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| Year : 2011 | Volume
: 22
| Issue : 2 | Page : 190-194 |
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| Effect of ProRoot MTA, Portland cement, and amalgam on the expression of fibronectin, collagen I, and TGFβ by human periodontal ligament fibroblasts in vitro |
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Sara Fayazi1, Seyed Nasser Ostad2, Hasan Razmi3
1 Department of Endodontics, Faculty of Dentistry, Rafsanjan University of Medical Sciences, Rafsanjan, Iran
2 Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
3 Department of Endodontics, Faculty of Dentistry, Tehran University of Medical Sciences, Tehran, Iran
Click here for correspondence address and email
| Date of Submission | 13-Dec-2008 |
| Date of Decision | 14-Jan-2010 |
| Date of Acceptance | 16-Jun-2010 |
| Date of Web Publication | 27-Aug-2011 |
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 Abstract | | |
Context: Today many materials have been introduced for root-end filling materials. One of them is mineral trioxide aggregate (MTA) that is mentioned as a gold standard.
Aims: The purpose of this in vitro study was to evaluate the reaction of human periodontal ligament fibroblasts to the root-end filling materials, such as ProRoot MTA, Portland cement, and amalgam.
Settings and Design: Eight impacted teeth were extracted in aseptic condition. The tissues around the roots were used to obtain fibroblast cells. After cell proliferation, they were cultured in the chamber slides and the extracts of the materials were added to the wells.
Materials and Methods: Immunocytochemical method for measuring the expression of Fibronectin, collagen I and transforming growth factor beta (TGF®) was performed by Olysia Bioreport Imaging Software.
Statistical Analysis Used: The results were analyzed by SPSS 13.0 and Tukey post hoc test with P<0.05 as the limit of significance.
Results: Collagen expression in MTA specimens was higher than the other groups in 24 h significantly. After 48 h, the Portland cement group showed the most expression of collagen significantly and after 1 week, Portland cement and MTA groups had the most expression of collagen but there was no significant difference between these 2 groups. After 1 week, the Portland cement group demonstrated a higher amount of TGF® and fibronectin.
Conclusions: The results suggest that Portland cement can be used as a less expensive root filling material with low toxicity. It has better effects than amalgam on the fibroblasts. Keywords: Amalgam, MTA, TGFβ
How to cite this article:
Fayazi S, Ostad SN, Razmi H. Effect of ProRoot MTA, Portland cement, and amalgam on the expression of fibronectin, collagen I, and TGFβ by human periodontal ligament fibroblasts in vitro. Indian J Dent Res 2011;22:190-4 |
How to cite this URL:
Fayazi S, Ostad SN, Razmi H. Effect of ProRoot MTA, Portland cement, and amalgam on the expression of fibronectin, collagen I, and TGFβ by human periodontal ligament fibroblasts in vitro. Indian J Dent Res [serial online] 2011 [cited 2023 Oct 5];22:190-4. Available from: https://www.ijdr.in/text.asp?2011/22/2/190/84278 |
Periradicular surgery is the most frequently performed endodontic surgical procedure. The aims of periradicular surgery are to remove the causes of disease and to provide a favorable environment for healing of the surgical wound. Newer root-end filling materials, among other advances, including developments in surgical armamentarium, the implementation of microsurgical techniques, enhanced illumination and magnification, have helped to improve the outcome of periradicular surgery. [1],[2],[3],[4],[5],[6] Management of the resected root-end during periradicular surgery is critical to a successful outcome. [7]
