INTRODUCTION:

Currently, a study regarding stem cells’ potential ability for treating diseases gains so much attention.¹ Stem cells may replace, restore or regenerate the damaged cell, tissue, organ into its normal functions.²Stem cells own a good potential proliferation rate, self-renewal, differentiation into mesenchymal lineage which are suitable for the regenerative dentistry approach.³In dentistry field, periodontitis, salivary gland defect, alveolar bone defect, and post orthodontics treatment relapse are problems which may be resolved through a regenerative dentistry approach.^4,5,6

Mesenchymal Stem Cells (MSCs) are one type of stem cells that can be isolated from various sources such as umbilical cords⁷, adipose tissue⁸, hair follicle⁹ and orofacial region. Nowadays, orofacial derived MSCs studies are emerging and promising. MSCs are usually from orofacial region, such as gingival derived mesenchymal stem cells (GDMSCs)¹⁰, dental pulp stem cell from permanent or deciduous tooth.^11-13 MSCs may secrete various growth factors that help the wound healing process.¹⁴

GDMSCs are unique and suitable for tissue engineering.¹⁵GDMSCs can be easily isolated with minimally invasive procedure. GDMSCs express the MSCs surface marker such as CD73, CD90, CD105 and lack of CD45 expression.¹⁰ GDMSCs also differentiate into mesenchymal lineage like osteogenic differentiation confirmed by the expressions of alkaline phosphatase, osteocalcin, osteopontin, osteonectin, Runt-related transcription factor 2 as bone marker.^16-20

The isolated MSCs exhibit the decreased proliferation, differentiation, and survival. In addition, the strategy to improve their ability are needed.²¹ The preconditioning of MSCs during culture maybe affect their stemness. Hypoxia preconditioning induces the adaptive state and maintain the stemness of MSCs.²² The MSCs culture in vitro niche do not refer to its normal niche of MSCs which is hypoxia in some tissue (4-7% of oxygen).²³ For that reason, before any clinical application, the MSCs characteristic should be maintained through preconditioning the culture in vitro.²² The Cobalt Chloride (CoCl₂) can be used as hypoxia mimicking agent (HMA) that induces the hypoxia inducible factor-1α (HIF-1α) expression in the cell culture.²⁴Therefore, the aim of this study is to confirm that CoCl₂ can stimulate HIF-1α expression in GDMSCs in vitro.

MATERIAL AND METHODS:

Study Design and Ethical Clearance Approval:

GDMSCs from rabbit (Oryctolagus cuniculus) were obtained from laboratory stock of Stem Cell Research and Development Center, Universitas Airlangga. GDMSCs were the sample for this study, randomly selected and divided into 4 groups GDMSC normoxia (24 hours and 48 hours) groups and GDMSC hypoxia (24 hours and 48 hours) groups respectively (N=20/n=5). In addition, this is a true experimental laboratory post-test only control group study design and ethically approved by the ethical Clearance Committee of Health Research from the Faculty of Veterinary, Universitas Airlangga, Surabaya, Indonesia with reference number of 2.KE.017.02.2020.

The Preparation of GDMSCs Treated with CoCl₂:

At fourth passage of GDMSCs cultured in M24 culture well was used in this study. To stimulate the hypoxia condition, CoCl₂(Sigma Aldrich, US) was prepared immediately prior to the administration in a 25mM stock solution in sterile DD water. The final concentration of 100μM CoCl₂was utilized in this study then added to the culture media of GDMSCs and incubated for 24 hours and 48 hours in the incubator (Esco Micro Pte. Ltd, Changi, Singapore) (37°C; 5% C0₂). Meanwhile, for the normoxia groups, GDMSCs was incubated in the incubator for 24 hours and 48 hours without treated with CoCl_2.²⁴

The Examination of Hypoxia Inducible Factor-1α Expression in the GDMSCs:

The post incubation lasted for 24 hours or 48 hours. Each group of GDMSCs in each well (n=5) was washed by using PBS and fixed with 4% paraformaldehyde. The examination of HIF-1α stimulated by CoCl₂ in the GDMSCs was using immunocytochemistry methods with Fluorescein isothiocyanate antibody labelling (Cat no. ab6717, Abcam, MA, USA). The observation of HIF-1α positive expression (green color) of GDMSCs used the inverted microscope equipped with fluorescence filter (Olympus, Tokyo, Japan) in 100x magnification.⁹

Statistical Analysis :

The Tukey Honest Significant Different (HSD) test from Statistical Package for Social Science (SPSS) 20.0 version (Chicago, Illinois, USA) was performed to compare the significant difference in HIF-1α expression between groups (p<0.01).

