Abstract
Objective:
To investigate the regeneration of rat’s salivary gland diabetic defect after intraglandular transplantation of Human Dental Pulp Stem Cells (HDPSCs) on acinar cell vacuolization and Interleukin-10 (IL-10).
Material and Methods:
HDPSCs isolated from the dental pulp of first premolars #34. HDPSCs from the 3rd passage was characterized by immunocytochemistry of CD73, CD90, CD105 and CD45. Twenty-four male Wistar rats, 3-month-old, 250-300 grams induced with Streptozotocin 30 mg/kg body weight to create diabetes mellitus (DM) divided into 4 groups (n=6); positive control group on Day-7; positive control group on Day-14; treatment group Day-7 (DM+5.105HDPSCs); treatment group on Day-14. On Day-7 and Day-14, rats were sacrificed. Histopathological examination performed to analyze acinar cells vacuolization while Enzyme-linked Immunoabsorbent Assay to measure IL-10 serum level. Data obtained were analyzed statistically using multiple comparisons Bonferroni test, Kruskal Wallis, Shapiro-Wilk and Levene’s test result
Results:
The highest acinar cell vacuolization found in control group Day 14 (0.239 ± 0.132), meanwhile the lowest acinar cell vacuolization found in treatment group Day 7 (0.019 ± 0.035) with significant difference (p=0.003). The highest IL-10 serum level found in treatment group Day 14 (175.583 ± 120.075) with significant difference (p=0.001)
Conclusion:
Transplantation of HDPSC was able to regenerate submandibular salivary gland defects in diabetic rats by decreasing acinar cell vacuolization and slightly increase IL-10 serum level.
Keywords:
Diabetes Mellitus; Totipotent Stem Cells; Interleukin-10; Acinar Cells
Introduction
Diabetes Mellitus (DM) is a progressive and chronic disease characterized by an increase in blood glucose levels. The prevalence of DM patients in the world was 8.8% and in Indonesia it was 6.2% [1[1] Ligita T, Wicking K, Francis K, Harvey N, Nurjannah I. How people living with diabetes in Indonesia learn about their disease: A grounded theory study. PLoS One 2019; 14(2):e0212019. https://doi.org/10.1371/journal.pone.0212019
https://doi.org/10.1371/journal.pone.021...
]. Salivary gland defect is one of the complications of DM in the oral cavity. Based on a recent meta-analysis study, the global prevalence of xerostomia in DM patients is 42.4%. Salivary gland defects can reduce the patient's quality of life because it causes difficulty in swallowing, speaking, chewing, dysgeusia, caries and an increase in plaque accumulation [2[2] Lessa LS, Pires PDS, Ceretta RA, Becker IRT, Ceretta LB, Tuon L, et al. Meta-analysis of prevalence of xerostomia in diabetes mellitus. Int Arch Med 2015; 8(224):1-13. https://doi.org/10.3823/1823
https://doi.org/10.3823/1823...
]. Clinically, salivary gland defects are characterized by decreased salivary secretion volume and histologically characterized by acinar cell vacuolization, which is an early sign of cell degeneration [3[3] Nugraha AP, Ernawati DS, Harijanti K, Parmadiati AE. Psychological stress induced xerostomia and hyposalivation: The case study in Indonesian female patient. J Int Dent Med Res 2019; 12(1):216-9.,4[4] Proctor GB. The physiology of salivary secretion. Periodontology 2000 2016; 70(1):11-25. https://doi.org/10.1111/prd.12116
https://doi.org/10.1111/prd.12116...
]. Nowadays, salivary gland defects are treated by performing salivary stimulation either through oral receptors using ascorbic acid, stimulation of mastication by chewing gum, and by pharmacological stimulation using pilocarpine. However, all of these therapies cause various complications and are only temporary relief; therefore, long-term definitive therapy is still needed [5[5] Villa A, Connell CL, Abati S. Diagnosis and management of xerostomia and hyposalivation. Ther Clin Risk Manag 2015; 11:45-51. https://doi.org/10.2147/TCRM.S76282
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[6] Ristevska I, Armata RS, D'Ambrosio C, Furtado M, Anand L, Katzman MA. Xerostomia: Understanding the diagnosis and the treatment of dry mouth. J Fam Med Dis Prev 2015; 1:008.-7[7] Cesar M, Justo R, Bonel M, Vidal J, Migowski E, Cisne R. The use of drugs for the treatment of xerostomia - Brief review. Int J Bas Appl Sci 2016; 5(4):238-44.].