The ideal healing response after periradicular surgery is the re-establishment of an apical attachment apparatus and osseous repair. [8],[9] Many materials, such as mineral trioxide aggregate (MTA), amalgam, glass ionomer, various of zinc oxide materials and Portland cement, have been used in endodontic treatment as the root-end filling materials. Traditionally, amalgam was the material of choice for root-end fillings. [10],[11],[12],[13],[14] Dentists are familiar with amalgam, and its usage for this purpose is also a reflection of its historical popularity as a restorative material. It is readily available, inexpensive, and easy to manipulate, radioopaque, and previously thought to be associated with a reasonable outcome. However, it has become clearly recognized that there are many disadvantages with amalgam. [15],[16],[17] Electrochemical corrosion of amalgam was reported to be responsible for failures of amalgam root-end fillings. [18] Scattering of excess amalgam particles during placement of the root-end filling can lead to tissue disfigurement. MTA was developed as a new root-end filling material at Loma Linda University, California, USA. A study on the physical and chemical properties of MTA investigated the composition, pH, radioopacity, setting time, compressive strength, and solubility of the material compared with amalgam and reinforced zinc oxide eugenol cements. [19] Unlike a number of dental materials that are not moisture-tolerant, MTA actually requires moisture to set. The MTA powder consists of fine hydrophilic particles. When mixed with sterile water, hydration of the MTA powder results in a colloidal gel that solidifies into a hard structure. It has a long setting time (2h 45min) so the material must be protected before it is fully set. The pH of MTA rises from 10.2 after mixing to 12.5 after 3h, remaining unchanged afterward. Likewise, the compressive strength of MTA increases with time, from 40.0 MPa after 24 h to 67.3 MPa after 21 days. Portland cement is another material that is mentioned as a substitute for MTA. It is more accessible than MTA with similar properties. [20],[21],[22] It was showed that MTA and Portland cement have antimicrobial effect against the tested strains ( Escherichia More Details coli, Candida, Actinomyces viscosus). [23] Steffen and Van Waes evaluated the scientific data on MTA and Portland cement from 1993 to 2009. This review of 50 papers conforming to the applied criteria showed that MTA and Portland cement have the same clinical, biological, and mechanical properties. It seems likely that MTA materials are based on industrial Portland cement mixed with bismuth oxide. The existing literature gives a solid base for clinical studies with Portland cement to replace MTA as an endodontic material. Portland cement could be a substitute for most endodontic materials used in primary teeth restoration. [24] But Portland cement is more accessible with lower price than MTA. The only difference is that bismuth oxide is added to MTA for better radioopacity. [25] The purpose of this in vitro study was to evaluate the effects of soluble compounds released from different materials (ProRoot MTA, Portland cement, and amalgam) on the cellular expression of 2 essential extracellular matrix proteins, fibronectin and type I collagen, as well as TGFβ in periodontal ligament fibroblast. For better evaluation we used immunocytochemistry (ICC), the technique that is used to detect the presence of proteins in cell suspension, cultured cells, or cytospin by use of a specific antibody.
| Materials and Methods | |  |
Human periodontal ligament tissue was obtained from the freshly extracted, healthy human unerupted third molars of patients who were referred to the oral and maxillofacial department of the dental school (Medical Science University of Tehran) with informed patient consent. Teeth with two-thirds of the whole root length formation, which needed no additional sectioning for extraction, were chosen as samples. Before tooth extraction, the patients were given a mouth rinse of iodine solution (Povidone iodine 10%; Tolid Daru Co., Tehran, Iran). The atraumatically extracted teeth were then immersed in Dulbecco's modified eagles medium (DMEM; Invitrogen-Gibco, UK) supplemented with 10% fetal Bovine serum, 125 U/mL penicillin, and 125΅g/mL streptomycin (Invitrogen-Gibco, UK). The samples were rinsed in Hank's Balanced Salt solution (Invitrogen-Gibco, UK) 3 times under laminar air flow. To avoid contamination from the gingiva, the periodontal ligament tissue from the middle third of the root surface was scraped with a sharp #12 scalpel. Then, the extracted tissue was immersed in 25-cm 2 culture dishes (Nunc, Roskilde, Denmark) that contained 4 mL of culture medium. The samples were then incubated at 36.5C, 5% CO 2 for a mean of 10 days to observe the human periodontal ligament fibroblast (HPDLF) cells (passage zero). After confluence (1 week), the cells were subcultured by using 0.25% trypsin/EDTA (Invitrogen-Gibco, UK). Passages number 3-6 were used in this study.
