INTRODUCTION:
The World Health Organization (WHO) had declared that the prevalence of the condition will increase from 135 million sufferers to 300 million sufferers by 20253.
Diabetes causes various complications within the oral cavity. The oral health disorders presented by individuals afflicted with diabetes mellitus vary greatly from mild to severe with various complications, including increased incidence of periodontal disease, caries, xerostomia, burning mouth syndrome, changes in oral flora, loss of oral mucosa resistance, bone resorption and impediments to the manufacture of dental prostheses that frequently result from bone loss4,5.
In diabetics, the pathological state of high blood glucose levels can accelerate a glycosylative reaction, resulting in large amounts of Advanced Glycation End products (AGEs). AGEs represent end products of amino-non-enzymatic catalytic reactions (proteins, lipids, and nucleic acids) and aldehyde groups of carbohydrates due to prolonged hyperglycemic conditions6. AGEs alone can induce a number of pathological changes. In addition, a number of studies have demonstrated that poor glycemic control in patients with type 1 DM is also associated with osteoporosis. This is because AGEs interfere with the activity of osteoblasts and osteoclasts which, if disrupted, will lead to inhibition of the osteogenesis process7.
Prosthodontic treatments relate closely to the condition of the alveolar bone. Loss of alveolar bone density may constitute a complicating factor during prosthodontic treatment and become an obstacle in certain treatments for tooth loss replacement such as implants. The prerequisites of successful implant treatment comprise bone regeneration, osseointegration, and bone formation associated with osteogenesis8.
Stem cells are defined as primitive cells that demonstrate the ability to continually divide and replicate themselves, thereby producing special cells that can differentiate into various types of cell or tissue. Stem cells carry out specific functions involving self-renewal of varing potential, while also possessing the capacity to differentiate into multilineages. Stem cells currently constitute a prominent contemporary subject of research, including within the field of dentistry9-12
The ideal cell source for tissue engineering must satisfy the requirements of sufficient quantity, ease of acquisition, and high immunocompability. One of the most intensively researched sources of mesenchymal stem cells (MSC) is that of Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) derived from umbilical cord tissue since they have the advantage of being comparatively readily available. hUCMSCs are considered to constitute invariably discarded biological waste whose use presents no ethical problems and which can be easily reproduced in a short period of time13-15.
Previous research has shown that diabetes-related AGEs significantly inhibit MSCproliferation in vitro through upregulation of oxidative stress markers and damage to the homeostasis of the intracellular environment16. High concentrations of oxygen in the niche can cause oxidative stress through the production of Reactive Oxygen Species (ROS) which are free radicals capable of damaging lipids, proteins and DNA, and influencing cell metabolism17,18.
AGEs accumulated in bone play a functional role in the incidence of diabetes-associated osteoporosis. Studies of AGEs have confirmed their ability to significantly inhibit proliferation and induce osteoblast apoptosis19. AGEs also increase ROS that can inhibit osteogenic molecular gene expression in osteoblasts, especially RUNX220. This study seeks to determine the effect of AGE products on hUCMSCs RUNX2 expression and the degree of calcification representative of a DM micro environment in vitro as a guide for the future research.
MATERIALS AND METHODS:
This experimental laboratory study involved the use of hUCMSCs obtained from the Stem Cell Research and Development Center, Universitas Airlangga. Ethical approval for this research was granted by the Ethical Research Commission, Faculty of Veterinary Medicine, Airlangga University. (2.KE.152.09.2018).
hUCMSCs Isolation:
Umbilical cords were extracted from the placenta of a healthy newborn delivered by Cesarean section with uncomplicated elective indications. The benefits of this study were explained to the research subjects who subsequently signed an informed consent form. The isolation and multiplication procedures relating to the hUCMSCs were conducted in accordance with the standard procedures of the Stem Cell Research and Development Center of Universitas Airlangga by modifying several stages that had previously been implemented by Hendrijantini et al. (2015). The umbilical cord was cut into 10cm lengths before being placed in sterile boxes lined with sterile gauze and washed with phosphate-buffered saline (PBS) three times in three different tubes to remove any residual blood. The umbilical cord was washed again with Ringer Lactate (RL) containing 2.5μg/mL gentamicin and 1000U/mL amphotericin for 20 minutes. It was then transported to the laboratory in a cooler box.
