Gingival Medicinal Signaling Cells Conditioned Medium effect on the Osteoclast and Osteoblast number in Lipopolysaccharide-induced Calvaria Bone Resorption in Wistar Rats’ (Rattus novergicus)
1Graduate Student of Dental Health Science, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.
2Dental and Biomaterial Research Group, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.
3Department of Orthodontics, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.
4Stem Cell Research and Development Center, Universitas Airlangga Surabaya, Surabaya, Indonesia.
5Dental Medicine Research Center, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.
6Laboratory of Virology, Department of Microbiology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia.
7Department of Oral Medicine, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.
8Department of Oral Biology, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.
9Doctoral Student of Veterinary Medicine, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia.
10Doctoral Student of Biology Science, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia.
11609 Armed Forces Dental Clinic, Kuching, Sarawak, Malaysia.
*Corresponding Author E-mail: alexander.patera.nugraha@fkg.unair.ac.id
The gingival medicinal signaling cells conditioned medium (GMSCs-CM) is a biocompatible material which possessed beneficial cytokine, anti-microbial peptide, growth factor that can be collected after culture. GMSCs- CM may inhibit bone resorption in order to improve the patient’s quality of life. In this study, the potential effect of GMSCs-CM on the number of osteoclasts and osteoblasts in Lipopolysaccharide (LPS)-induced calvaria bone resorption in wistar rats (Rattus novergicus) has been analyzed. Twenty-eight male and healthy wistar rats (R. novergicus) at the age of 1-2 months old with 250-300 grams body weight were divided into 4 groups, namely PBS group: 100μg PBS day 1-7; LPS group: 100μg LPS day 1-7; LPS and GMSCs group: 100μg LPS + 100μg GMSCS-CM day 1 1-7, GMSCs group: 100μg M-GMSCs day 1-7. Escherichia Coli LPS was used to induce the bone resorption on the calvaria with subcutaneous injection. GMSCs-CM was collected after passage 4-5 then injected subcutaneously on the calvaria. All samples were examined on the. 8th day through cervical
dislocation. The number of osteoblasts and osteoclasts in calvaria was then observed under 400x magnification. One Way ANOVA and Tukey HSD were conducted to analyze differences between groups (p<0.01). The number of osteoclasts in calvaria decreased significantly in the LPS + GMSCs-CM group compared to LPS group (p<0.01). The number of osteoblasts in calvaria increased significantly in the LPS + GMSCs-CM group compared to LPS group (p<0.01). GMSCs-CM can reduce the amount of osteoclast significantly and increases the production of osteoblast in LPS-induced calvaria bone resorption in wistar rats (R. novergicus).
KEYWORDS: Gingival Mesenchymal Stem Cells Conditioned Medium, Dentistry, Lipopolysaccharide, Medicine, Osteoblast, Osteoclast.
The mineralized structure of the bone is thought to hard and last in long duration. Nevertheless, one of most dynamic tissue was bone. Bone constantly undergo remodeling through the cellular interaction, bone formation was done by osteoblast and bone resorption was done by osteoclast.1 Osteoclasts are multinucleated cells formed by the fusion of hematopoietic myeloid precursors typically in adjacent bone marrow to the bone surface.2 Osteoclasts play an important role in bone resorption in several degenerative diseases of bone influenced by chronic inflammatory condition.3,4 Nuclear Factor Associated T cell-1 (NFATC1) as transcription factor of osteoclast have an important role during osteoclastogenesis and osteoclast maturation.5 There are some cytokines and receptors which have an important role in osteoclastogenesis both in vitro and in vivo, such as tumor necrosis factor (TNF)-α, macrophage colony stimulating factor (M-CSF), Receptor activator of NF- kB ligand (RANKL).6-8 RANKL is expressed on the osteoblast surface that can bind Receptor activator of NF-kB that expressed on the pre-osteoclast surface that may initiate osteoclastogenesis and maturation of osteoclasts that can lead to bone resorption.9
Many inflammatory diseases such as periodontitis can occur as a result of an imbalance between bone formation and resorption, leading to decreased bone quality.10 The chronic metabolic disease such as diabetes mellitus (DM) may affect the bone remodeling and quality due to impairment of inflammation response. The pro-inflammatory cytokine in DM is very dominant.11 Inflammatory diseases such as periodontitis, rheumatoid arthritis, osteomyelitis, and degenerative diseases such as osteoporosis.12-15 Patients that suffering from bone resorption due to inflammatory diseases and degenerative diseases usually have an increased risk of morbidity and mortality, which can seriously impair their quality of life and thus impose an enormous economic burden.16-19 Therefore, it is necessary to carry out an intervention strategy through regenerative therapy using medicinal signaling cells or mesenchymal stem cells conditioned medium (MSCs-CM) to inhibit bone resorption due to inflammatory and degenerative diseases in order to improve the quality of life of the patients.