Bone Marrow Mesenchymal Stem Cells (BMMSC) which are considered to be a solution to problems in various tissue damage related to the tissue regeneration capacity by differentiating into various cell lines, having a paracrine effect and having immunomodulation capacity. However, the isolation of BMMSC is very invasive. Human Dental Pulp Stem Cells (HDPSC) are considered as a better source of MSC because their extraction is less invasive than BMMSC, possess the ability of self-renewal, higher plasticity, and proliferation compared to BMMSC, but has a similar pattern of gene expression, fenotip and protein in vitro with BMMSC [8[8] Shi S, Robey PG, Gronthow S. Comparison of human dental pulp and bone marrow stromal stem cells by cDNA microarray analysis. Bone 2001; 29(6):532-9. https://doi.org/10.1016/S8756-3282(01)00612-3
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[9] Pierdomenico L, Bonsi L, Calvitti M, Rondelli D, Arpinati M, Chirumbolo G, et al. Multipotent mesenchymal stem cells with immunosuppresive activity can be easily isolated from dental pulp. Transplantation 2005; 80(6):836-42. https://doi.org/10.1097/01.tp.0000173794.72151.88
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[10] Ledesma-Martínez E, Mendoza-Núñez VM, Santiago-Osorio E. Mesenchymal stem cells derived from dental pulp: A review. Stem Cells Int 2016; 2016:4709572. https://doi.org/10.1155/2016/4709572
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-11[11] Kanwal A, D'souza J, Muthiah L, Srividya S. The current status of stem cell regeneration in intra oral applications - A systematic review. Open J Stomatol 2017; 7(4):197-224. https://doi.org/10.4236/ojst.2017.74015
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].
DPSC allegedly has a good salivary gland regeneration capacity by being a component of epithelial cells (acinar) itself or by being a salivary gland mesenchymal component to induce and support the regeneration process of the salivary glands. Therefore, stem cell-based regenerative therapy is expected to produce replacement tissue that can mimic the natural structure and function of the salivary glands and provide long-term benefits for salivary gland defects [12[12] Lombaert I, Movahednia MM, Adine C, Ferreira JN. Concise review: Salivary gland regeneration: Therapeutic approaches from stem cells to tissue organoids. Stem Cells 2017; 35(1):97-105. https://doi.org/10.1002/stem.2455
https://doi.org/10.1002/stem.2455...
[13] Bronckaers A, Hilkens P, Fanton Y, Struys T, Gervois P, Politis C, et al. Angiogenic properties of human dental pulp stem cells. PLoS One 2013; 8(8):e71104. https://doi.org/10.1371/journal.pone.0071104
https://doi.org/10.1371/journal.pone.007...
[14] Yoo C, Vines JB, Alexander G, Murdock K, Hwang P, Jun HW. Adult stem cells and tissue engineering strategies for salivary gland regeneration: A review. Biomater Res 2014; 18:9. https://doi.org/10.1186/2055-7124-18-9
https://doi.org/10.1186/2055-7124-18-9...
-15[15] Gonzalez R, Woynarowski D, Geffner L. Stem cells targeting inflammation as potential anti-aging strategies and therapies. Cells Tissue Transp Ther 2015; 7:1-8. https://doi.org/10.4137/CTTT.S19477
https://doi.org/10.4137/CTTT.S19477...
].
Material and Methods
Ethical Clearance
This study received ethical clearance for the use of animal subjects, number 047/HRECC.FODM/V/2018. Ethical permission was obtained in accordance with the guidelines of the Board for Animal Experiments at the Faculty of Dental Medicine, Airlangga University, which related to the Guidelines on the Care and Use of Animals for Scientific Purposes [16[16] Tan B. Guidelines on the Care and Use of Animals for Scientific Purposes. National Advisory Committee for Laboratory Animal Research; 2004.].
