• Users Online: 113
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 13  |  Issue : 4  |  Page : 208-212

Effect of mineral trioxide aggregate with or without propolis extract on the proliferation of fibroblast cell line


Department of Endodontics, Faculty of Oral and Dental Medicine, Cairo University, Cairo, Egypt

Date of Submission21-Sep-2016
Date of Acceptance27-Oct-2016
Date of Web Publication14-Dec-2016

Correspondence Address:
Marwa M Bedier
Building No. 1, Street 72, Maadi, Cairo 11728
Egypt
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1687-8574.195717

Rights and Permissions
  Abstract 

Objectives
The aim of the study was to assess the cellular reaction of baby hamster kidney fibroblast cell line to mineral trioxide aggregate (MTA) and mineral trioxide aggregate mixed with propolis ethanolic extract (MTA-P) at different observation points using the crystal violet proliferation assay.
Materials and methods
Extracts of the materials were prepared at a surface area-to-volume ratio of 79 mm 2 /ml and collected 24, 72 h and 7 days after mixing. Cells were seeded into 96-well plates at 2 × 10 4 cell/well for 24 h and then incubated with 100 μl extracts from each group (n = 6). Cell proliferation was assessed using the crystal violet assay. Cells cultured in culture medium only without the extracts served as control. Statistical analysis was performed using one-way analysis of variance, two-way analysis of variance and independent t-test, P value less than 0.05.
Results
At 24 h, MTA-P showed higher cell number ratio than MTA (P < 0.05). At 72 h and 7 days, results showed no statistically significant difference among the groups.
Conclusion
The early biocompatibility of MTA-P seems better than MTA, yet remains constant. MTA biocompatibility, however, seems to improve by time.

Keywords: baby hamster kidney fibroblast, cell proliferation, crystal violet, mineral trioxide aggregate, propolis


How to cite this article:
Bedier MM. Effect of mineral trioxide aggregate with or without propolis extract on the proliferation of fibroblast cell line. Tanta Dent J 2016;13:208-12

How to cite this URL:
Bedier MM. Effect of mineral trioxide aggregate with or without propolis extract on the proliferation of fibroblast cell line. Tanta Dent J [serial online] 2016 [cited 2023 Feb 3];13:208-12. Available from: http://www.tmj.eg.net/text.asp?2016/13/4/208/195717


  Introduction Top


Pulp therapy is based on the premise that the pulp tissue has the capacity to heal. Pulp preservation by direct pulp protection includes pulp capping and pulpotomy. Both procedures permit preservation of the pulp tissue to continue its functions. Various factors, including pulpal status, stage of root formation, periodontal condition, age and time elapsed between pulp exposures and protection, pulp protecting materials, the nature and size of exposure and bacterial microleakage affect the ultimate result of pulp preservation [1].

It is widely accepted that the ultimate goal of the application of a capping material is to induce the dentinogenic potential of pulpal cells [2,3]. The dentinogenic potential can be induced directly as a specific biological effect of the capping material on pulpal cells, or indirectly as a part of the stereotypic wound healing mechanism in the traumatized pulp [4,5].

Several pulp capping materials have been proposed, yet, the most frequently used materials have been calcium hydroxide, and most recently, mineral trioxide aggregate (MTA). The major disadvantage of calcium hydroxide is its dissolution over time. As most dentin bridges form under calcium hydroxide may contain tunnels and allow the pulp to become infected due to microleakage [6]. MTA has been introduced for its excellent sealing ability, its biocompatibility and its ability to stimulate hard tissue formation.

In spite of its advantageous properties, MTA still has some drawbacks as the poor handling characteristics, physicochemical properties (e.g., setting time) and the initial necrosis and cytotoxicity. Several investigations have used additives for improving the handling characteristics and physicochemical properties of MTA [7-9], but few studies evaluated the effect of additives on biological properties of MTA.