MSCs-CM are biocompatible material and it able to accelerate cell proliferation in vitro and it may beneficial for enhance rate of wound healing or tissue defect.20-23
During culture period of MSCs, beneficial exosome and secretome can be collected such as cytokine, anti- microbial peptide, growth factor that may have potential role to control excessive inflammatory responses.24-27 MSCs-CM is often removed and it can be a medical waste. MSCs-CM can be purified and can be used due to it has advantageous bioactive factors released by MSCs during cell culture. Previous studies have revealed that MSCs-CM can modulate the immune response and protect against inflammation-induced tissue lesions. The
consideration of MSCs use for clinical application are inexpensive, practice, less tumorigenicity, easy to collect, no time consuming for prolong duration cell expansion.28-30 In this study, the potential effect of gingival mesenchymal stem cells-conditioned medium (GMSCs-CM) to inhibit inflammation-induced bone resorption was investigated. GMSCs-CM was chosen because it is technically easy to obtain, can be collected through minimally invasive procedure compared to bone marrow stem cells, relatively safe and free from ethical problems.31-32
The effect of GMSCs-CM on calvaria bone resorption was investigated by means of Escherichia Coli lipopolysaccharide (LPS)-induced wistar rat (Rattus novergicus) calvaria bone resorption model by analyzing the number of osteoblasts and osteoclasts by histopathological examination. E. coli LPS has been shown to be a potential agent for inducing inflammatory cytokines and pathological bone loss.33 LPS-induced inflammatory cytokines such as TNF-α and Interleukin- 1ß play an important role in the maturation of osteoclast progenitors.34,35 These cytokines are known to be involved in LPS-induced osteoclastogenesis and bone resorption in vivo and in vitro. Furthermore, LPS can initiate osteoclastogenesis, maturation, facilitate osteoclast fusion and survival.36 In addition, RANKL expression in osteoblasts is stimulated by LPS.37 However, to date, no studies have investigated the potential effect of GMSCs-CM on the number of osteoclast and osteoblast in LPS-induced calvaria bone resorption in vivo, and thus we did here using an animal model.
The ethical health committee of the Faculty of Dental Medicine, Universitas Airlangga for animal laboratory used was approved this study protocol. True experimental laboratory with analytical post-test control group design was conducted. The number of samples is determined by the minimum sample formula from lameshow's, obtained a total sample size of 28 samples (n = 7/group). The sample selection was using blind simple random sampling technique. The sample was male wistar rats (Rattus Novergicus), 1-2 months old, 250-300grams. The experimental animal condition is healthy and there are no systemic complaints that can be seen with fine hair and normal mobility.
The 28 experimental animals were divided into 4 groups, namely PBS group: 100µg PBS day 1-7; LPS group: 100 μg LPS day 1-7; LPS and GMSCs group: 100μg LPS + 100μg GMSCS-CM day 1 1-7, GMSCs group: 100μg M- GMSCs day 1-7. Escherichia Coli LPS (Sigma Aldrich,
US) was used to induce bone resorption on the calvaria of animal model through subcutaneous injection. GMSCs-CM was obtained from a patent formulation owned by Pusat Pengembangan dan Penelitian Sel Punca (stem cell research and development center) Universitas Airlangga Surabaya, East Java, Indonesia. The dose of GMSCS-CM given is 100μg once a day. All samples were sacrificed on day 8 through cervical dislocation.