HDPSC Isolation and Culture
HDPSC was isolated from the healthy premolar tooth pulp #34 patients undergoing orthodontic therapy. The extracted teeth were immediately included in the Dulbecco’s Modified Eagle Medium (DMEM) (D5796, Sigma Aldrich Corp., St. Louis, MO, USA) and it was sent to the laboratory. The teeth were washed with Phosphate-Buffered Saline (PBS) solution. The teeth were washed and cut to separate the crown and roots; then the pulp tissue is taken. The pulp tissue was digested in a solution of 3 mg / ml collagenase type 1 (C9891, Sigma Aldrich Corp., St. Louis, MO, USA) and 4 mg / ml dispase (D4818, Sigma Aldrich Corp., St. Louis, MO, USA) for 30-60 minutes at 37ºC. HDPSC was obtained by filtering the digested tissue with 70 μm cell filters. 1 cell suspension (1x105 cells / flask) planted in α-minimum essential medium (α-MEM) (M8292, Sigma Aldrich Corp., St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 μM L-ascorbic acid-2-phosphate, 100 U / mL penicillin-G, 100 μg / mL streptomycin and 0.25 μg / mL fungizone (A9528, Sigma Aldrich Corp., St. Louis, MO, USA). Cells were conditioned at a temperature of 37ºC in 5% CO2 medium was replaced every 2-3 days. The cell culture was passaged three times to get the desired cell number [17[17] Jang JH, Lee HW, Cho KM, Shin HW, Kang MK, Park SH, et al. In vitro characterization of human dental pulp stem cells isolated by three different methods. Restor Dent Endod 2016; 41(4):283-95. https://doi.org/10.5395/rde.2016.41.4.283
https://doi.org/10.5395/rde.2016.41.4.28...
[18] Luisi SB, Filho MSA, Pranke P. Isolation, immunophenotypic characterization and pluripotency of dental pulp stem cells. Dent Oral Craniofac Res 2017; 3(5):1-3. https://doi.org/10.15761/DOCR.1000216
https://doi.org/10.15761/DOCR.1000216...
-19[19] Suciadi SP, Nugraha AP, Ernawati DS, Ayuningtyas NF, Narmada IB, Prahasanti C, et al. The efficacy of human dental pulp stem cells in regenerating submandibular gland defects in diabetic wistar rats (Rattus novergicus). Res J Pharm Tech 2019; 12(4):1573-9. https://doi.org/10.5958/0974-360X.2019.00261.0
https://doi.org/10.5958/0974-360X.2019.0...
].
Immunocytochemistry Analysis
HDPSC in the 3rd passages was then examined for cluster differentiation markers to ensure positive MSC markers, namely CD73, CD90, CD105 and negative CD45 markers. Cells were coated with coverslips, and after incubation at 37ºC for 1-2 hours, fixation was done with 10% formaldehyde (F8775, Sigma Aldrich Corp., St. Louis, MO, USA) for 15 minutes. Coverslips were rinsed four times. Monoclonal antibodies labeled FITC (Santa Cruz Biotechnology, Dallas, Texas, USA) CD105 (anti CD 105 sc-71042) positive, CD73 (anti CD73 sc-18849) and CD90 (anti CD90 sc-53116) positive and CD45 (anti CD45 sc-53665) negative [20[20] Nugraha AP, Narmada IB, Ernawati DS, Dinaryanti A, Hendrianto E, Ihsan IS, et al. Osteogenic potential of gingival stromal progenitor cells cultured in platelet rich fibrin is predicted by core-binding factor subunit-α1/Sox9 expression ratio (in vitro). F1000 Res 2018; 7:1134. https://doi.org/10.12688/f1000research.15423.1
https://doi.org/10.12688/f1000research.1...
]. Monoclonal antibodies were applied to cells and incubated for 60 minutes then rinsed with PBS twice and the cells were analyzed using the Olympus FSX100TM fluorescence microscope (Olympus Corporation of the Americas, Center Valley, PA, USA) with 200x magnification in 5 fields of view by two observers.
Diabetic Rats Model
All animals (Wistar Rats) were housed in polycarbonate cages, subjected to a 12-hour light-dark cycle at the constant temperature of 23ºC, and fed a standard pellet diet (expanded pellets; Stepfield, Witham, Essex, UK) with tap water ad libitum at a temperature of 22ºC ± 2ºC [21[21] Furman BL. Streptozotocin-induced diabetic models in mice and rats. Curr Protoc Pharmacol 2015; 70(5):5.47.1-5.47.20. https://doi.org/10.1002/0471141755
https://doi.org/10.1002/0471141755...