The umbilical cord was cut into 1 mm3 sections, cleaned, separated from arteries, veins, and adventitia. After that, the umbilical cord wasimmersed in a 0.75mg/mL collagenase IV cone tube and 37℃ 0.075mg/mL of DNAse I for 40 minutes in a medium hot plate stirrer for 15 minutes. The subsequent steps involved filtering the umbilical cord through a cell filter and collecting the pellets. The supernatant was removed and centrifuged at 1,800 rpm for six minutes in a procedure repeated twice.
The pellets were transferred to a petri dish and stored in a 5% CO2 incubator at 37ºC. Daily observation of cell growth through an inverted microscope was undertaken until it reached the confluent stage when the cell population reached a size enabling the transition process to be initiated9.
Flowcytometry Assessment:
Flowcytometry assessment of passage 4 was performed by tripination and suspension of hUCMSCs in αMEM medium, after which it was washed with PBS and fixed in 10% formaldehyde solution for ten minutes. It was covered with AGE-BSA solution for one hour prior to the cells being incubated for 40 minutes by means of a Human MSC Analysis Kit (BD StemflowTM, BD Biosciences) with the addition of anti-human antibodies CD 73, CD 90, CD 105, and negative cocktails CD45 and CD34. Unbonded antibodies were removed by PBS washing. Primary antibodies bound were labeled using FITC conjugated anti-human antibodies following a 30-minute period of incubation. The cells were then analyzed using an FACSCaliburflowcytometer (BD Biosciences, Franklin Lakes, NJ, USA).
AGE-BSA Preparation:
Modified AGE-BSA was produced by reacting BSA with glycolaldehyde under sterile conditions followed by extensive dialysis and purification. The doses were 0.39 mM.
Assessment of RUNX2 Expression:
Monolayer cells are transformed into single cells through a process of trypsination. Centrifugation was completed at a speed of 1,800 rpm for six minutes. Pellets were added to 1ml αMEM/f12 of growth medium, resuspended and planted on a 24-well microplate until confluent. The medium was replaced with maintenance medium for up to 21 days, at the end of which period the cells present on the slip cover were ready to be harvested. Fixation with 3% formaldehyde was undertaken for 15 minutes at room temperature, followed by four washes with PBS twin and drying. 10% PBS blocking was added for 15 minutes and washed again with twin PBS. RUNX2 antihuman antibodies labeled with FIT-C (Biologend, CA, USA) were added to each sample, incubated at 37°C for two hours, washed with PBS and dried with tissue paper. 50% glycerin was added to a glass object in order to enable immediate observation of the results through a fluorescent microscope at 40x magnification.
Alizarin Red:
The fixative solution was removed and the culture washed with water up to three times with the objective of preventing damage to the monolayer. One milliliter of red alizarin was added to each well and incubated for 20 minutes. The coloring agent was then disposed of and the incubated material washed thoroughly with sterile aquades. A differentiated cell contained a deposit mineral bright red in color (9).
Data Analysis:
Statistical analysis was performed using a Statistical Package for the Social Sciences Software (SPSS) edition 24.0 (SPSSTM, Chicago, United States) and the data obtained subsequently analyzed by means of a one way analysis of variance (ANOVA) test and an independent T-test.
RESULTS:
Isolation and Culture:
During the first 24 hours, almost all the cells were oval in shape. Twenty four hours later, the cells were attached to the tube and had assumed a spindle or fibroblast-like form. After a further three days, these cells achieved 90% confluence (see Figure 1).
Figure 1. hUCMSCs culture. A. 24 hours, almost all cells are oval in shape. B. After three days, the cells assume a spindle or fibroblast-like shape.
hUCMSCs Characterization:
This procedure was performed to examine antigen expression on the surface using immunocytochemistry and flowcytometry in passage 4. The results indicated that hUCMSCs expressed positive CD73, CD90 and CD105 but negative CD45.
Figure 2. Flowcytometry results for CD90 with Neg PE
From Figure 2, it can be seen that the flowcytometry results for the CD90 control were 53% and 22.63%. This meant that the cells expressed positive CD90. For the Neg PE control, the results were 0% and 22.63%, meaning that the cell expresses Negative Neg PE.
