Keywords
diabetes mellitus - osteoporosis - bone remodeling - medicine
Introduction
Implant failure can occur due to low bone mineral density which results in imperfect
osteointegration between the implant and the bone. Many factors can cause a decrease
in bone mineral density, one of which is systemic diseases, such as diabetes mellitus
(DM) and osteoporosis. Based on previous studies, the percentage of implant failure
in DM conditions is around 14.3% and osteoporosis is around 10.9%.[1]
[2]
[3]
DM is a chronic disease that occurs when the pancreas is unable to produce enough
insulin or when the body is unable to effectively use the insulin it produces. The
prevalence of DM worldwide in 2014 reached 8.5% of the entire adult population, and
in Indonesia, the prevalence of DM in 2013 in adult individuals reached 6.9%. This
condition that can lead to failure of dental implant treatment for it is associated
with prolonged wound healing, the prevalence of microvascular disease (chronic hyperglycemia),
and impaired response to infection. Histological analysis showed that bone implant
contact in diabetic experimental animals was smaller than nondiabetic. The rates of
mineral apposition on new bone formation and bone density around dental implants were
significantly lower in uncontrolled diabetic experimental animals.[4]
[5]
[6]
[7]
[8]
[9]
DM causes changes in bone remodeling and reduced remineralization which results in
poor osseointegration, therefore, lead to osteoporosis.[10]
[11]
[12]
[13]
[14] Osteoporosis is a progressive systemic bone disease characterized by reduced bone
mass or density and microarchitecture deterioration of bone tissue. The Indonesian
Osteoporosis Association in 2007 reported that the proportion of osteoporosis sufferers
in the population over 50 years of age ranges from 32.3% in women and 28.8% in men.
The etiology of osteoporosis is an increase in bone resorption and a decrease in bone
formation, an imbalance between osteoclast and osteoblast activity. The activity of
osteoclasts and osteoblasts is influenced by the balance between osteoprotegerin (OPG),
receptor activator of nuclear factor κB ligand (RANKL), and receptor activator of
nuclear factor-κB (RANK) which directly plays an important role in the physiological
process of bone remodeling. This complex of three molecules functions as a receptor
and a ligand. The interaction between RANK–RANKL signifies the initiation of osteoclastogenesis
and activation of osteoclasts. The interaction between OPG and RANKL serves to offset
the effect of the RANK–RANKL interaction.[5]
[15]
[16]
[17]
Bone resorption is related to osteoclast and osteoblast activity. Many biomarkers
can be used to see the activity of these cells. A decrease in the osteoblast/osteoclast
ratio, an increase in the RANKL/OPG ratio, and the level of tartrate-resistant acid
phosphatase (TRAP) secreted by osteoclasts during the bone resorption process are
important biomarkers that can indicate the activity of these cells.[18]
[19]
In an in vivo study, hyperglycemia, one of the characteristics of DM, was associated with decreased
expression of Runt-related transcription factor 2 (RUNX2), where the transcription
factor of this gene is the “master regulator” of osteoblast development.[20] Osterix is another transcription factor that plays important role in osteoblast
differentiation and matrix mineralization. Osterix promotes osteoblasts in the late
maturation stage and inhibits their proliferation by regulating the canonical Wnt/β-catenin
pathway.[21]
One of the important steps in the success of bone formation is angiogenesis at the
wound site. Vascularization provides not only a supply of nutrients and growth factors
but also the migration of osteoprogenitors and osteoclasts from hematopoietic precursors.
The process of angiogenesis is impaired in a diabetic mouse model. Decreased vascular
endothelial growth factor (VEGF) expression is also an indication of impaired angiogenesis.
Skeletal vascularization and bone remodeling including differentiation of osteoblasts
and bone resorption by osteoclasts are important keys to bone healing. VEGF is known
to be a major angiogenic and osteogenic promoter to facilitate bone regeneration.[22]
This study will inspect at the differences in osteoblast/osteoclast ratio, RANKL/OPG
ratio, TRAP, RUNX2, Osterix, and VEGF expressions in Wistar rats with DM and osteoporosis.