]. Mice are fastened for approximately 12 hours before induction to empty the stomach and accelerate the occurrence of DM conditions. Diabetic rats model was induced by STZ (Bioworld Merchandising Inc., Irving, TX, USA) with a dose of 30 mg / kg which was dissolved in citrate buffer (CV. Gamma Scientific Biolab, Malang, Indonesia) 30 mg/mL (pH 4.5) injected intraperitoneally in the area beside the midline between two nipples or in right / left mouse umbilication. Induction was done once; the mouse was held and the part to be injected is rubbed with 70% alcohol. The needle was injected perpendicular to the right / left umbilical to the peritoneal cavity; then the ingredients are injected slowly. Mice were given 10% sucrose solution or 10% dextrose (PT Otsuka, Jakarta, Indonesia) during the first night after induction to avoid sudden hypoglycemic post [22[22] Knas M, Maciejzcyk M, Daniszewska I, Klimiuk A, Matczuk J, Kolodziej U, et al. Oxidative damage to the salivary glands of rats with streptozotocin-induced diabetes-temporal study: Oxidative stress and diabetic salivary glands. J Diabetes Res 2016; 2016:4583742. https://doi.org/10.1155/2016/4583742
https://doi.org/10.1155/2016/4583742...
]. Mice were declared diabetic if on the 7th day after induction of blood sugar levels ≥ 200 mg / dL checked using Accu check (0197, EasyTouch GCU, Bioptik Technology Inc., Taiwan) [22[22] Knas M, Maciejzcyk M, Daniszewska I, Klimiuk A, Matczuk J, Kolodziej U, et al. Oxidative damage to the salivary glands of rats with streptozotocin-induced diabetes-temporal study: Oxidative stress and diabetic salivary glands. J Diabetes Res 2016; 2016:4583742. https://doi.org/10.1155/2016/4583742
https://doi.org/10.1155/2016/4583742...
,23[23] Mahmoud EF, Mahmoud MF. Effect of pomegranate peel extract on submandibular salivary glands of streptozotocin-induced diabetes in ratsHistological, immunohistochemical and ultrastructural study. J Adv Biol Biotech 2017; 13(3):1-15. https://doi.org/10.9734/JABB/2017/34101
https://doi.org/10.9734/JABB/2017/34101...
].
HDPSC in Vivo Injection
HDPSC was injected in rats in the treatment group with a single dose of 5.105 cells / 250 gr BW in 0.2 mL intraglandular PBS solution in the submandibular gland. The control group mouse has injected 0.2 mL of PBS intraglandularly in the submandibular gland. The mice were sacrificed on the 7th and 14th day using rodent anesthesia (Ketamine 70 mg / kg BW and Xylazine 5 mL). The sub-mandibular gland and blood tissue were extracted for further analysis [19[19] Suciadi SP, Nugraha AP, Ernawati DS, Ayuningtyas NF, Narmada IB, Prahasanti C, et al. The efficacy of human dental pulp stem cells in regenerating submandibular gland defects in diabetic wistar rats (Rattus novergicus). Res J Pharm Tech 2019; 12(4):1573-9. https://doi.org/10.5958/0974-360X.2019.00261.0
https://doi.org/10.5958/0974-360X.2019.0...
].
Paraffin Block Preparations
Preparations were obtained from salivary gland incisions, which were fixed with 10% neutral buffered formalin (NBF) (HT501128, Sigma Aldrich Corp., St. Louis, MO, USA) with solution volume of 10 times the size of the specimen. After fixation, dehydration was done through the extraction of water from the tissue with alcohol (30%, 50%, 70%, 80%, 96% and absolute) for 60 minutes each. Clearing was then carried out, which was to clear the tissue to be transparent by inserting the tissue into xylol solution 2 times each for 60 minutes. Impregnation was done consequently by infiltration with soft paraffin for 60 minutes at 48ºC and went through block process in hard paraffin in the mold and let set for a day. Paraffin blocks were attached to the holder and were cut using a microtome rotary for 4-6 μm.
