|Year : 2015 | Volume
| Issue : 2 | Page : 87-91
The effect of Emdogain and Simvastatin on periodontal ligament stem cells differentiation to osteoblasts: An in vitro study
Behzad Houshmand1, Iraj Amiri2, Mehrdad Hajilooi3, Mohsen Dalband4, Sara Soheilifar5
1 Department of Periodontology, School of Dentistry, Shahid Beheshti University of Medical Science, Tehran, Iran
2 Department of Research for Endometria and Endometriosis, Hamadan University of Medical Science, Hamadan, Iran
3 Department of Immunology, School of Medicine, Hamadan, Iran
4 Department of Oral and Maxillofacial Surgery, School of Dentistry, Hamadan, Iran
5 Department of Periodontology, School of Dentistry, Hamadan University of Medical Science, Hamadan, Iran
|Date of Web Publication||20-Apr-2015|
Department of Periodontics, Faculty of Dentistry, Hamadan University of Medical Science, Shahid Fahmideh Street, Hamadan
Source of Support: Hamadan University of Medical Science, Hamadan, Iran, Conflict of Interest: None
Context: Regeneration of periodontium is one of the main goals of periodontal treatment. This may be achieved by differentiation of stem cells to periodontal tissue cells. Aims: This study sought to assess the differentiation of human periodontal ligament stem cells (PDLSCs) cultured in osteogenic medium supplemented with Emdogain or Simvastatin. Settings and Design: This is an experimental study. Materials and Methods: After teeth extraction, the PDL attached to the middle third of the root surface was removed. Cells were expanded in culture medium and used for osteogenic differentiation and added to the test materials (different combinations of the basic medium, dexamethasone [DEX], Simvastatin and Emdogain). Osteogenic differentiation was evaluated using Alizarin Red staining (ARS), alkaline phosphatase (ALP) activity and calcium content tests. Statistical Analysis Used: Statistical analysis was performed using SPSS version 17 software. Deliberation was done with 6 × 3 factorial (six groups × three periods). Then, the data were submitted to ANOVA, and the differences between the groups were compared using Tukey post-hoc test. Results: Quantitative analysis of ARS demonstrated that the frequency of mineralized nodule formation was the highest in the enamel matrix protein derivatives (EMD) + DEX group. The highest ALP activity on day 7 was in DEX + Simvastatin group. Calcium content was the lowest in the control group and the highest in EMD + DEX group at all tested time points. Conclusions: This study showed that 100 μg/ml Emdogain had a significant effect on osteogenic differentiation of human PDLSCs to osteoblasts and 10−8 M Simvastatin had an effect comparable to that of DEX.
Keywords: Differentiation, enamel matrix protein, osteoblast, periodontal ligament, Simvastatin, stem cells
|How to cite this article:|
Houshmand B, Amiri I, Hajilooi M, Dalband M, Soheilifar S. The effect of Emdogain and Simvastatin on periodontal ligament stem cells differentiation to osteoblasts: An in vitro study. SRM J Res Dent Sci 2015;6:87-91
|How to cite this URL:|
Houshmand B, Amiri I, Hajilooi M, Dalband M, Soheilifar S. The effect of Emdogain and Simvastatin on periodontal ligament stem cells differentiation to osteoblasts: An in vitro study. SRM J Res Dent Sci [serial online] 2015 [cited 2022 Aug 13];6:87-91. Available from: https://www.srmjrds.in/text.asp?2015/6/2/87/155462
| Introduction|| |
The periodontium is the supporting tissues of the teeth and constitutes a complex, with each of its components (gingiva, periodontal ligament [PDL], cementum, and bone) undergoes certain changes due to periodontal disease.  The final goal of periodontal treatment is to prevent progressive attachment loss and regenerate the lost periodontal supporting tissues. Although conventional periodontal treatments may reduce the probing depth and stop the attachment loss,  histologically, results in proliferation of epithelial tissue at a faster rate than the underlying mesenchymal tissue. , Several treatments or techniques have been introduced and used in clinical practice, but the results were not so acceptable. Currently, our understanding of molecular and cellular processes involved in periodontal regeneration has been greatly enhanced and tissue engineering is now a possible alternative to conventional treatments. PDL contains pluripotent stem cells as a source of periodontal tissue regeneration. These mesenchymal stem cells can differentiate into cementoblasts, osteoblasts and fibroblasts and synthesize periodontal tissue extracellular matrix.  These cells originate from bone capillaries and the PDL space, ultimately differentiating in the PDL space. ,