In osteoporosis and DM conditions, there is a change in the remodeling process in
the bones that leads to a decrease in bone density. This decrease can affect the success
of dental treatment, especially dental implants. In these two systemic conditions,
we will compare how the bone remodeling process is seen based on markers of osteoblastogenesis
and osteoclastogenesis so that we can find out which conditions are most at risk for
dental implant failure. The results of this study are expected to help determine the
optimal dental implant treatment plan in this condition.
Materials and Methods
Materials and Instrumentations
Materials for the preparation of experimental animal models of DM: streptozotocin
(STZ), citrate buffer. Materials for termination of experimental animals and taking
of research specimens: 10% ketamine, 1 mL xylazine 2%, 4% paraformaldehyde, 10% buffered
formalin, ethylenediaminetetraacetic Acid (EDTA). Materials for staining and reading
hematoxylin–eosin (HE) are slide, xylol solution, absolute ethanol, 70% ethanol, Meyer's
HE solution, eosin solution, adhesive liquid (DPX), cover slip, and fluorescent microscope.
Materials for immunohistochemical (IHC) staining are xylol solution, absolute ethanol,
70% ethanol, aquabidest, H2O2 3%, phosphate buffered saline solution, 0.025% trypsin, ultra V block, monoclonal,
primary antibody enhancer, and horseradish peroxidase.
The instruments used in the study were a 3-mL syringe, 10-mL syringe, sterile cotton,
gauze, dental tweezers, chirurgie tweezers, surgical scissors, a scalpel handle, scalpel
blade, sewing needle, absorbable sewing thread, arterial clamp, and Nikon H600L light
microscope.
Animals and Study Design
All animal experiments and procedures were approved by Faculty of Dental Medicine
Health Research Ethical Clearance Commission, Universitas Airlangga (553/HRECC.FODM/VIII/2019).
Eight- to 10-week-old female Wistar rats (n = 27 [determination of the number of samples in our study using Lameshow. From the
calculation, the minimum number of samples per group of 8.9 is fulfilled to 9]; weighing
150–200 g) were acclimatized in Stem Cell Research and Development Centre, Institute
of Tropical Disease animal facility under a 12-hour light–dark cycle with ad libitum
access to standard chow and water. Wistar rats were randomly divided into three groups:
diabetic, osteoporotic, and control groups. Diabetic group (P1; n = 9) was injected with STZ intraperitoneally (20 m/kg body dissolved in sodium citrate
buffer pH 4.5) for 5 consecutive days. Based on our previous experiments using STZ
and alloxan, the lowest animal mortality rate was found with multiple low doses of
STZ. To calibrate the hyperglycemic condition in the model, blood glucose checks are
performed periodically every day using a glucometer through blood taken from rat tails.
Blood glucose was monitored 1 day after the STZ injection using glucometer. Animals
were considered diabetic when their blood glucose levels were > 300 mg/dL, they were
maintained for 4 weeks. After injection of STZ with multiple low doses for 5 days
and waiting for 4 weeks, the general condition of the experimental animals was in
good condition. There was no significant change in weight loss. Osteoporotic group
(P2; n = 9) was underwent bilateral ovariectomy and maintained for 12 weeks to induce osteoporosis
and have blood glucose level < 100 mg/dL. Control group (n = 9) was left without any intervention and have blood glucose level < 100 mg/dL This
group was maintained for 1 week. All rats were killed and mandibles were obtained
for histological staining and IHC evaluation.
Hematoxylin–Eosin Staining
Harvested mandibles (first molar region) were fixed in 10% buffered formalin, then
were decalcified in 10% EDTA for 2 months. After dehydration, mandibles were embedded
in paraffin and cut into sagittal section of 5 µm for HE staining, HE examination
to see the number of osteoclasts and osteoblasts seen in five visual fields. Image
acquisition was performed using a Nikon H600L microscope under ×200, ×400, and ×1,000
magnification, DS-Fi2 camera, and Nikon image system software.