Staining and Interpretation of Hematoxylin and Eosin
Deparaffinization was done by submerging the sample in xylol for 5 minutes. The sample was re-hydrated using alcohol from high to lower concentrations (96%, 80%, 70%) for 2 minutes and at the end using water for 10 minutes. Staining was done with Mayer's Hematoxylin (MHS1, Sigma Aldrich Corp., St. Louis, MO, USA) for 15 minutes and washed with running water until samples appeared blue for 5 minutes or less. The samples were submerged in 1% acidic alcohol (1% HCL in 70% alcohol) for 5-10 seconds. Rinsing with water until appeared blue again for 10-15 minutes or submerse in alkaline solution (ammonia water) followed by washing for 5 minutes. The sample was stained with 1% eosin Y for 10 minutes. Wash it with water for 1-5 minutes. The sample was dehydrated with alcohol 70%, 80% and 96% for 2 minutes of each. Clearing was performed by submerging the sample in xylol for 5 minutes and mounting with EZ Mount. Results showed a nucleus with a blue / black stain and cytoplasm with a pink stain. The examination was carried out by two observers in 5 different visual fields and using Nikon H600L light microscope (Nikon Imaging Japan Inc., Tokyo, Japan) at 400x magnification with a 300 megapixels Fi2 DS digital camera and image processing software Nikon Image System. Vacuolization observed by the number of acinar cells that showed vacuolization compared to the total acinar cells in 1 field of view by two observers.
IL-10 ELISA Examination
IL-10 level examination used an ELISA kit (Cat. No E0108Ra, Bioassay Technology Laboratory, Birmingham, UK) according to the manufacturer’s instructions. Serum was left to form clots first for 10-20 minutes at room temperature before use, then centrifuged in 2000-3000 RPM for 20 minutes. All reagents, standard solutions and samples were prepared at room temperature before use. The process was carried out at room temperature and began by pouring 50μl standard in standard well, 40 μl samples and 10 μl anti-IL-10 antibodies into the sample wells, and 50 μl streptavidin-HRP (Horseradish Peroxidase) into the sample wells and standard wells and mixed them carefully. The plates were covered with a sealer and incubated for 60 minutes at 37ºC. The sealer was removed and plates were rinsed 5 times with a wash buffer. Submersion was done using at least 0.35 ml wash buffer for 30 seconds to 1 minute for every washing. The plates were dried with paper towels. The substrate solution was added 50 μl and 50 μl of substrate solution B to each well. The plates ware covered with a sealer and incubated for 10 minutes at 37ºC in the dark. Stop solution was added to each well, to make the color changed from blue to yellow. Optical density (OD) readings were performed using a microplate reader with a 450nm wavelength 10 minutes after stop solution was added.
Statistical Analysis
Data obtained were analyzed statistically using Kruskal Wallis (p<0.05) based on a Shapiro-Wilk normality test and Levene's variance Homogeneity test (p>0.05) and then a multiple comparisons Bonferroni test (p<0.05) was done with IBM SPSS Statistics for Windows, Version 20.0 (IBM Corp., Armonk, NY, USA).
Results
After isolation and culture, DPSC was the passage for 3 times to obtain desired cell numbers (Figure 1). In 3rd passage, immunocytochemistry characterization was done on DPSC to determine the marker MSC expression - CD73, CD90, CD105(+) and not expressing HSC marker - CD45(-) (Figure 2).
The morphology of HDPSCs (yellow arrow). A) HDPSC first isolation. B) HDPSC first passage did not show a fibroblast-like appearance. C) HDPSC second passage showed a fibroblast-like appearance. D) HDPSC third passage showed more fibroblast-like structures. 200x magnification.
HDPSCs expressed positively MSCs marker CD73(+), CD90(+), CD105(+), while HDPSCs did not express HSCs marker CD45(-) (yellow arrow) with ICC examination with FITC using fluorescence microscope. 200x magnification. The left column showed a non-contrast appearance and the right column showed contrast appearance. A1) Non-contrast appearance from ICC CD45 examination. A2) Contrast appearance from ICC examination which indicated negative CD45. B1) Non-contrast appearance from ICC CD73 examination. B2) Contrast appearance from ICC examination, which indicated positive CD73. C1) Non-contrast appearance from ICC CD90 examination. C2) Contrast appearance from ICC examination, which indicated positive CD90. D1) Noncontrast appearance from ICC CD105 examination. D2) Contrast appearance from ICC examination, which indicated positive CD105.
Acinar cell vacuolization appears in HE staining as rounded-white colored with defined border acinar cells (Figure 3).
Acinar cells vacuolization (yellow arrow) in histopathological examination using HE staining. 400x magnification. Vacuolizations were clearly observed in control groups. A) Histopathological appearance of acinar cells in control group on 7th day. B) Histopathological appearance of acinar cells in the control group on 14th day. C) Histopathological appearance of acinar cells in treatment group on 7th day. D) Histopathological appearance of acinar cells in treatment group on 14th day.