Propolis is a new natural preparation, which is produced by bees from poplar and coniferous trees or clusia flowers to strengthen and join the hive cells [10,11], in addition to protection the beehives from microbial infection [12]. The general composition of propolis is resin (50%), vegetable balsam, wax, essential aromatic oils (30%), salivary secretions (10%), pollen (5%), other substances (5%) including amino acids, ethanol, vitamin A, B complex and E, minerals, steroids and flavonoids [13-15]. The precise chemical composition of propolis is variable according to the plant source, to the climate, the season, year, and location [16,17]. Propolis is an anti-inflammatory agent, has shown to inhibit synthesis of prostaglandins, support the immune system by promoting phagocytic activities, stimulate cellular immunity and augment healing effects. Additionally, it contains elements such as iron and zinc, which are important for the synthesis of collagen [13,18].

The biological response of the materials used for endodontic therapy are of particular concern, because damage and irritation could cause degeneration to pulp or periapical tissue and cause delayed wound healing, thus the purpose of this study was to assess the effect of addition of propolis extract to MTA on the proliferation activity of baby hamster kidney fibroblast (BHK) cell line.


  Materials and methods Top


Culturing procedure

BHK cell line (Invitrogen, Carlsbad, California, USA) was used in this study; medium in which the cell line was preserved was removed from the flask. Cells were washed with PBS, and then monolayer of the cells were covered with 1-2 ml 0.05% trypsin and 0.02% EDTA mixture for 2 min at 37°C. The action of the trypsin was stopped by adding 3 ml modified Eagle's medium (α-MEM, BioWhittaker; Lonza, Brussels, Belgium), supplemented with 5% fetal bovine serum and supplemented with antibiotics (10 000 μg penicillin-G + 10 000 μg streptomycin and 25 μg amphotericin B) and 2 mmol/l l-glutamine (BioWhittaker; Lonza), cells were collected at the bottom of 15 mm single sterile falcon tubes, centrifuged at 250g for 5 min and then washed twice with fresh complete medium and resuspended with 10 ml. The cell suspension was transferred into two sterile 75-cm polystyrene, filter-cap cell flasks labeled with cell type and date, then 10 ml fresh, complete α-MEM medium was added and the cells were incubated at an atmosphere of 5% CO 2 and 95% air at 37°C. Cells were incubated until adequate number of cells was obtained for the study and the cell cultures were periodically checked for growth, cell viability and the culture media was periodically changed every 48 h.

Preparation of the materials' extracts

Two types of samples were prepared: samples in which MTA (ProRoot MTA; Dentsply Tulsa, Tulsa, Oklahoma, USA) powder was mixed with sterile water (MTA) and samples in which MTA powder was mixed with propolis (100 μg/ml); prepared from 30% ethanolic extract of propolis (MTA-P). MTA was mixed according to the manufacturers' instructions in a laminar flow hood with a final water/propolis-to-powder ratio of ∼0.3 (1:3 g). Freshly prepared materials were placed at the bottom of flat-bottomed wells of 24-well cell culture plates to achieve a thickness of ∼1-2 mm.

The extracts of the test materials were prepared as follows: 600 μl of the culture medium was placed in each well containing the freshly mixed materials. The extraction medium of the tested materials was collected at each observation point: 24, 72 h and 7 days, then the extracts were diluted (1: 4) using complete medium to achieve quarter concentration.

Crystal violet proliferation assay

BHK cell line were seeded on 96-well culture plates at a density of 2 × 10 4 cells/well in 100 μl of culture medium and were incubated for 24 h in CO 2 incubator. The culture medium was then replaced by 100 μl aliquots of the extract of tested materials at the different observation points. Cells with complete medium without the extracts served as negative control. After 24 h, exposure of the cells to the extract was stopped, then cells were fixed by addition of 100 μl 1% glutaraldhyde and shaken on rotating platform at 300 cycles/min for 30 min, the fixation solution was removed and the plates were washed by deionized water to remove any cell debris, then air-dried. The cells were stained by addition of 100 μl of 0.02% crystal violet solution (SERVA Electrophoresis GmbH, Heidelberg, Germany). Plates were shaken on a rotating platform for 30 min, and then unbound dye was removed by extensive washing with distilled water.