Immunohistochemical Evaluation
The demineralized and paraffin-embedded mandibles were cut into 5-μm sections and
treated with 3% H2O2 for 5 minutes at room temperature. Then, sections were blocked with ultra V block
and incubated with prediluted RUNX2 monoclonal antibody (RUNX2 [27-K]: sc-101145)
(Santa Cruz Biotechnology, Inc., United States), Osterix monoclonal antibody (OSX
[F-3]: sc-393325) (Santa Cruz Biotechnology, Inc.), VEGF monoclonal antibody (VEGF
[C-1]: sc-7269) (Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-TRAP antibody
(1:100, ABIN6654430, antibodies-online GmbH), mouse monoclonal anti-RANKL antibody
(1:100, ABIN5611143, antibodies-online GmbH), and mouse polyclonal anti-OPG antibody
(1:100, ABIN925985, antibodies-online GmbH) for 25 to 30 minutes at room temperature.
All slides were incubated for 15 minutes at room temperature with secondary antibodies
and counterstained with hematoxylin. The expression of RUNX2, Osterix, VEGF, TRAP,
RANKL, and OPG were evaluated using the Remelle scale index (immunoreactive score
[IRS]) which was the result of multiplying the percentage score of immunoreactive
cells with the color intensity score of immunoreactive cells. The data for each sample
are the IRS mean value in five fields of view on a ×200, ×400, and ×1,000 magnification.
All examinations used a Nikon H600L light microscope equipped with a 300-megapixel
DS-Fi2 digital camera and image processing software Nikon Image System ([negative = 0,
low intensity = 1, moderate intensity = 2, and strong intensity = 3] × [negative = 0,
positive cell < 10% = 1, positive cell 11–50% = 2, positive cell <51–80% = 3, positive
cell > 80% = 4]).
Statistical Analysis
All data are presented as the mean ± standard deviation. Statistical analysis was
performed using the statistics package SPSS 22.0 (IBM, Armonk, New York, United States).
Comparison between the groups was determined using one-way analysis of variance with
least significant difference multiple comparison test and Kruskal–Wallis' test with
Mann–Whitney's test. A p < 0.05 was considered to be statistically significant.
Results
Osteoporosis and DM Reduced RUNX2 Expression
RUNX2 expression observed by IHC staining is shown in [Fig. 1]. The osteoporosis group showed significantly lower RUNX2 expression than the control
and DM groups. The DM group showed a lower, but not statistically significant, expression
of RUNX2 than the control group ([Fig. 2]).
Fig. 1 Representative images of immunohistochemical RUNX2 expression in the mandible of
Wistar rats in diabetic group with ×200 magnification (A), ×400 magnification (B), ×1,000 magnification (C); osteoporotic group with ×200 magnification (D), ×400 magnification (E), ×1,000 magnification (F); and control group with ×200 magnification (G), ×400 magnification (H), ×1,000 magnification (I); and RUNX2-positive cells were observed (red arrow). RUNX2, Runt-related transcription
factor 2.
Fig. 2 Quantitative analysis of immunohistochemical staining cell expressions. Data are
presented as mean ± standard deviation. n = 9. DM, diabetes mellitus; OPG, osteoprotegerin; RUNX2, Runt-related transcription
factor 2; RANKL, receptor activator of nuclear factor κB ligand; TRAP, tartrate-resistant
acid phosphatase; VEGF, vascular endothelial growth factor.
Osteoporosis and DM Reduced Osterix Expression
Osterix expression was determined by IHC staining as shown in [Fig. 3]. The osteoporosis and DM groups showed significantly lower Osterix expression than
the control group. The DM group showed lower, but not statistically significant, expression
of Osterix than the osteoporosis group ([Fig. 2]).