The acinar cells vacuolization data obtained were not homogenous and not normally distributed (p<0.05). The highest acinar cell vacuolization found in control group Day 14 (0.239 ± 0.132), meanwhile the lowest acinar cell vacuolization found in treatment group Day 7 (0.019 ± 0.035) with significant difference (p=0.003, p<0.05) (Table 1).
There were significant differences between K+14 and K+7, P+7 and K+14, P+14 and K+14 group (p<0.05) (Table 2). The IL-10 serum level data obtained were not homogenous and not normally distributed (p<0.05) with a significant difference (p=0.001, p<0.05).
The highest level of IL-10 serum was found in the treatment group on the 14th day (175.583 ± 120.075), meanwhile the lowest level of IL-10 serum found in the control group on the 7th day (24.071 ± 9.316) (Table 3).
There were significant differences between groups (p=0.001, p<0.05). There were significant differences between K+7 and K+14, K+7 and P+14, P+7 and P+14 groups (p<0.05) (Table 4).
Discussion
In this present study found that acinar cell vacuolization between the control and treatment groups Day 7 were not significantly different. This is indicated that the tissue regeneration process by DPSC on the 7th day had not occurred well. This is supported by previous research that that on the 7th day of the HDPSC injection post there were no significant results on the microvascular structure, which is one of the important factors in the regeneration process [24[24] Nam H, Kim GH, Bae YK, Jeong DE, Joo KM, Lee K, et al. Angiogenic capacity of dental pulp stem cell regulated by SDF-1α-CXCR4 axis. Mol Cells 2017; 40(6):386-92. https://doi.org/10.1155/2017/8085462
https://doi.org/10.1155/2017/8085462...
].
Acinar cells vacuolization between the control and treatment groups Day 14 were significantly decreased. It can occur because the acinar cell vacuolization value was observed by the number of acinar cells that showed vacuolization compared to the total acinar cells in 1 field of view, so a decrease on acinar cell vacuolization can occur because acinar cells vacuolization actually decreased or the number of normal acinar cells increased. The decrease in vacuolization value indicated that there was an improvement in the conditions of the treatment group compared to the control group in the 2nd week. This is consistent with the experiment conducted previously, which revealed that in the 2nd week of post-transplantation DPSC in co-transplanted in salivary glands showed that there was a significant increase in Alpha-Amylase-1 markers (AMY-1) marker of differentiation of acinar cells and FGF7 which are mesenchymal markers. This showed that the salivary gland regeneration by DPSCs occurs very well in the 2nd week and it is able to increase the differentiation of the salivary glands in vivo [25[25] Janebodin K, Reyes M. Neural crest-derived dental pulp stem cells function as ectomesenchyme to support salivary gland tissue formation. Dentistry 2012; 13(001):5. https://doi.org/10.4172/2161-1122.S13-001
https://doi.org/10.4172/2161-1122.S13-00...
].
Acinar cells vacuolization tends to increase in both groups Day 7 and Day 14 due to blood glucose did not controlled; therefore, the progressive diabetic complication worsens each day. However, the acinar cells vacuolization increased significantly in the control group than the treatment group. This indicated that HDPSC only able to decrease and inhibit the progression of acinar cells vacuolization.
The literature shows that IL-10 inhibits macrophage activation [26[26] Kyurkchiev D, Bochev I, Ivanova-Todorova E, Mourdjeva M, Oreshkova T, Belemezova K, et al. Secretion of immunoregulatory cytokines by mesenchymal stem cells. World J Stem Cells 2014; 6(5):552-70.]. Meanwhile, MSC will increase IL-10 expression through macrophages, dendritic cells, and peripheral blood mononuclear cells [27[27] Li J, Zhu H, Liu Y, Li Q, Lu S, Feng M, et al. Human mesenchymal stem cell transplantation protects against cerebral ischemic injury and upregulates interleukin-10 expression in Macacafascicularis. Brain Res 2010; 1334:65-72. https://doi.org/10.4252/wjsc.v6.i5.552
https://doi.org/10.4252/wjsc.v6.i5.552...
]. It means that the administration of MSC will increase IL-10 levels comparable to levels of macrophages in serum.