Extract preparation
ProRoot MTA 100 mg (Dentsply Tulsa, USA) was mixed with 35 mg H 2 O and placed in 2mL DMEM in 3 plates. [26] Portland cement (Ciman Shargh, Mashhad, Iran) was prepared similar to ProRoot MTA. Amalgam (100 mg) was placed in 2mL DMEM. The specimens were allowed to set in each time (24h, 48h, and 1 week) at 37C in a humidified CO 2 incubator prior to add the cells plate. After 24 h, 48 h, and 1 week, the medium was removed and centrifuged at 6000 rpm for 5 min. The top standing solution in the centrifuge tube was used for the tests (MTA: test 1, Portland cement: test 2, and Amalgam: test 3).
Immunocytochemistry
HPDLF cells were cultured in 4-well chamber slides (Nunc, Denmark). A total of 4Χ10 4 cells were cultured into each well. After 24 h, the medium was removed and tests medium was added to the wells. The examination of samples was performed after 24 h, 48 h, and 1 week. In each experiment, at least 1 well of the 4-well chamber slide was used as control (no treatment with test material in medium) and 1 as the negative control (containing test material but without primary antibody against test antigen during ICC protocol) in each time. After this, the specimens were fixed and stained according to the ICC protocol given by the company. As secondary antibody and staining system, LSAB2 kit (Dako, Denmark) for fibronectin, collagen, and TGFβ was used. All the assays were repeated 3 times to guarantee reproducibility. After that, digital imaging from the wells was obtained by confocal microscope (Olympus, Tokyo, Japan) and evaluated by Olysia Bioreport Imaging software (Olympus, Tokyo, Japan). The average and standard deviation were calculated and compared with those of the controls. The data were analyzed by one-way ANOVA followed by a multiple comparison Tukey post hoc test using Sigma Stat software (SPSS13.0, Chicago, IL, USA). Statistical significance of the data was determined at P≤0.05.
| Results | | |
The data obtained by using the software were related to the cell staining and the bigger the number the lesser will be the expression. The materials that were grouped in one subset have not shown any significant differences between each other, but among different subsets, a significant difference was observed.
Collagen
Collagen expression in MTA specimens was higher than the other groups in 24 h significantly. After 48 h, the Portland cement group showed the most expression of collagen significantly and after 1 week, Portland cement and MTA group have the most expression of collagen but there was no significant difference between these 2 groups [Table 1]. | Table 1: Comparison of the collagen expression in different times with statistical subsets - Tukey post hoc test
Click here to view |
Fibronectin
There was no significant difference in fibronectin expression between the groups in 24 h. After 48 h there was no significant difference between MTA, Portland cement, and amalgam groups and after 1 week, Portland cement and MTA groups showed higher expression of fibronectin but there was no significant difference between these 2 groups [Table 2]. | Table 2: Comparison of the fibronectin expression in different times with statistical subsets - Tukey post hoc test
Click here to view |
TGFβ
After 24 h TGFβ expression was higher in amalgam and MTA groups but after 48 h, MTA group showed higher expression and after 1 week, the Portland cement group demonstrated a higher amount of TGFβ [Table 3]. | Table 3: Comparison of the TGFâ expression in different times with statistical subsets - Tukey post hoc test
Click here to view |
| Discussion | | |
The effect of root-end filling materials on the periradicular tissue is very important in healing. The effects of MTA on cementoblast growth and osteocalcin production were investigated in a tissue culture experiment. [27] Results suggested that MTA permitted cementoblast attachment and growth, whereas the production of mineralized matrix gene and protein expression indicated that MTA could be considered cementocoinductive. MTA was found to stimulate extracellular regulated kinases, members of the mitogen-activated protein kinase pathway, which are involved with bone cell proliferation, differentiation, and apoptosis. [28] Our study showed that MTA stimulates collagen expression more than other tested materials in 24 h. The results of this study are similar to other studies that showed MTA as the most biocompatible filling material and that it is non toxic to periodontal ligament (PDL) fibroblast in vitro. [29] According to the study by Keiser et al., MTA in freshly mixed state and in 24 h set state has the least cytotoxicity in comparison with the other filling materials by using