In the last years, the effects of different growth and differentiating factors on stem cells differentiation have been evaluated. In this field, enamel matrix protein derivatives (EMD) was introduced, and its application was studied. Emdogain (EMD) is a commercially available enamel matrix derivative in polyglycol alginate and is successfully used in clinical practice to achieve periodontal regeneration. , Surgical periodontal regeneration of deep intra-bony defects with EMD may lead to significantly greater improvements in clinical parameters  and the results are comparable to guided tissue regeneration which can be maintained over a 10-year period.  In vitro studies had suggested that it can regulate cells in the osteoblastic lineage, prolong the growth of cultured primary mouse osteoblasts,  influence bone metabolism through the activation of growth factors, increase the number of osteoblasts,  and enhance the osteogenic capacity of bone marrow with an increase in cell numbers, alkaline phosphatase (ALP) activity and mineralized nodule formation.  Furthermore, EMD was capable of stimulating trabecular bone biosynthesis and regeneration. 
On the other hand, statins were also found to induce bone formation under in vitro and in vivo conditions in osteoporotic animal models.  They may serve as a treatment option for osteoporosis especially in cases suffering from a significant loss of trabecular bone.  Statins induce osteoblast differentiation and mineralization in MC3T3-E1 cells.  Studies have shown that local and systemic administration of statins could induce bone formation. ,,
This study sought to assess the differentiation of PDL stem cells (PDLSCs) to osteoblasts in standard osteogenic medium in comparison with cells cultured in osteogenic medium supplemented with Emdogain or Simvastatin.
| Materials and Methods|| |
In this experimental study, PDLSCs were isolated from the roots of 17 clinically healthy first premolars of young individuals extracted for orthodontic reasons. All the participants signed the informed consent, and the study protocol was approved in Ethics Committee of the Hamadan University of Medical Science. A week before tooth extraction, patients received scaling and polishing and were advised to brush their teeth thoroughly. The tooth to be extracted was polished again immediately before extraction if visible plaque was observed. Prep and drape were done, and local anesthesia was induced. In order to minimize the risk of infection transmission, patients rinsed their mouth with diluted povidone-iodine, instruments were precisely sterilized, sterile gloves were washed with normal saline and surgeon and assistants wore mask and surgical gown.
After the extraction, the PDL tissues attached to the middle third of the root surface were removed using a surgical scalpel. Coronal and apical portions of the ligament were not used to avoid contamination by gingival and pulpal cells. Stem cells were cultured according to Gay et al., as follows: 
Periodontal ligament cells were scraped from premolars and transferred to a plate containing α-modified eagle medium (α-MEM) and 15% fetal bovine serum (FBS), then enzymatically digested for 1-h at 37°C in a solution of 3 mg/ml collagenase Type I. The samples were then centrifuged at 400 ×g for 10 min and plates were expanded with α-MEM containing 15% FBS and 1% antibiotics in six-well plates and cultured at 37°C with 5% CO 2 . After 3 days of culture, numerous fibroblastoid cells migrated from the explants. At day 7, adherent cells, which were 80-90% confluent, were washed twice with phosphate-buffered saline (PBS) and released from the culture surface by using 0.25% trypsin-EDTA solution (Gibco, Germany) and plated in tissue culture polystyrene flasks at 5 × 10 3 cells/cm 2 . Primary cultures of PDLSCs mainly consisted of colonies of bipolar fibroblastoid cells, which, after subcultivation, proliferate with a population-doubling time of 48 h, reaching a confluent growth-arrested condition.