The procedure resulted in cells with stained nuclei, whereas the cytoplasm remains clear. Finally, the bound dye was solubilized by the addition of 100 μl of 70% ethyl alcohol for 10 min and then microplate spectrophotometer was used to read the absorbance at a wavelength of 570 nm. The optical density (OD) measured in the crystal violet assay was used as a surrogate for cell proliferation (cell number). The OD value of each culture well exposed to each of the test materials was related to the mean of the OD of the untreated culture wells (100%) and were expressed as the cell number ratio. In all the experiment, the extract of each tested materials at the different observation points was tested in six well and repeated three times to ensure reproducibility, then data was statistically analyzed using the statistical package for the social science (version 21; SPSS Inc., IBM Corporation, New York, New York, USA); Two-way analysis of variance (ANOVA) was used to show the effect of materials and observation points on mean cell number ratio, one-way ANOVA was used to compare between the different observation points on mean cell number ratio and independent t-test was used to compare between different groups. Significant level set at P less than 0.05.


  Results Top


The mean (SD) values of the cell number ratio for MTA started 87.12 (8.10) after 24 h, which increased to 105.15 (12.23) after 72 h and 104.88 (9.68) after 7 days. One-way ANOVA test showed a statistically significant difference among the points (P = 0.011, P < 0.05). The value of the mean cell number ratio was lowest for the 24 h. For the MTA-P, the mean (SD) values of the cell number ratio after 24, 72 h and 7 days were 111.57 (7.87), 114.25 (10.91) and 113.10 (8.54), respectively. One-way ANOVA test showed no statistically significant difference among the points (P = 0.880, P > 0.05). At 24 h, results showed statistically significant difference between groups (P = 0.001, P < 0.05). The value of the mean cell number ratio was lower for MTA than MTA-P, while at 72 h (P = 0.204, P > 0.05) and 7 days (P = 0.150, P > 0.05), results showed no statistically significant difference between the groups [Figure 1].

Two-way ANOVA showed, there was a statistically significant effect of the between-subjects factor (material) on the cell number ratio (P = 0.001, P < 0.05). Regardless of the observation point, MTA showed significantly lower cell number ratio compared with MTA-P. Also, the within-subjects factors (observation point) had a statistically significant effect on the cell number ratio (P = 0.001, P < 0.05). Regardless of the material, 24 h showed significantly lower cell number ratio [Table 1].
Table 1 The mean (SD) values of the cell number ratio for comparison between observation points and between materials


Click here to view



  Discussion Top


Several studies have suggested that vital pulp-therapy treatments can be permanent, because the pulp has enough vital tissue, it was advocated that pulp-capping procedures could be performed successfully on an asymptomatic carious exposure [1]. The development of newer materials that are biocompatible, bactericidal, inductive of a reparative process, and have better sealing properties could render long-term vital-pulp therapy. MTA gained numerous clinical applications in endodontic procedures and was recommended for direct pulp protection. Several reagents have been used to improve the working properties of MTA; chlorhexidine gel, sodium hypochlorite gel, and K-Y jelly [7-9].

In the present study, addition of propolis to MTA was considered to do its antimicrobial, anti-inflammatory, antioxidant and immunomodulation properties. Propolis are known to play an important role in reducing the inflammatory response by inhibiting the lipoxygenase pathway of arachidonic acid [19] and in producing transforming growth factor β-1, which is important for differentiation of odontoblast, synthesis of collagen by dental pulp cells and formation of reparative dentine [20].