Figure 3. Flowcytometry results for CD105 withNeg PE
The flowcytometry results contained in Figure 3 were 90.28% for CD 105 and 21.16% for the control. These results indicated that those cells expressed positive CD105.
Figure 4. Flowcytometry results for CD105 and CD73
The flowcytometry results contained in Figure 4 showed that cells expressing CD73 were positive at 6.08% and 3.83% in the control.
Data Collection and Sample Characteristic:
Data collection was started during passage 5, comprising the amount of AGE secreted by hUCMSCs into the medium. Every change of medium color was observed and examined with an ELISA reader. Seven media were collected on each of days 3, 6, 9, 12, 14, 17, and 21, the corresponding data of which can be seen in Figure 6.
Figure 6. Bar graph of the differences in each group (significant differences shown in*) using a Tukey HSD and between groups (significant differences showed in *) using an independent t-test (sig < 0.05).
The data was analyzed using an ANOVA which indicated differences between the treatment group (sig = 0.00) and the control group (sig = 0.00). The data was then analyzed by means of a Tukey HSD (sig < 0.05) in order to identify the differences. The calculations performed indicated that in the AGE-BSA group, the peak level of RUNX2 expression occurred on days 7 and 14 and that there was a decrease in the level of RUNX2 on day 9. The peak RUNX2 expression level in control group was found to have occurred on day 14.
Osteogenic Differentiation with Alizarin Red:
After 21 days immersed in osteogenic media, the cells assume Alizarin red coloring indicating that osteogenic differentiation had occurred and calcium-rich tissue had been formed. In the control media, mineralization is clearly observable. The osteogenic media added to the AGE experienced mineralization which produced a bright color.
Figures 5.3 Microscopic review of Alizarin red
staining. A. Osteogenic medium of control group containing a number of brightly
colored regions which indicate the presence of mineral deposits. B. In
osteogenic medium of treatment group, added by AGE
DISCUSSION:
Diabetes mellitus (DM) is a metabolic disease which has assumed the proportion of a global epidemic due to its being widespread within the population causing high levels of morbidity and mortality22. Moreover, its prevalence increases with age as is also the case with osteoporosis, a higher risk of which is associated with Diabetes mellitus (DM) type 2. Several factors have been considered as possible mechanisms responsible for this disease, among which are changes in bone remodelling that may be induced by altered glucose circulation and also by the existance of AGEs. The latter form gradually with age, as well as in response to physiological levels of sugar and more concentrated hyperglycemic environments.
The latest prosthodontic treatment is that of dental implants, some of the keys to the success of which include: bone regeneration, osseointegration, and bone formation associated with osteogenesis (8). Stem cell technology-based bone engineering is becoming an increasingly-employed technique to increase the success rate of this treatment option in diabetic patients23,24. On the other hand, the results of this study indicate both the source of stem cells as originating from hUCMSCs together with their osteogenic potential in vitro. Secondly, the findings produced by this initial study of hUCMSCs could potentially represent an enhanced form of palliative therapy for patients with a medical history of diabetes.
The material used in this study consisted of hUCMSCs derived from Wharton’s Jelly. Mesenchymal stem cells (MSCs) are characterized by means of flowcytometry assessment, as this is a hematopoietic marker, marked by positive expression of CD105, CD73, and CD90 and the absence of CD14, CD31, CD34, CD45, CD106, and HLA-DR (25). From the flowcytometry assessment results, it was ascertained that the isolated Wharton’s Jelly utilised in this study expressed positive CD105, CD73 and CD90 and negative CD45, thereby indicating that the cells were positive Mesenchymal Stem Cells.
In cases of Diabetes Mellitus, not only are changes in the amount and activity of the bone forming cells impressive, but also the response of these cells to local and systemic factors which contribute to bone remodeling process. During the processes of bone formation and remodelling, signal factors determine the differentiation, replication and survival of osteoblast cells which are critical to enhancing bone metabolism.
One of the most important factors in determining bone formation is RUNX2, one of the multifunctional transcription factors which plays an important role in osteoblastic differentiation and osteogenic specifications. RUNX2 controls bone development and directs the multipotential mesenchymal cells towards the osteoblast cell lineage, while also improving early stage osteoblast differentiation and inhibiting osteoblast differentiation in the final stages26.