Fig. 3 Representative images of immunohistochemical Osterix expression in the mandible of
Wistar rats in diabetic group with ×200 magnification (A), ×400 magnification (B), ×1,000 magnification (C); osteoporotic group with ×200 magnification (D), ×400 magnification (E), ×1,000 magnification (F); and control group with ×200 magnification (G), ×400 magnification (H), ×1,000 magnification (I); and Osterix-positive cells were observed (red arrow).
Osteoporosis and DM Reduced VEGF Expression
VEGF expression was determined by IHC staining as shown in [Fig. 4]. The osteoporosis group showed significantly lower VEGF expression than the control
and DM groups. The DM group showed lower, but not statistically significant, VEGF
expression than the control group ([Fig. 2]).
Fig. 4 Representative images of immunohistochemical VEGF expression in the mandible of Wistar
rats in diabetic group with ×200 magnification (A), ×400 magnification (B), ×1,000 magnification (C); osteoporotic group with ×200 magnification (D), ×400 magnification (E), ×1,000 magnification (F); and control group with ×200 magnification (G), ×400 magnification (H), ×1,000 magnification (I); and VEGF-positive cells were observed (red arrow). VEGF, vascular endothelial growth
factor.
Osteoporosis, but not DM, Promoted the Expression of RANKL/OPG Ratio
RANKL/OPG ratio has a pivotal role in osteoclast formation and activation. The numbers
of RANKL and OPG-positive osteoblast were determined with IHC staining as shown in
[Figs. 5] and [6]. Compared with control group, either osteoporosis or DM led to decrease the number
of OPG-positive cells in the mandible. However, the number of RANKL-positive cells
was also downregulated in both osteoporotic and diabetic groups as compared with control
group. Therefore, only in osteoporotic group, but not in diabetic group, the expression
of RANKL/OPG ratio was increased ([Fig. 2]).
Fig. 5 Representative images of immunohistochemical RANKL expression in the mandible of
Wistar rats in diabetic group with ×200 magnification (A), ×400 magnification (B), ×1,000 magnification (C); osteoporotic group with ×200 magnification (D), ×400 magnification (E), ×1,000 magnification (F); and control group with ×200 magnification (G), ×400 magnification (H), ×1,000 magnification (I); and RANKL-positive cells were observed (red arrow). RANKL, receptor activator of
nuclear factor κB ligand.
Fig. 6 Representative images of immunohistochemical OPG expression in the mandible of Wistar
rats in diabetic group with ×200 magnification (A), ×400 magnification (B), ×1,000 magnification (C); osteoporotic group with ×200 magnification (D), ×400 magnification (E), ×1,000 magnification (F); and control group with ×200 magnification (G), ×400 magnification (H), ×1,000 magnification (I); and OPG-positive cells were observed (red arrow). OPG, osteoprotegerin.
Both Osteoporosis and DM Did Not Have Any Effect in the Expression of TRAP
TRAP is abundantly expressed in osteoclasts and considered as a marker of osteoclast
differentiation.[12] Therefore, we also observed TRAP-positive osteoclast using IHC staining as stated
in [Fig. 7]. Contrary to our expectations, neither osteoporosis nor DM upregulated the number
of TRAP-positive osteoclasts ([Fig. 2]).
Fig. 7 Representative images of immunohistochemical TRAP expression in the mandible of Wistar
rats in diabetic group with ×200 magnification (A), ×400 magnification (B), ×1,000 magnification (C); osteoporotic group with ×200 magnification (D), ×400 magnification (E), ×1,000 magnification (F); and control group with ×200 magnification (G), ×400 magnification (H), ×1,000 magnification (I); and TRAP-positive cells were observed (red arrow). TRAP, tartrate-resistant acid
phosphatase.