Various cell lines are commonly used in cell culture response evaluations. An established cell line has the advantage of easier culture procedures and is recommended by the ISO 7405 guidelines [21,22]. BHK cell line is one of the most widely used cell lines [23]. Fibroblasts were selected because they are more often in contact to the material in clinical conditions such as pulp capping. This is in addition to their great rate of proliferation and sensitivity as reported by Ragnarsson et al. [24].

The decision to use a particular test in detecting the cell culture response is based on its consonance with the chemical nature of the materials [21,25]. As MTA is a hydrophilic material, it is likely to release ionic components; this could interfere with the intercellular enzyme activities than affecting the membrane permeability, which are more suitable for lipophilic materials [21, 26, 27]. Cell proliferation was determined quantitatively, by the crystal violet assay [28,29]; it is a method for measurement of the cell number in monolayer. It is based on the observation that, in fixed cells, crystal violet binds to nuclear proteins and the amount of bound chemical correlates in linear relationship with the cell number in culture [30].

The extracts of the materials were used to avoid the limitation of the direct contact of the materials with the cells which may affect the proliferation of the cells; the extract excludes the effect of the surface topography on the cells and it allows evaluation of the effect of the materials on the surrounding as well as distant cells, in addition it allows evaluation effect of the chemical components leaching out of the materials [21].

The present study, MTA showed early adverse effect on cell proliferation, this could be explained on the basis that MTA is rich in calcium oxide, which convert to calcium hydroxide on contact with tissue fluids. The calcium hydroxide separates into calcium and hydroxide ions, resulting in increase pH and calcium ion release, the initial high surface pH of MTA, which can cause denaturation of proteins in both adjacent cells and the medium [31]. Cell proliferation of MTA tends to improve gradually over time up to 7 days, these findings were in agreement with those reported by Camilleri et al. [25], Ko et al. [31], and De Deus et al. [32]. These findings were in contrast with Karimjee et al. [33] who reported greater cell lysis at 72 h, De Deus et al. [34] who found decrease on mitochondrial dehydrogenase activity by time, this could be attributed to the difference in the methods of extraction preparation, type of cells and cell culture tests used.

This study revealed that samples in which MTA powder was mixed with propolis (100 μg/ml) showed higher cell proliferation ratio, which was in agreement with the finding of Jacob et al. [35] where the Brazilian red propolis showed slight increase in cell migration and viability at 10 and 100 μg/ml concentrations and Al-Shaher et al. [18], where the exposure of PDL or pulp fibroblasts to 4 mg/ml or lower concentrations of propolis resulted in greater than 75% viability of cells, while the results contradicted those of De Funari et al. [36], where; as the propolis concentration reached 62.5 μg/ml, NIH-3T3 cell viability approached 20%, this could be attributed to the use of different types of propolis, cell line or different cytotoxicity test.

Addition of propolis improved the early cell proliferation of MTA; this could be attributed to the fact of its ability to accelerate tissue healing which has been attributed for its use in periodontal disease and surgical wound healing [37]. It has been suggested by Jahromi et al. [38] that the anti-inflammatory properties might be the cause of lower cytotoxicity of propolis through the suppression of immune cells, activation of macrophage derived nitric oxide and cytokine production, neutrophil activation and synthesis of collagen. The presence of iron and zinc in propolis are important for synthesis of collagen and increasing the healing effect of epithelial tissue [39].

Acknowledgements

The author express his deepest appreciation to the head and staff members of the Department of Applied Research and Development Center in the Holding Company for Biological Products and Vaccines (VACSERA) who helped the author to conduct this work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Badland LK. Endodontic considerations in dental trauma. In: Ingle JI, Bakland LK, editors. Endodontics. Toronto: BC Decker Inc.; 2002. 795-844.  Back to cited text no. 1
    
2.
Schröder U. Effects of calcium hydroxide-containing agent on pulp cell migration, proliferation and differentiation. J Dent Res 1985; 64:541-548.  Back to cited text no. 2
    