Calvaria was cleaned with running tap water for 10-15 minutes, then dried, then washed again with 1x PBS (OneMed, Indonesia) 3 times for 5-10 minutes each time and dried again. After the calvaria was washed and cleaned, the sample was fixed in 10% neutral buffer formalin (Sigma Aldrich, US) for 4-7 days. Calvaria was washed again with 1x PBS (OneMed, Indonesia) 3 times for 5-10 minutes each time. EDTA was used as decalcifier to decalcify calvaria for 14-28 days. Calvarias have been softened after decalcification process, continued to tissue processing, embedding, and section with a microtome to obtain HPA slide. Hematoxylin eosin (HE) staining was applied on the HPA slide to investigate the number of osteoblasts and osteoclasts. The calculations of the number of osteoblasts and osteoclasts in calvaria were observed using an inverted light microscope (Nikon, Tokyo, Japan) with 400x magnification in 5 fields of view observed by 2 observers. The results obtained were then recapitulated and analyzed.
Analysis of the data that obtained was preceded by the normality test with the Kolmogorov-Smirnov test followed by the Levene's homogeneity test. If the results of the normality and homogeneity tests showed that the data were normally distributed and homogeneous (p> 0.05), the data were analyzed using the One-way Analysis of Varience (ANOVA) test to analyze the differences between groups (p<0.01). The difference between each treatment group was carried out by the Tukey Honest Significant Different (HSD) test (p<0.01). If the results of the normality and homogeneity tests showed that the data were normally distributed but not homogeneous, so the data obtained were analyzed using the Kruskal-Wallis test to find out whether there were differences between the three treatments, the difference in each treatment group was carried out by the Mann- Whitney test (p<0.01). The statistical package for social science (SPSS) software 20.0 edition for mac (IBM corporation, Illinois, Chicago, US) was used to analyze the data.
In this study, data on the number of osteoclasts and the number of osteoblasts in the calvaria were found to be homogeneous and normally distributed (p>0.05). The number of osteoclasts in the calvaria decreased significantly in the LPS GMSCs-CM group compared to the other groups (p=0.0001; p<0.01). LPS injection in the calvaria increased the number of osteoclasts the most compared to other groups (p=0.0001; p<0.01). There was no significant difference in the number of osteoclasts in PBS group and GMSCs-CM group (p=0.0099; p> 0.01) (Figure 1).
The number of osteoblasts in the calvaria increased significantly in the LPS + GMSCs-CM group compared to LPS group (p=0.001; p<0.01). LPS injection in the calvaria significantly decreased the number of osteoblasts compared to the other groups (p=0.001; p<0.01). There was no significant difference in the number of osteoblasts in PBS group and GMSCs-CM group (p=0.099; p>0.01). However, there was a significant difference between the PBS group and GMSCs-CM group compared to LPS + GMSCs-CM group and the LPS group (p<0.01).
Figure 1. Osteoclasts in the calvaria in each group (black box) were observed by means of light inverted microscope at 100x and 400x magnification. The number of osteoclasts in each group (* significant between groups (p <0.01).
Figure 2. Osteoblast in the calvaria in each group (black box) were observed at 100x and 400x magnification. The number of osteoclasts in each group (* significant between groups (p <0.01).