At the third passage, cells in the developing adherent layer were used for osteogenic differentiation and the test materials were added after cell removal with 0.25% trypsin-EDTA solution. Then the collected cells were divided into six culture plates and cultured for 24 h. When the cells were 80% confluence, the culture medium was changed to six differential media, 5 of which contained basic medium (α-MEM containing 10% FBS, 5 mM β-glycerophosphate and 50 μg/ml l-ascorbic acid) and were supplemented with 10 -8 M dexamethasone (DEX) (Group 1), Simvastatin (Group 2), DEX and Simvastatin (Group 3), Emdogain (Group 4), DEX and Emdogain (Group 5). Negative control cultures were maintained in α-MEM containing 10% FBS without osteogenic supplements (Group 6). Cells were re-fed every 3 days and Alizarin Red staining (ARS), ALP activity and calcium tests were done at days 7, 14, 21 and 28 of culture.
Mineralization of the cell layer was evaluated using ARS. Briefly, the cells were rinsed twice with PBS and fixed with ice-cold 70% ethanol. After rinsing in deionized water, the plates were stained with 40 mM Alizarin Red S (Sigma-Aldrich, Germany) solution, pH 4.2, for at least 10 min, rinsed with water and washed in PBS for 15 min to remove nonspecific stains. The plates were then air dried and analyzed by inverted microscopy.
In addition to ARS, assessment of osteoblast differentiation was done by measuring ALP activity in culture (Pars Azmun Co.). PDLSCs were cultured as described above and at the mentioned time points, cells were washed with PBS and scraped into a solution containing four parts of buffer solution (1 mol/l diethanolamine and 0.5 mmol/l MgCl 2 ) and one part of substrate solution (10 mmol/l p-nitrophenyl phosphatase). ALP activity was determined colorimetrically with p-nitrophenyl phosphatase as a substrate. Subsequently, the protein content was measured.
The calcium content of samples was measured by the cresolphthalein complexone method at the mentioned time points. The test was done according to the instructions of Pars Azmun Co.
Statistical analysis was performed using SPSS version 17 (SPSS Inc, USA). Deliberation was done with 6 × 3 factorial (six groups × three periods). Then the data were submitted to ANOVA, and the differences between the groups were compared using Tukey post-hoc test. The distribution of data in normal range was tested as in nonparametric method using Kolmogorov-smirnov test.
| Results|| |
In this study, a pluripotent stem cell population was derived from human PDL. These cells were visible like mesenchymal type stem cells and adherent spindle-shaped and fibroblast-like cells at the bottom of the plates.
Results of ARS showed that the deposits observed were calcified. Extensive osteogenic differentiation was noticeable in PDLSCs exposed to osteogenic conditions or tested materials, which indicates calcium deposition [Figure 1]; whereas, no mineralization was observed in the cells cultured in control medium.
|Figure 1: Deposits observed in bone nodule-like structures stained with Alizarin Red: (a) ×4 magnification of cells in dexamethasone group. (b) ×100 magnification of cells in enamel matrix protein derivatives group|
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As shown in [Figure 2], quantitative analysis of ARS demonstrated that mineralized nodule formation was the highest in the group supplemented with Emdogain and DEX. The differences between groups were significant (P < 0.05).
|Figure 2: Quantitative analysis of alizarin red staining in different groups|
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Alkaline phosphatase activity was measured after 7 and 14 days of subculture. As shown in [Figure 3], there were significant differences between the control group and the experimental groups (P < 0.05).
|Figure 3: Alkaline phosphatase activity in different groups on tested days|
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After 7, 14, 21 and 28 days of subculture, calcium content was measured in addition to ALP activity. As observed in [Figure 4], the amount of calcium was the lowest in the control group and the highest in EMD + DEX group on all the tested days (P < 0.05).