Fig 1. The HIF-1α expression in GDMSCs (green color) was successfully stimulated by 100mM CoCl₂for 24 hours incubation (hypoxia) and the control (normoxia) observed by using fluorescence inverted microscope (Olympus, Tokyo, Japan) with 100x magnification.

The HIF-1α expression in GDMSCs was successfully stimulated by 100mM CoCl₂ (see Figure 1). The greatest expression of HIF-1α was found in GDMSCs treated with CoCl₂for 24 hours. The lowest expression of HIF-1α was found in GDMSCs normoxia condition in 24 hours. There was significant difference in HIF-1α expression between GMSCs normoxia (24 hours and 48 hours) and GMSCs hypoxia of 24 hours groups but not 48 hours group (p<0.01) as shown in Figure 2.

Fig 2. The assessment of HIF-1α expression of GDMSCs with CoCl₂(hypoxia) or without CoCl₂(normoxia) for 24 hours and 48 hours of incubation described as average and standard deviation. Information: *significant different at p<0.01.

This study finds that CoCl₂can stimulated the HIF-1α expression of GDMCs. The greatest expression of HIF-1α is found in GDMSCs treated with CoCl₂ (hypoxia condition) for 24 hours. In comparison, the lowest HIF-1α expression is detected in GDMSCs normoxia condition in 24 hours. There was significant different in HIF-1α expression between GMSCs normoxia condition (24 hours and 48 hours) and GMSCs hypoxia condition of 24 hours groups but not with 48 hours group. Our study result is in line with Lambertini et al. and Wu’s studies that mentioned the HIF-1α expression is significantly increased in the MSCs post administration of CoCl₂as HMA.^24,25Teti et al study also supported this study result which showed that the hypoxic condition is successfully induced in dental pulp MSCs and umbilical Cord MSCs by utilizing CoCl₂.²⁶Additionally, CoCl₂did not affect the cell viability percentage but successfully induce hypoxic preconditioning in Human Adipose Derived Mesenchymal Stem Cells.²⁷Hypoxia preconditioning is the characteristic of MSCs endogenous niche. Hypoxia preconditioning of MSCs is able to delay the cell aging, prolongs the cellular life span and survival, and also maintains the stemness of MSCs related genes.^22,28,29

The MSCs transplantation in the targeted tissue is an essential step of MSCs regenerative treatment. The hypoxia preconditioning of MSCs can improve the skeletal regeneration through the enhancement of neovascularization compared to MSCs in normoxic condition.³⁰ MSCs secrete the homing factors which play important role for movement/migration towards the targeted tissue that is stimulated with hypoxic conditions.³¹

Beside the enhancement of others molecular markers, HIF-1α is the most important cellular regulator in the hypoxia condition. HIF-1α can enhance the genes that involve in the metabolism of glucose, iron transport, neovascularization, and erythropoiesis.³² The ability of CoCl₂ as a hypoxia mimicking agent through the inhibition of FIH and HIF-P4Hs causes the occupation of Fe₂⁺ binding site and the blockade of HIF-1α degradation.³³

Despite GDMSCs are unique and have numerous advantages for regenerative dentistry, the hypoxia preconditioning of GDMSCs may be necessary before the transplantation. In addition, the successfulness of GDMSCs transplantation for MSCs-based regenerative approach depends on the maintenance and the stabilization of the physiological oxygen condition in MSCs.³⁴ Based on the result above, we can conclude that CoCl₂can enhance significantly HIF-1α expression of GDMSCs for 24 hours in vitro. Further studies are still needed to examine the mechanism CoCl₂on HIF-1α expression in others MSCs.

ACKNOWLEDGEMENT:

This study was supported by research grant of Penelitian Dasar Unggulan Perguruan Tinggi (PDUPT) number:306/UN3.15/PT/2021 from Ministry of Research, Technology and Higher Education, Republic of Indonesia. The authors thank the Stem Cell Development and Center, Publication and Research Center, Faculty of Dental Medicine, Universitas Airlangga for supporting our study.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 26.05.2020 Modified on 28.06.2020

Research J. Pharm. and Tech. 2021; 14(5):2639-2642.

DOI: 10.52711/0974-360X.2021.00465