The Efficacy of Human Dental Pulp Stem Cells in regenerating Submandibular Gland Defects in Diabetic Wistar Rats (Rattus novergicus)

 

Septiana P. Suciadi1, Alexander P. Nugraha2,3,4, Diah S. Ernawati5, Nurina F.  Ayuningtyas5,  Ida B. Narmada2, Chiquita Prahasanti6, Aristika Dinaryanti4, Igo Syaiful Ihsan4, Eryk Hendrinto4, Helen Susilowati4, Fedik Abdul Rantam4,7

1Faculty of Dentistry, Universitas Airlangga, Surabaya, Indonesia.

2Orthodontics Department, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.

3Doctoral Student of Medical Science, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia.

4Stem Cell Research and Development Center, Universitas Airlangga, Surabaya – Indonesia.

5Oral Medicine Department, Faculty of Dental Medicine, Universitas Airlangga, Surabaya – Indonesia.

6Periodontics Department, Faculty of Dental Medicine, Universitas Airlangga, Surabaya – Indonesia.

7Virology and Immunology Laboratory, Microbiology Department, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya – Indonesia.

*Corresponding Author E-mail: diah-s-e@fkg.unair.ac.id

 

ABSTRACT:

Chronic hyperglicemia in Diabetes Mellitus caused microangiopathy in salivary gland. Human Dental Pulp Stem Cells (HDPSCs) suspected can regenerate the defect. The aim of this study was to analyze the efficacy of HDPSCs in stimulating angiogenesis, acinar cell numbers and Transforming Growth Factor-β (TGF-β) serum to regenerate submandibular gland defects in diabetic Wistar rats. Twenty-four male Wistar (250-350 g) rats 3-months-old were used. Rats were divided into 4 groups (n=6 each: a positive control group on Day 7 (DM) (C+7), a positive control group on Day 14 (DM) (C+14), a treatment group on Day 7 (DM+5.105 HDPSCs transplantation intraglandular) (T7) and a treatment group on Day 14 (DM+5.105 HDPSCs transplantation intraglandular) (T14). Wistar Rats were administered with 30 mg of Streptozotocin per kg of bodyweight to induce Diabetes Mellitus (DM). Histopathological examination with HE staining was performed to analyse neovascularization and acinar cell numbers. ELISA was performed to measure TGF-β serum.  Statistical analysis used: A Tukey HSD or Bonferroni test after ANOVA or Kruskal Wallis test was performed (p<0.05) based on a Saphiro Wilk and Levene’s test (p>0.05). The highest acinar cell number was found in the T7 group [513.167±136.17] with no significant difference [p=0.136, p<0.05]. The highest capillaries were found in T14 [10.667±4.54] and TGF-β serum level [168.87±37.38] with significant difference [p=0.006; p<0.05] and [p=0.008, p<0.05]. HDPSCs can regenerate submandibular gland defects in Diabetic Wistar rats by stimulating angiogenesis, acinar cells number and TGF – β serum.

 

KEYWORDS: Human Dental Pulp Stem Cells, Submandibular Gland Defect, Diabetes Mellitus, Acinar Cells, Angiogenesis, Transforming Growth Factor-β.

 

 

 

 

INTRODUCTION:

Diabetes Mellitus (DM) is a systemic disease that affects many people. It is the most common endocrine disorder, affecting mankind all over the world, prevalence of which is increasing, daily.1 In 2017, the International Diabetes Federation (IDF) declared the global prevalence of DM to be 8.8%, making it the seventh largest cause of death in the world.2 In the absence of intervention, this number is likely to have more than doubled by 2030.3 The needs of diabetic patients are not only limited to adequate glycemic control, but also correspond to definitive therapy: preventing complications, disability limitation and rehabilitation.4 Early detection of type II diabetes can be justified because diabetes is an important health problem has a relatively long asymptomatic phase, interventions are available that have a proven beneficial effect on clinically meaningful outcomes and screening procedures are safe.5 Patients with DM, a hyperglycemic condition, when sugar cannot enter cells, high levels of sugar build up in the blood, may suffer various forms of complications.6 For example, chronic hyperglycemia will cause endothelial dysfunction which, in turn, induces microangiopathic conditions, one of which affects the salivary glands. In these cases the structural changes are clearly oxidative in nature and are associated with development of vascular disease in diabetes.1,7 Salivary gland defects are characterized by altered nuclei resulting in pyknosis, karyorrhexis, or even karyolysis due to reduced oxygen supply (ischemia) in acinar cells caused by microangiopathic conditions.8-10 Oral health is an important part of overall health.11 The reduced number of acinar cells, if not compensated by cell regeneration, can cause xerostomia, a decrease in saliva production. Xerostomia can induce susceptibility to oral conditions such as caries and periodontal disease leading to ineffective mastication that gradually reduces the quality of life of diabetics.12-14 The idea of promoting neovascularization and improving the perfusion of ischemic tissue via angiogenesis constitutes a promising form of treatment for xerostomia. Angiogenesis can stimulate tissue repair through endothelial cells and growth factors present in the salivary glands.15