The elevated IL-10 levels in control group will reduce the levels of existing macrophages, so that the administration of MSC increases in IL-10 levels did not appear to be too significant. This can explain why in this experiment, there was no significant increase in the treatment group when compared with the control group both on day 7 and 14. This result did not in accordance with the previous study, which said that DPSC was able to significantly increase IL-10 production when DPSC was conditioned with Peripheral Blood Mononuclear Cells (PBMSC) [28[28] Ding G, Niu J, Liu Y. Dental pulp stem cells suppress the proliferation of lymphocytes via transforming growth factor-ß1. Human Cell 2015; 28(2):81-90. https://doi.org/10.1007/s13577-014-0106-y
https://doi.org/10.1007/s13577-014-0106-...
].
It has been shown that DPSC transplanted in the sciatic nerve was able to decrease IL-10 levels in the sciatic nerve sample [29[29] Omi M, Hata M, Nakamura N, Miyabe M, Kobayashi Y, Kamiya H, et al. Transplantation of dental pulp stem cells suppressed inflammation in sciatic nerves by promoting macrophage polarization toward anti-inflammation phenotypes and ameliorated diabetic polyneuropathy. J Diabetes Investig 2016; 7(4):485-95. https://doi.org/10.1111/jdi.12452
https://doi.org/10.1111/jdi.12452...
]. When compared between this study and others [28[28] Ding G, Niu J, Liu Y. Dental pulp stem cells suppress the proliferation of lymphocytes via transforming growth factor-ß1. Human Cell 2015; 28(2):81-90. https://doi.org/10.1007/s13577-014-0106-y
https://doi.org/10.1007/s13577-014-0106-...
,29[29] Omi M, Hata M, Nakamura N, Miyabe M, Kobayashi Y, Kamiya H, et al. Transplantation of dental pulp stem cells suppressed inflammation in sciatic nerves by promoting macrophage polarization toward anti-inflammation phenotypes and ameliorated diabetic polyneuropathy. J Diabetes Investig 2016; 7(4):485-95. https://doi.org/10.1111/jdi.12452
https://doi.org/10.1111/jdi.12452...
], the results of the study showed that there were no differences between the control group and treatment groups could occur due to differences in the injection location of DPSC and the samples used for IL-10 levels, namely DPSC injected intraglandularly in the submandibular gland while the IL-10 examination sample originated from peripheral blood serum.
However, the effect may be seen systemically if the dose from DPSC is increased as in an experiment carried out previously [30[30] Datta I, Bhadri N, Shahani P, Majumdar D, Sowmithra S, Razdan R, et al. Functional recovery upon human dental pulp stem cell transplanatation in a diabetic neuropathy rat model. Cytotherapy 2017; 19(10):1208-24. https://doi.org/10.1016/j.jcyt.2017.07.009
https://doi.org/10.1016/j.jcyt.2017.07.0...
], which demonstrated that expression of IL-10 serum levels would recover in repeated transplants from DPSC at a dose of 1x106 cells intra-muscular. In this experiment, only single-dose DPSC was used (5x105 cells intraglandularly). Hence, the absence of significant differences in this study could be due to the dose being too low and not being given repeatedly dose of DPSC, which caused intraglandular administration could not provide a systemic effect on serum levels of IL-10.
Conclusion
Transplantation of HDPSC was able to regenerate submandibular salivary gland defects in diabetic rats by decreasing acinar cell vacuolization and slightly increase IL-10 serum level.
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Financial Support: Penelitian Dasar Unggulan Perguruan Tinggi (PDUPT) of the Ministry of Research, Technology and Higher Education of the Republic of Indonesia (Kemenristekdikti RI) (Grant No. 893/UN3/2018).
Acknowledgement: The authors would like to thank the Doctral of Medical Science, Faculty of Medicine, Faculty of Dental Medicine, Stem Cell Research and Development Centre, Universitas Airlangga for its support.
References
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» https://doi.org/10.1371/journal.pone.0212019 -
[2]Lessa LS, Pires PDS, Ceretta RA, Becker IRT, Ceretta LB, Tuon L, et al. Meta-analysis of prevalence of xerostomia in diabetes mellitus. Int Arch Med 2015; 8(224):1-13. https://doi.org/10.3823/1823
» https://doi.org/10.3823/1823 -
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Publication Dates
-
Publication in this collection
13 Jan 2020 -
Date of issue
2019
History
-
Received
31 May 2019 -
Accepted
19 Sept 2019 -
Published
02 Oct 2019