HPDLF, in vitro. [30] Portland cement has the main components similar to MTA. The amount of gypsum in MTA is half of the Portland cement. Portland cement is composed of particles with a wide range of size, whereas ProRoot MTA showed a uniform and smaller particle size. [21] The biocompatibility of Portland cement has previously been documented. [22] Matsuda et al. [31] have shown that the behavior of HPDLF cells is different from human gingival fibroblasts (HGF). Despite the difference reported between HPDLF and HGF and the ease of HGF cell culture, it was decided to culture the HPDLF cells to achieve more accurate results that correlate to the same tissue (PDL). Fibroblasts originated from PDL showed different proliferative behavior compared with those from gingival cells at confluences in vitro. [32] Mariotti and Cochran [32] found that it took 4 days for fibroblasts that were derived from the gingival cells to become confluent and 6 days for the fibroblasts derived from the PDL. In this study, the HPDLF cells reached confluence in T-25 flasks after 1 week. According to the technique used in this study for HPDLF cell culture, it seems that cultivation of these cells in a sterile condition is not always successful. In our study, a total of 8 extracted teeth were used, but most of the samples, although not infected by fungi, did not show any sign of cell growth. This can be related to the number of initial fibroblasts within the scraped tissue and/or the state of the cell cycle during sample extraction. The function of the fibroblasts is under the influence of age, trauma, and inflammation; in this study, therefore, young patients with healthy unerupted, third-molar teeth were chosen. In a pilot study, the proper number of HPDLF in each well of the chamber slide that make good image on ICC staining was 4Χ10 4 and the proper dilution of each antibody was evaluated (for collagen I (1/100), fibronectin (1/400), and TGFβ (1/100). After 1 week, Portland cement group had more collagen and fibronectin expression than the others [Figure 1] and [Figure 2]. There was no significant difference between MTA and Portland cement groups. It appears that Portland cement is a proper substitute for MTA, because of its good effect on fibroblast cells. It is similar to MTA in some properties, such as collagen expression. TGFβ plays a significant role in regulating the synthesis of extracellular matrix. TGFβ has also been shown to increase the formation of the extracellular matrix components: collagen, fibronectin, elastin, and glycosaminoglycan. [33] It was found that Portland cement has the best effect on fibroblasts by increasing TGFβ expression in 1 week. There was no sign of staining in the negative control group because of the lack of primary antibody [Figure 3]. In a study by Guven et al, it was shown that ProRoot MTA can increase the TGFβ levels at 24 and 72 h (P <0.05) in human gingival fibroblasts in vitro. In their study, amalgam had no favorable effects onthe synthesis of TGFβ[34] . It is similar to our study where amalgam had no constructive influence on the protein synthesis that was evaluated. | Figure 1: The image of collagen expression after 1 week in Portland cement group (400×)
Click here to view |
 | Figure 2: The image of fibronectin expression after 1 week in Portland cement group (400×)
Click here to view |
 | Figure 3: The image of negative control group without adding primary antibody
Click here to view |
Generally, amalgam specimens do not have the same effects as that of Portland cement or MTA, therefore, it is not an appropriate substitute for MTA. In the study by Pistorious et al, it was shown that gingival fibroblast showed a highly significant decrease in protein synthesis and cell proliferation when in contact with amalgam. On the contrary, MTA and titanium had no irritative effect on these parameters. [35] Given the low cost of Portland cement and similar properties when compared with ProRoot MTA, it is reasonable to consider Portland cement as a possible substitute for MTA in endodontic applications. However, industrially manufactured Portland cement is not approved currently for use and therefore no clinical recommendation can be made for its use in the human body. More in vivo studies are needed to approve Portland cement as the substitute for MTA.
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Correspondence Address:
Sara Fayazi
Department of Endodontics, Faculty of Dentistry, Rafsanjan University of Medical Sciences, Rafsanjan
Iran
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0970-9290.84278
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3] |
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