 

Mesenchymal Stem Cells (MSCs) are progenitor cells that can differentiate into mesodermal, endodermal or ectodermal derivatives, rendering them suitable for use in tissue engineering. Factors such as multipotent ability, the presence of immunomodulators and the capacity to migrate directly to the tissue can initiate tissue regeneration through angiogenesis. Thus, in turn, can enhance neovascularization which improves the microangiopathic conditions that occur due to DM, especially in the salivary glands.16-18 MSCs can be recruited and mobilized to inflammation sites, as well as those resulting from injury, where they can be incorporated into the microenvironment of ischemic tissue. Angiogenic factors produced by MSCs include Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor-β (TGF-β), Placental Derived Growth Factor (PGF), angiopoietin-1, Interleukin-6 and Monocyte Chemotactic Protein-1 (MCP-1) which facilitate tissue regeneration.14

 

 

One example of MSCs that can be used is the Human Dental Pulp Stem Cell (HDPSCs) which has cell differentiation and renewal capabilities. It is enabling them to regenerate acinar cells damaged by DM through various methods. HDPSC has similar capabilities with bone marrow mesenchymal stem cell, but not invasive. However, research into the use of stem cells originating from the oral cavity remains limited and rarely applied even though it has encouraging potential for tissue regeneration. Consequently, further research needs to be undertaken.18 The aim of this study was to investigate the efficacy of HDPSCs in stimulating angiogenesis, acinar cell numbers and TGF-β serum to regenerate submandibular gland defects in diabetic Wistar rats.

 

MATERIAL AND METHODS:

Study design:

24-male wistar rat, 3-month old, healthy, weighing 250-300 grams were used. All experimental procedures were conducted with the approval of the local ethics committee (047/HRECC.FODM/V/2018), having received a Home Office license. A healthy rat is characterized by shiny fur, glowing eyes and agile movements. All experimental subjects were maintained in a laboratory environment at 30% to 60% humidity and temperatures ranging from 22±20C while being subject to 12-hour dark/light cycles.19 Sample size was determined using Lemeshow’s formula and selected through a simple random sampling method. The animal models were divided into four groups (n = 6: a positive control group on Day 7 (DM) (C + 7), a positive control group on Day 14 (DM) (C + 14), a treatment group on Day 7 (DM + 5.105 HDPSCs transplantation intraglandular) (T7) and a treatment group on Day 14 (DM + 5.105 HDPSCs transplantation intraglandular) (T14).

 

Induction of Diabetes Mellitus:

The subjects were obliged to fast for approximately 12 hours before induction to empty the stomach and accelerate the occurrence of DM conditions. Diabetic rat subject creation was achieved by STZ (bioWORLD, US) at a dose of 30 mg/kg which was dissolved in 30 mg/ml (pH 4.5) of citrate buffer (CV. Gamma Scientific Biolab, Malang, Indonesia) injected intraperitoneally in the area adjacent to the midline between two nipples or in the right/left umbilication. Induction was performed once with the rat being held and the part to be injected rubbed with 70% alcohol. The needle was inserted perpendicular from the right/left umbilical to the peritoneal cavity and the contents injected slowly. Subjects were given 10% sucrose solution or 10% dextrose (Otsuka, Indonesia) during the first night after induction to avoid sudden hypoglycemia.20 Subjects were declared diabetic on Day 7 after induction if their blood sugar levels were measured at ≥200 mg / dl using Accu check (0197, EasyTouch GCU, Taiwan).21,22

Isolation and culture of Human Dental Pulp Stem Cell:

HDPSC was isolated from the premolar dental pulp of patients undergoing orthodontic therapy. The extracted teeth were immediately immersed in Dulbecco’s modified eagle medium (DMEM) (D5796, Sigma Aldrich, US) and forwarded to the laboratory. The tooth was washed with Phosphate-Buffered Saline (PBS) solution, cut in half and the pulp tissue removed. The pulp tissue was digested in a solution of 3 mg/ml collagenase type 1 (C9891, Sigma Aldrich, US) and 4 mg/ml dispase (D4818, Sigma Aldrich, US) for 30-60 minutes at 370C. HDPSC was obtained by filtering the digested tissue with 70μm cell filter. 1 cell suspension (1 x 105 cells / flask) was planted in α -Minimum Essential Medium (α-MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L - glutamine, 100 μM L-asorbic acid-2-phosphate, 100 U / mL penicillin - G, 100μg / mL streptomycin and 0.25μg / mL fungizone (A9528, Sigma Aldrich, US). Cells were conditioned at a temperature of 370C in 5% CO2 medium which was replaced every 2-3 days. The cell culture was passaged three times to obtain the desired number of cells.23,24