During the early stages, RUNX2 is known to have the opposite effect when compared with the later stages of osteoblastic differentiation. During early differentiation, RUNX2 directs multipotent mesenchymal cells to the osteoblastic lineage and inhibits them from differentiating to the chondrocyte and adipocyte lineage 27. The existence of this process is confirmed by the results of this study since RUNX2 expression increased between days 3 and 7.
After multipotent mesenchymal cells have
differentiated into preosteoblasts, RUNX2 induces them to become immature
osteoblasts, subsequently inhibiting their further maturation and transition
into osteocytes and maintaining osteoblasts in an immature stage28.
Moroever, the findings of this research indicated that the level of RUNX2
expressed by hUCMSCs in AGE medium increased from day 12, peaking on days 14
and 21. These research findings were very similar to those of Notsu et al.
(2014) which confirmed that the AGE-TGF 
signal increased RUNX2 mRNA expression in
ST2 cells on day 14 after treatment with AGE. This caused the suppression of
osteocalcin and osterix expression and the enhancement of RUNX2 expression at
the late osteoblastic differentiation stage, as these would probably inhibit
the differentiation of preosteoblast necessary to mineralize mature
osteoblasts. The increase in RUNX2 expression during the late stage of
osteoblastic differentiation inhibits bone maturation28.
In the study by Notsu et al. (2014), AGE – receptor for advanced glycation endproducts (RAGE) signaling induces the suppresses mineralization of mouse stromal ST2 cells in addition to hMSCs by increasing TGFβ expression and secretion. These findings showed that AGE-TGFβ signaling produces negative effects which impair osteoblastic differentiation in both cell types during the maturation stage. Certain accumulated evidence also indicates that the TGFβ signal suppresses osteoblast differentiation in vitro.28
AGEs induce apoptosis and suppress cell growth in osteoblasts, while AGE-RAGE signaling expressed in osteoblasts brings about TGFβ expression and secretion. TGFβ, in turn, promotes RUNX2 expression during the late stage of cell differentiation, thereby reducing bone formation. According to the study by Kanazawa (2017), this process enhances the calcification of vascular smooth muscle cells in rats, in contrast to its effect on osteoblastic cells. These findings suggest that the biologic activities of AGE result in bone fragility and vascular calcification29.
The level of RUNX2 in the AGE-BSA group decreased earlier than in the control group. In the AGE-BSA treatment group, the amount of RUNX2 declined earlier on day 8. This finding may be caused by AGE-BSA in the treatment group which affects the number of hUCMSCs through the production of ROS. The reaction between ROS and the lipid membrane will form MDA that possesses cellular damage properties. On the other hand, ROS may react with Fe/Cu ions with the result that they produce hydroxyl radicals (OH*). These radicals can translocate into the cell nucleus causing the destruction of genetic components/DNA fragmentation culminating in cell death, a process known as apoptosis 30. As the level of hUCMSCs decreases, that of RUNX2 also falls.
Osteogenic differentiation of hUCMSCs Alizarin Red staining is performed to detect osteoblastic differentiation in hUCMSCs by identifying the presence of extracellular calcium deposits. Osteoblasts will change color to bright red, whereas undifferentiated MSC does not produce a certain coloration31. In this study, cultures placed on the osteogenic medium showed red staining indicating that hUCMSCs can differentiate into osteoblastic lineage in an osteogenic environment. This was also reported in various studies where stem cells isolated from hUCMSCs presented multilineage differentiation, one of which developed into an osteoblast13. In the osteogenic medium to which AGE was added, the red staining was not as significant as in the medium without AGE. The red staining was still visible because of the osteogenic medium used. On the other hand, it was less significant because the AGE added to the medium inhibited the osteoblast differentiation.
CONCLUSION:
From this study, it can be concluded that AGE increases expression in hUCMSC at the initial and maturation stages on the seventh and the fourteenth days This is also proven by the Alizarin red examination during which an increase in RUNX 2 on day 21 indicated a lower level of mineralization.
CONFLICT OF INTEREST:
The authors declare no conflict of interest
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Received on 09.05.2020 Modified on 20.07.2020
Accepted on 23.08.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(8):4019-4024.
DOI: 10.52711/0974-360X.2021.00696