Osteoporosis and DM Reduced Osteoblast/Osteoclast Ratio
As shown in [Fig. 8], both osteoporosis and DM decreased the osteoblast/osteoclast ratio. The number
of osteoblasts did not show any significant difference between osteoporosis, DM, and
control groups. On the contrary, the number of osteoclasts was markedly increased
in the mandible of diabetic group ([Fig. 9]).
Fig. 8 Representative images of HE stained. HE stained of osteoblast (blue arrow) and osteoclast
(red arrow) in the mandible of Wistar rats in diabetic group with ×200 magnification
(A, G), ×400 magnification (B, H); osteoporotic group with ×200 magnification (C, I), ×400 magnification (D, J); and control group with ×200 magnification (E, K), ×400 magnification (F, L). The arrow showed osteoblast at the bone surface. HE, hematoxylin–eosin.
Fig. 9 Quantitative analysis of histological staining for osteoblasts and osteoclasts. Data
are presented as mean ± standard deviation. n = 9. DM, diabetes mellitus.
Discussion
Hyperglycemic conditions initiate the formation of advanced glycation end-product
(AGE) that can induce osteoblast apoptosis through the mitogen-activated protein kinase
pathway. Receptor of AGE expression is also increased in osteoblasts in DM, thereby
increasing the sensitivity of cells to the effects of AGEs. In addition, AGE was able
to inhibit the differentiation of mesenchymal stem cells (MSCs) which are osteoblast
precursors.[23] In addition, hyperglycemia can inhibit the Wnt/β-catenin signaling pathway where
it also plays an important role in RUNX2-stimulated osteogenesis.[24]
The reduction of RUNX2 could be due to the decrease in estrogen levels affecting the
suppression of MSC production and the number of preosteoblasts so that the number
of osteoblasts also decreases. The process of osteogenesis in osteoporosis has entered
the final phase of mature bone formation so that the amount of RUNX2 expression has
decreased. Under conditions of hyperglycemia, AGE was able to inhibit the differentiation
of MSCs into osteoblasts. It is possible that the effect of estrogen on the suppression
of MSC production is stronger than that of AGE which inhibits MSC differentiation.
However, this finding needs to be performed in more in-depth research to be able to
confirm this.[2]
In a previous study conducted by Hendrijantini et al[2] (2019), low levels of estrogen in the osteoporosis group affected the decrease in
the number of osteoblasts. One of the number of osteoblasts depends on transforming
growth factor (TGF)-β1. The role of TGF-β1 in the process of bone formation inhibits
the expression of RUNX2 thereby preventing maturation of osteoblasts from differentiating
into osteocysts.[2]
The diabetes group had lower Osterix expression than the control group. This is in
accordance with research conducted by Rios-Arce et al (2020) showing that the diabetes
group had lower Osterix expression than the control group. High blood glucose in the
diabetic group decreased extracellular signal-regulated kinase (ERK) phosphorylation.
The ERK pathway is known as a key stimulator of osteoblast differentiation.[25]
Osteoporosis model group was ovariectomized which causes estrogen levels in the body
to decrease so that the proliferation and differentiation of human bone marrow–derived
MSCs also decreases through the Notch signaling pathway. Notch signaling plays a role
in the proliferation and early differentiation of osteoblasts.[26] The condition of DM can cause a decrease in MSC differentiation. Postmenopausal
estrogen deficiency may also lead to decreased MSC differentiation. This is the reason
why there may be no difference in Osterix expression values in the DM and osteoporosis
groups.[27]
[28]
[29]
VEGF is a specific endothelial growth factor that stimulates endothelial cell proliferation
and differentiation, increases vascular permeability, mediates endothelial vasodilation,
and participates in interstitial matrix remodeling.