 

Characterization of Human Pulp Stem Cell:

HDPSC in the third passages was then examined for cluster differentiation markers to confirm the presence positive MSC markers, namely: CD73, CD90, CD105 and negative CD45 markers. Cells were coated with coverslips and, after incubation at 370C for 1-2 hours, fixation was performed with 10% formaldehyde (F8775, Sigma Aldrich, US) for 15 minutes and the coverslips were rinsed four times. Monoclonal antibodies were labelled FITC (Santa Cruz Biotechnology TM, Dallas, Texas, United State) 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.25 Monoclonal antibodies were applied to cells and incubated for 60 minutes before being rinsed twice with PBS and the cells were analysed in five different visual fields observed by 2 persons (EH, APN) using an Olympus FSX100TM fluorescence microscope (Center Valley, PA).

 

Human Pulp Stem Cell In vivo Injection:

After a month injection of STZ, subjects in the treatment group were injected with a single 5.105 cells/250-gram BW dose of HDPSC in 0.2 ml intraglandular PBS solution in the submandibular gland. The control group subjects were injected with 0.2 ml of PBS in the submandibular gland. The subjects were sacrificed on Day 7 and Day 14 using rodent anaesthesia (Ketamine 70 mg/kg BW and xylazine 5 ml). The sub-mandibular gland and blood tissue were extracted for further analysis.20

 

Staining and Interpretation of Hematoxylin and Eosin:

Staining was performed with Mayer's Hematoxylin (MHS1, Sigma Aldrich, US) for 15 minutes followed by rinsing with running water for five minutes or less until the samples appeared blue. The results featured a nucleus with blue/black stain and cytoplasm with pink stain. The examination was carried out by two observers (JL and SPS.) in five different visual fields using a Nikon H600L light microscope (Japan) at 1000x magnification with a 300megapixel Fi2 DS digital camera and Nikon Image System image processing software. Angiogenesis was observed from the number of capillaries (cavities containing erythrocytes and surrounded by red endothelial cells). Acinar cell formation constitutes a central cell nucleus found in the eosinophilic cytoplasm.

 

Detection of TGF-β ELISA:

TGF ELISA (Bioassay Technology Laboratory, Cat. No E0108Ra) is an enzyme immunoassay for the quantitative determination of TGFin human serum, animal serum and cell culture supernatant. During pre-testing of the standards, all samples were diluted in assay buffer, acidified with HCl and subsequently neutralized with Neutralization Buffer. Thereafter, the neutralized standards and samples were added to the antibody-coated (polyclonal) microtiter wells. After incubation, unbonded sample material was removed by washing. In a second step, monoclonal mouse anti TGF antibody, a biotinylated anti mouse IgG antibody and the Streptavidin-HRP (Horseradish Peroxidase) Enzyme complex were successively incubated, forming an immune enzyme sandwich complex. After incubation, the unbonded conjugate was washed off. Having added the substrate solution, the intensity of colour developed was proportional to the concentration of TGF in the sample.27

 

Statistical analysis:

Angiogenesis and acinar cell numbers were analysed statistically using multiple comparisons: a Tukey HSD test (p<0.05) after ANOVA (p<0.05) analysis based on a Saphiro - Wilk and a Levene’s test result (p>0.05), TGF-β were analysed statistically using multiple comparisons of a Bonferroni test (p<0.05) after a Kruskal Wallis test (p<0.05) had been performed, with Statistical Package for the Social Sciences Software (SPSS) 20.0 edition (SPSS™, Chicago, United States).

 

HDPSC was passaged three times to obtain the desired cell numbers (see Figure 1). It was transplanted 500.000 cells in each rat and calculated by cell counting software tools. In the third passage, HDPSC expressed positive MSCs markers CD73 (+), CD90 (+), CD105 (+), while it did not express HDPSCs marker CD45 (-) (see Figure 2). The acinar cell constituted a central cell nucleus found in the eosinophilic cytoplasm. The neovascularization was observed as capillaries (cavities containing erythrocytes) (see Figure 3). Capillaries showed enhancement in the Treatment Group with significant difference (p=0.006, p<0.05). Meanwhile, acinar cell numbers analysed by means of ANOVA showed enhancement, but it was not significant (p=0.136, p<0.05). TGF-β showed enhancement with significant difference (p=0.008, p<0.05) as confirmed by a Kruskal Wallis test.