|Figure 4: Intra-cellular calcium deposition in different groups on tested days|
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| Discussion|| |
In this study, osteogenic differentiation of PDLSCs in osteogenic medium supplemented with Emdogain and Simvastatin was evaluated. As mentioned in the results section, the negative control group showed the minimum amounts of tests. During 2 weeks of subculture, effect of Emdogain on ALP activity was more than that of DEX. At day 7, all groups supplemented with DEX, Emdogain, Simvastatin and combinations of them, had similar range of ALP activity. Although, on day 14, the amount of ALP activity increased in all groups, EMD group showed significantly higher values. This finding may be explained by the different time of action of the materials' effects on cell differentiation. Combining Emdogain with DEX had a significant effect on intra-cellular calcium deposition of the cultured cells. The effect of DEX on calcium content was greater than that of Simvastatin. Combination of Emdogain and DEX resulted in a high number of mineralized nodule formation. Therefore, the effect of Emdogain on osteogenic differentiation was noticeable.
Sonobe et al. reported that only after 16 days of subculture, ALP activity was significantly higher in the bone marrow mesenchymal stem cells (BMMSCs) cultured in standard medium supplemented with Simvastatin, than the control group.  In our study, ALP activity in the group supplemented with Simvastatin was significantly higher than the control group on both tested days. This difference may be attributed to different characteristics of stem cells derived from bone marrow or PDL.
In Keila et al. study incubation of BMMSCs with 25 μg/ml EMD for 3 weeks resulted in significantly more mineralized nodule formation compared to the control cultures consisted of the basic medium supplemented with DEX.  Similar results were obtained in our study with 100 μg/ml Emdogain at day 28 of subculture, but in their study 100 μg/ml EMD did not produce any effect. They showed that ALP activity at day 10 increased by two folds with 25 μg/ml EMD. In our study, the group supplemented with 100 μg/ml Emdogain had higher ALP activity on days 7 and 14, compared to the DEX group.
Study of van den Dolder et al. showed no consistent effect of EMD on cell growth or differentiation of rat bone marrow cells. This difference between their finding and ours may be attributed to the fact that they added EMD to culture in their study by coating the discs with 100 μg/ml EMD. 
Lee et al., in their study in 2012 evaluated the effect of the addition of EMD and Simvastatin on growth and odontoblastic differentiation of hDPSCs induced by Portland cement with bismuth oxide. They assessed the differentiation of cells and reported increased odontogenic potential, ALP activity and mineralized nodules due to Simvastatin and Emdogain.  These results are in agreement with our findings.
Karanxha et al. studied the efficacy of treatment with a combination of Simvastatin and EMD for human dental pulp stem cells odontoblastic differentiation. They concluded that the combined application of Simvastatin and EMD was more effective in inducing differentiation than a single therapy.  These findings are in agreement with our study results.
Based on the available literature, the effect of EMD on proliferation and differentiation varies among different cell types and sources. Also, variation in experimental designs may also be responsible for controversial results of different studies. For example, in the study by van den Dolder et al. plates were coated with EMD diluted in 0.1% acetic acid, while other studies used diluted EMD in their medium and they suggested that diluting EMD in culture medium might give a more predictable result than using EMD as a coating. Thus, we added Emdogain to medium in the present study. Furthermore, various concentrations of Emdogain were used in studies. Some researchers have reported that 100 μg/ml EMD is the optimal concentration for promoting the osteogenic potential of PDL cells, while others  reported that only 25 μg/ml EMD stimulated the osteogenic potential of bone marrow stromal cells. In our study, we evaluated the effect of Emdogain at 100 μg/ml concentration on PDLSCs. Evaluating different doses of the tested materials and comparing their effects to identify the most effective dosage can be the subject of future studies.
Ultimately, it may be concluded that 100 μg/ml Emdogain had a significant effect on osteogenic differentiation of PDLSCs and 10−8 M Simvastatin had an effect comparable to that of 10−8 M DEX.
| Acknowledgments|| |
We gratefully acknowledge Dr. Hosein Mahjoub for assisting in statistics analysis and also Hamadan University of Medical Science for financial support of this study.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]