The decrease in the hormone estrogen caused by ovariectomy or menopause can increase
body fat mass. Visceral fat area is significantly increased in postmenopausal women
compared with premenopausal women. It is known that glucocorticoids have an important
role in the distribution of body fat mass. Glucocorticoids inhibit the production
of cyclooxygenase-2 and prostaglandin E2 (PGE2). So that PGE2 which has the ability
to synthesize VEGF is inhibited.[30]
Accordance with research conducted by Zhang et al (2018), chronic hyperglycemia can
stimulate the synthesis and secretion of VEGF.[31] VEGF accumulation can lead to microvascular complications. The main stimulus for
VEGF production is cellular hypoxia. Hyperglycemia acts as a toxin to the endothelium
by increasing oxidative stress. The high concentration of glucose in the blood increases
the production of vasoconstrictors, especially endothelin-1. In osteoporosis, there
is a decrease in the hormone estrogen which causes high levels of glucocorticoids
in the body. Glucocorticoids can inhibit the production of PGE2, where PGE2 plays
a role in VEGF synthesis. These findings can explain why the DM expression value is
higher than the osteoporosis group.[30]
[31]
The process of bone remodeling is also related to the RANKL/OPG ratio. The interaction
between RANK and RANKL signifies the initiation of osteoclastogenesis and activation
of osteoclasts. OPG is known as an osteoclast inhibiting factor. OPG is a binding
receptor and a competitor for RANK receptors to bind to RANKL. OPG binding to RANKL
inhibits osteoclastogenesis, thereby inhibiting bone resorption. The OPG–RANKL bond
offsets the effect of the RANK–RANKL bond. Bone formation can be associated with increased
OPG expression or reduced RANKL expression as indicated by a decrease in the RANKL/OPG
ratio. Conversely, a decrease in OPG expression or an increase in RANKL expression
can lead to pathological bone resorption characterized by an increase in the RANKL/OPG
ratio.[16]
[18]
[32]
This study found that the value of the RANKL/OPG ratio in the DM group was significantly
lower than in the osteoporosis and control groups. This is probably caused by hyperglycemia
which can cause a significant decrease in the RANKL/OPG ratio produced by osteoblasts.
Sassi et al found that there was a decrease in RANKL levels in DM patients. Hu et
al also found that high glucose levels inhibited RANKL-induced osteoclastogenesis
which significantly reduced RANKL gene expression during bone regeneration.[33]
[34]
[35]
This study found that the TRAP expression in the three study groups did not show a
significant difference. Several previous studies on DM also showed varied results,
Lu et al found that mice treated with DM showed a higher TRAP expression value than
the control group. In contrast, Hu et al found that mature osteoclast biomarkers,
including TRAP, were lower in DM group mice. Different results were also shown by
Xu et al who found that TRAP serum values in DM mice did not have a significant difference
with non-DM mice. The diversity of the results of this study is likely due to various
factors that can affect the variability of the study, such as differences in research
design, methodology, and other unknown factors.[33]
[36]
[37]
[38]
Several previous studies on osteoporosis have also shown varied results. Halleen et
al found that serum TRAP in postmenopausal women was higher than in premenopausal
women. In contrast, Verit et al found no significant changes in serum TRAP in both
postmenopausal women with osteoporosis, postmenopausal women without osteoporosis,
and premenopausal women. Kim et al also did not find any significant changes in the
expression of TRAP in the ovariectomy group mice with the control mice. This is probably
due to the duration of osteoporosis after the ovariectomy. Miyazaki et al found that
differences in expression of TRAP activity were more pronounced at study duration
of less than 8 weeks. In this study, specimen collection from experimental animals
was performed at week 12, and this factor is probably the reason that TRAP expression
in the osteoporosis group did not differ significantly from that of the control.[39]
[40]
[41]
[42]
The limitation of this study was the difference of observation time between osteoporosis
and DM condition; therefore, the development of those diseases can be different. The
future study was expected to have same and longer observation time so that the pathway
and mechanism of DM and osteoporosis in mandible can be more understandable and prevented.
Conclusion
The results of this study indicate that DM and osteoporosis can induce changes in
the bone remodeling pathway which are represented by a decrease in osteoblast biomarkers
and an increase in osteoclast biomarkers.