 

Fig. 1: The morphology of HDPSCs (yellow arrow). A) HDPSCs first isolation. B) HDPSCs first passage did not have a fibroblast-like appearance. C) HDPSCs second passage showed fibroblast-like appearance. D) HDPSCs third passage showed more fibroblast-like structures when observed through a Nikon TMS Inverted Microscope (US) 100x magnification.

 

Fig. 2: HDPSCs expressed positive MSCs marker CD73 (+), CD90 (+), CD105 (+), while they did not express HSCs marker CD45 (-) (yellow arrow) as proven through ICC examination with FITC using a fluorescence Olympus FSX100TM microscope at 100x magnification (Center Valley, PA).

 

Fig. 3: Histological comparison of the positive control and treatment groups at 400x magnification. Acinar Cells and Capillary (Yellow Arrow) in the positive Control Group on Day 7 (A.), in the positive Control Group on Day 14 (B.), in the Experimental Group on Day 7 (C.) and in the Experimental Group on Day 14 (D.). Acinar cells and capillaries were analysed with HPA examination using HE staining at 1000x magnification (Nikon H600L light microscope and a DS Fi2 digital camera with 300 megapixels and Nikon Image System image processor software).

 

Fig. 4: The Mean ± Standard Deviation (SD) of acinar cells in each group. The highest number of acinar cells were found in the T7 group, while the lowest was found in the C+14 group.

 

Fig. 5: The Mean ± Standard Deviation (SD) of capillaries in each group. The highest number of capillaries was found in the T14 group, while the lowest was found in the C+7 group.

 

Fig. 6: The Mean ± Standard Deviation (SD) of Serum TGF-β in each group. The highest level of Serum TGF-β was found in the T14 group, while the lowest was found in C+7 group.

 

Table 1. Multiple Comparisons of Tukey HSD test results of acinar cells and capillaries between groups.

Variable

Group

C+ 7

C+ 14

T 7

T 14

Acinar Cells

C+ 7

 

 

 

 

C+ 14

0.968

 

 

 

T 7

0.346

0.168

 

 

P+14

0.977

1.000

0.183

 

Capillaries

C+ 7

 

 

 

 

C+ 14

0.714

 

 

 

T 7

0.072

0.428

 

 

T 14

0.006*

0.059

0.656

 

*information: significant at p< 0.05.

 

Table 2. Multiple Comparisons of Bonferroni test results TGF-β between groups.

Variable

Group

C+ 7

C+ 14

T 7

T 14

TGF β

C+ 7

 

 

 

 

C+ 14

0.003*

 

 

 

T 7

1.000

0.006*

 

 

T 14

0.002*

0.483

0.003*

 

*information: significant at p< 0.05.

 

Stem cells are unspecialized cells that can be differentiated into any type of cell in the body, by two important characteristics.28 First, they are unspecialized cells capable of renewing themselves through cell division, sometimes after long period of inactivity, second, under certain physiologic or experimental conditions they can be induced to become tissue or organ specific cells with special functions.29 Healing process can be observed optimally on day 7 and 14. On those day, the number of capillaries reaching the peak and gradually decrease. HDPSC expressed positive MSCs markers CD73 (+), CD90 (+), CD105 (+), while it did not express HDPSCs marker CD45 (-). CD73 is expressed in wide variety of cell types including endothelial cells, lymphocytes, and fibroblasts. CD90 expression has been identified in endothelial cells (both vascular and lymphatic), hematopoietic stem cells, lymphocytes, and fibroblasts. CD105 is highly expressed in vascular endothelial cells.27 HDPSCs show great promise for regenerative medicine, give an idea about the growth and development of an organism throughout the whole life cycle.30,31 It can regenerate acinar cells in the submandibular gland by increasing their numbers, as well as those of capillaries and the amount of TGF-β serum. No significant difference existed between acinar cell numbers in the positive control group and the treatment group. Rather, there was considerable coincidence between the groups in this regard. Cell regeneration did not achieve the complete phase of cell formation on either day 7 or day 14. Therefore, it did not show a significant increase when observed with the HE stained microscope. However, if the observation performed by means of ki-67 immunohistochemistry was evaluated, it showed cell proliferation in the form of nucleus division between day 3 and day 5.32The process of cell formation in salivary gland is called branching morphogenesis which consists of cell proliferation, gap formation, differentiation, cell migration, apoptosis, and reciprocal interactions between epithelial, mesenchymal, neuronal, and endothelial cells. The formation of cells can be observed about day 18 to 20 because there has been proliferation in the terminal branches. Whereas in this research, proliferation has not yet reached the terminal branch so that the number of new acinar cells is not maximal.33