Excessive osteoclastogenesis is often found in chronic inflammatory conditions, one of which is of its etiology is bacterial LPS-induced bone loss.38 In this study, the number of osteoclasts in the calvaria after LPS induction increased the most and significantly different compared to other groups. LPS is a potensial endotoxin that has been frequently used to induce pro-inflammatory cytokines in the immune system which are important factors to induce osteolytic bone resorption.39 Various inflammatory cytokines induced by LPS such as TNF-α and Interleukin-1ß play an important role in maturation of osteoclast cell progenitors.35,36 The increased TNF-α level by LPS can induce RANKL and M-CSF expression that enhance the osteoclastogenesis and osteoclast maturation.7-8 LPS increases osteoclastogenesis, differentiation, osteoclast maturation through the activation of mitogen-activated protein kinases (MAPKs) pathways such as c -Jun N-terminal kinases (JNK) and extracellular signal regulated kinases (ERK1/ 2).40
The number of osteoblasts in the LPS group also decreased significantly compared to the other groups. Previous research stated that LPS can inhibit alkaline phosphatase (ALP) activity, osteocalcin and COL1A1 production, thereby inhibiting osteoblastic differentiation from periodontal ligament stem cells (PDLSCs).41 LPS can significantly reduce metabolism, viability and ALP activity of MC3T3-E1 cells which is osteoblast cell line. LPS also significantly decreased mRNA expression associated with osteoblast genes on MC3T3-E1 cells. However, LPS significantly increased mRNA expression and protein levels of bax and caspase-3, but decreased Bcl-2 expression in MC3T3-1 cells. LPS can induce osteoblast apoptosis as. Furthermore, activation of JNK pathway can restrain the osteoblastogenesis or differentiation of osteoblast.41
The degradation of bone tissue due to osteolysis from increased pro-inflammatory cytokines can be inhibited using biomaterials with immunoregulatory and immunomodulatory capabilities. One of the biomaterials that have potential abilities as immunoregulators and immunomodulators is GMSCs. GMSCs were identified on the gingiva. GMSCs have promising capabilities in terms of regeneration, immunoregulatory and immunomodulatory abilities.42-44 GMSCs are able to differentiate into osteogenic, and chondrogenic lineage.45 However, the application of GMSCs has limitations, one of which is the long duration for cell expansion so that it is quite expensive. Therefore, conditioned medium was chosen from GMSCs due to its inexpensive and quite easy to applicate in clinical practice.21,31
In this study, the number of osteoclasts decreased drastically in the LPS + GMSCs-CM group. Thus, it was significantly different from the other groups. Previous studies have revealed that MSCs-CM can modulate the immune response and protect against inflammation- induced tissue lesions.36,37,46 MSCs-CM can be a strategic biomaterial for cytotherapy in various chronic inflammatory diseases of bone tissue. Previous research using human palatine tonsil-derived mesenchymal stem cells (T-CM) could inhibit the RANK-RANKL interaction on RAW 264.7 which is osteoclast precursor cell.47 Our results were supported by previous research by Li et al (2018) using an LPS-induced murine model. found that injection of adipose mesenchymal stem cells - conditioned medium (AMSC-CM) effectively decreased LPS-induced bone destruction (in vivo) was analyzed using micro-computed tomography and histopathology. Pro-inflammatory serum TNF-α, IL-1 and IL-6 levels associated with bone damage decreased significantly after AMSC-CM injection in the LPS. Furthermore, level of TNF-α, IL-1 and IL-6 mRNA expression in LPS-induced macrophage was decreased post AMSCs- CM administration.48 Nevertheless, GMSCs-CM may
inhibit LPS-induced calvaria bone resorption, this study is still limited due to only investigate the cellular aspect such as osteoclast and osteoblast number. Further study is still needed to examine the molecular aspect of LPS- induced bone resorption and analysis of growth factor, cytokine and chemokine level that possessed in the GMSCs-CM.
Based on the results of this study, it can be concluded that gingival medicinal signaling cells conditioned medium (GMSCs-CM) can significantly reduce the number of osteoclasts and increase the number of osteoblasts in LPS-induced calvaria bone resorption in wistar rats (Rattus novergicus).
The authors are grateful to the authorities of Stem Cells Research and Development Center; Faculty of Dental Medicine Research Center, Universitas Airlangga, Surabaya, East Java, Indonesia for the support and the facilities. This study was supported by Hibah Internal Penelitian Dosen Pemula (PDP) 2020 with appointment number 346/UN3/2020, Universitas Airlangga, Surabaya, East Java, Indonesia
The authors declare no conflict of interest.
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Received on 04.10.2020 Modified on 23.11.2020
Accepted on 26.12.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(10):5232-5237.
DOI: 10.52711/0974-360X.2021.00911