The one-way ANOVA showed significantly difference between groups (p = 0.000). The Tukey HSD test showed TRAP expression was significantly reduced in
the SC4 and SC8 groups compared with the other groups. The lowest TRAP expression
was observed in the SC4 and SC8 groups (SC4 = 0.000 to G4 and G8) (SC8 = 0.034 to
G8).
Fig. 5 Graph of mean value and standard deviation (SD) in the tartrate-resistant acid phosphatase
(TRAP) expression of each group.
Discussion
Estrogen deficiency, resulting in postmenopausal disorders, causes a decrease in stem
cells and triggers disruption in MSCs, which is the source of osteoprogenitor and
osteoblasts in bone formation.[15 ] Reduced bone density in osteoporosis occurs due to MSCs defects, leading to lower
osteoblast proliferation and differentiation.[16 ] MSCs, which are sourced from hUCMSCs, undergo several changes, transforming them
into osteoprogenitors and, eventually, osteoblast cells. The differentiation between
MSCs and osteoblasts includes certain growth factors as well as transcription factors
such as BMP-2 and Osx.
The results of this study support those of previous research, which reported significant
differences in TGF-β1 and RUNX-2 expression between the administration of hUCMSCs
and osteoporosis.[8 ] BMP-2 is known to be a cytokine group within the TGF-β subgroup. Osx constitutes
a downstream of RUNX-2. The increase in BMP-2 and Osx expression in osteoporotic subjects,
induced by hUCMSCs, shows an increase in osteoblastogenesis activity.
The increase expression in BMP-2 and Osx expression induced by hUCMSCs is in line
with other studies, indicating that BMP-2 expression in the osteoporosis group is
significantly lower than the normal control group. Another study showed that in the
osteoporotic group, a decrease in the osteoblastogenetic process occurred, which was
characterized by a lower Osx expression value than the normal control group.[17 ] This finding is in line with those of previous studies, showing that Osx expression
in the osteoporosis group was significantly lower than in the normal control groups.[18 ]
BMP-2 works in a matrix and is inactive during the homeostatic phase. The occurrence
of defects in the bone triggers remodeling commencing with resorption by osteoclasts,
accompanied by a quiescent period (silence) during which the osteoblastogenetic process
begins. The increase of BMP-2 expression is caused by BMP-2 which initiates the differentiating
of mesenchymal cells into preosteoblasts and toward mature osteoblasts. During this
phase, the concentration, location, and expression time of the BMP-2 gene must be
regulated appropriately.[19 ]
Osx will be produced by stem cells and osteoprogenitor to ensure that stem cells differentiate
specifically into osteoblasts. Stem cells sourced from hUCMSCs can replace bone defects
that occur with the result that it becomes a source of osteoprogenitor and osteoblasts.
The increase in the number of stem cells will promote increased bone regeneration.[20 ] Previous studies have mentioned that the high-expression of Osx after bone marrow
mesenchymal stem cell (induction) will intensify osteoblastogenesis during in vivo bone regeneration.[21 ]
The decreased of TRAP expression is caused by the stem cells inhibiting osteoclast
differentiation, as indicated by TRAP expression in chondroclasts, which is capable
of degenerating skeletal phosphoproteins including OPN.[12 ] TRAP is strongly expressed in osteoclasts. TRAP staining has been generally employed
to identify osteoclasts and undifferentiated monocytes. TRAP is considered an important
biochemical marker for osteoclasts, and its serum concentration serves as a biochemical
marker of osteoclast function and the degree of bone resorption.[22 ]
A previous study demonstrated that decellularized extracellular matrix (ECM) stem
cells strongly inhibit the osteoclastogenesis of primary BMMs. This inhibitory effect
of ECM-derived cells on osteoclast differentiation has already been indicated by fewer
TRAP-positive multinucleated cells and lower levels of osteoclast-specific gene expression.[22 ]
Labeling of PKH 26 serves to prove the existence of hUCMSCs that are injected into
the mandibular bone and enable observation of its internal distribution. The presence
of hUCMSCs produce a powerful florescent green color. Strong luminescence indicates
the greater presence of such cells. To date, no studies have been conducted on such
a method which involves direct, local injection into the osteoporotic mandibular bone.
While several similar studies have involved the local administration of MSCs, these
relate to different organs.
Previous studies into rat muscles involved analysis of a crush injury, administering
a local injection of MSC, confirming the presence of stem cells in the MSC injection,
and their migration from the local injection to all of the subject’s muscles.[23 ] The research undertaken by Wang involves attempts to administer MSC to the distal
femur, in order to promote an overall increase in the thickness of the trabecular
femur compared with those in the controls.[24 ] Several studies have shown that localized therapy will produce a strong localized
response. If closely associated with osteoporosis, stem cells can be systemically
administered in order that they identify and move to sites requiring improvement in
a process often referred to as homing.
Certain disadvantages of this systemic use include differences in the effects of MSC
in specific sites, with the result that further research is required to enable regulation
of the ability of cells in vivo within the body.[25 ] This labeling of cells indicated two important facts. The first was that exogenous
hUCMSCs were obtained externally to the mandibular bone in addition to the host MSC
that was indeed in the body. The second was the ability to locally migrate to the
bone tissue around the injection site, with the resulting possibility of this MSC
being obtained for therapy at the injection site and its surroundings.
Conclusions
Augmented BMP-2 and Osx expression in osteoporotic mice induced by hUCMSCs produced
an increase in osteoblastogenetic activity. The lowest TRAP expression in osteoporotic
mice induced by hUCMSCs indicated a decrease in osteoclastogenesis activity. The result
was that bone regeneration therapy could be managed by the induction of hUCMSCs.
Suggestion
Researchers suggest doing research with other types of markers that are not yet studied.
To obtain better results and improve the use of hUCMSCs as a degenerative disease
therapy, for example, diabetes mellitus.