 

Branching morphogenesis is interaction of mesenchymal epithelium and regulated by extracellular matrix and growth factor. Extracellular matrix consists of collagen, laminin, proteoglycans, fibronectin which is important for salivary morphogenesis. Growth factors play a role for organogenesis of salivary glands and are synthesized by the ductus. The formation of acinar cells includes stalk elongation, cleft formation, and dichotomization. Stalk elongation is an extension of the stem by elongation of the mesenchymal zone, then a gap at the end of the stem is formed called the cleft formation. The gap is divided into two clear parts called dichotomization. On day 13, there is a synthesis of laminin which is important for gland morphogenesis. On day 15 a gland lumen is formed and day 18 formed terminal differentiation of acinar cells. Cell proliferation in submandibular glands is mainly localized at the end of peripheral branches, which shows progenitor cell proliferation.34-36Normally, the damage of salivary gland can be regenerated through the autologous division mechanism. However, in progressive disease conditions such as DM, autologous division cannot compensate the damage and have impact on tissue function. Therefore, regeneration of additional progenitor cells is needed. In the body, there are endogenous mesenchymal stem cells found in the interstitial zone near the intercalary duct. In DM conditions, the volume of the ductus is reduced and replaced by fibrous and adipose tissue so that the ability of cell proliferation by endogenous stem cells is disrupted.35

There was a significant difference between groups with regards to capillaries. The injection of HDPSCs can regenerate salivary gland cells by increasing the number of capillaries. HDPSCs therapy can enhance salivary gland function by trans-differentiation into endothelial cells (ECs) to replace the damaged tissue and promote angiogenesis. This release soluble autocrine/paracrine factors, thereby activating endogenous adult cells involved in cell renewal/protection and neovascularization and stimulating endogenous resident endothelial cell (ECs) proliferation and differentiation by soluble paracrine factors. HDPSC secreted various growth factors such as VEGF which can induce the endothelial dierentiation of MSCs and has been shown to regulate ECs migration and dierentiation and promote recruitment of ECs for angiogenesis and endothelialisation in injured tissue.14

 

Angiogenesis increased on day 3, with both Vascular Endothelial Growth Factor-2 (VEGFR-2) and VEGF receptor expression rising. Furthermore, between day 5 and day 7 VEGF and VEGFR-2 reached a simultaneous peak of expression. After day 7, the lowest value for the expression of VEGFR-2 occurred together with descending concentration of systemic VEGF. Thereafter, on days 10 through 14, the level of expression of VEGFR-2 increased.37-38 The number of capillaries on days 7 and 14 increased which meant that the process of angiogenesis could be stimulated by HDPSC injection. TGF - β experienced a slight increase in this research. Certain studies conducted by Maring et al., and Massague have shown that injury-activated TGFβ members control the migration of HDPSCs.39,40 HDPSCs are recruited to the injury site by homing mainly through the vascular network. TGF-β can recruit vascular cells and promote the function of endothelial cells and neovascularization.41 Members of the TGF–ß subfamily regulate a variety of cellular functions, such as cell fate, growth, proliferation, apoptosis, differentiation, polarity, movement, invasion, and adhesion. Normally expressed TGF-ß plays critical roles in numerous biological behaviours such as inflammation and immune response, embryonic development, wound healing, extracellular matrix (ECM) formation and remodelling and epithelial–mesenchymal transition.36,42 Moreover, TGF-ß also modulates the expressions and/or activities of several biochemical cues that are important for acinar cell regeneration, thus indirectly affecting the regenerative process.42,43 HDPSCs can regenerate submandibular gland defects in Diabetic Wistar rats by stimulating angiogenesis, acinar cells number and TGF–β serum.

 

ACKNOWLEDGEMENT:

The research was funded by the Penelitian Dasar Unggulan Perguruan Tinggi (PDUPT) of the Ministry of Research, Technology and Higher Education of the Republic of Indonesia (Kemenristekdikti RI) (letter of appointment grant number 893/UN3/ 2018). The authors would like to thank the Faculty of Medicine, Faculty of Dental Medicine, Faculty of Veterinary Medicine, Stem Cell Research and Development Centre, Universitas Airlangga for its support of the research reported here.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 30.01.2019           Modified on 18.02.2019

Accepted on 20.03.2019         © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(4):1573-1579.

DOI: 10.5958/0974-360X.2019.00261.0