3.
Stanley HR. Pulp capping: conserving the dental pulp - Can it be done? Is it worth it? Oral Surg Oral Med Oral Pathol 1989; 68:628-639.  Back to cited text no. 3
    
4.
Baume LJ. The biology of pulp and dentine. In: Meyers HM, editor. Monographs in oral science. Basel: S. Krager AG Inc.; 1980. 67-82.  Back to cited text no. 4
    
5.
Tziafas D, Pantelidou O, Alvanou A, Belibasakis G, Papadimitriou S. The dentinogenic effect of mineral trioxide aggregate (MTA) in short-term capping experiments. Int Endod J 2002; 35:245-254.  Back to cited text no. 5
    
6.
Cox CF, Bergenholz G, Heys DR, Syed SH, Fitzderald M, Heys RJ. Pulp capping of dental pulp mechanically exposed to oral microflora: a 1-2 year observation of wound healing in the monkey. J Oral Pathol 1985; 14:156-168.  Back to cited text no. 6
    
7.
Ber B, John FH, Gregory PS. Chemical modification of ProRoot MTA to improve handling characteristics and decrease setting time. J Endod 2007; 33:1231-1234.  Back to cited text no. 7
    
8.
Wiltbank KB, Schwartz SA, Schindler WG. Effect of selected accelerants on the physical properties of mineral trioxide aggregate and Portland cement. J Endod 2007; 33:1235-1238.  Back to cited text no. 8
    
9.
Parirokh M, Torabinejad M. Mineral trioxide aggregate: comprehensive literature reviews-part I: chemical, physical and antibacterial properties. J Endod 2010; 36:16-27.  Back to cited text no. 9
    
10.
Gojmerac W. Bees, beekeeping, honey and pollination. Westport, CT: A VI publishing Inc.; 1980. 115-116.  Back to cited text no. 10
    
11.
Valcic S, Montenegro G, Mujica AM, Franzblau S, Singh MP, Maiese WM, et al. Phytochemical, morphological and biological investigation of Propolis from Central Chile. Z Naturforsch 1999; 54:406-416.  Back to cited text no. 11
    
12.
Elkins R. Healing from the hive. In: Elkins R, editor. Bee pollen, royal jelly, propolis and honey. Pleasant Grove, UT: Woodland Publishing Inc.; 1996. 49.  Back to cited text no. 12
    
13.
Burdock GA. Review of the biological properties and toxicity of bee Propolis. Food Chem Toxicol 1998; 36:347-363.  Back to cited text no. 13
    
14.
Sonmez S, Kirilmaz L, Yucesoy M, Yucel B, Yilmaz B. The effect of bee Propolis on oral pathogens and human gingival fibroblasts. J Ethnopharmacol 2005; 102:371-376.  Back to cited text no. 14
    
15.
Yaghoubi MJ, Ghorbani GH, Soleimanian ZS, Satari R. Antimicrobial activity of Iranian Propolis and its chemical composition. DARU 2007; 15:45-48.  Back to cited text no. 15
    
16.
Markham KR, Mitchell KA, Wilkins AL, Daldy JA, Lu Y. HPLC and GC-MS identification of the major organic constituents in New Zealand Propolis. Phytochemistry 1996; 42:205-211.  Back to cited text no. 16
    
17.
SForcin JM, Fernandes AJ, Lopes CA, Bankova V, Funacol SR. Seasonal effect on Brazilian Propolis antibacterial activity. J Ethnopharmacol 2000; 73:243-249.  Back to cited text no. 17
    
18.
Al-Shaher A, Wallace J, Agarwal S, Bretz W, Baugh D. Effect of Propolis on human fibroblasts from the pulp and periodontal ligament. J Endod 2004; 30:359-361.  Back to cited text no. 18
    
19.
Parolia A, Kundabala M, Rao NN, Acharya SR, Agrawal P, Mohan M, et al. A comparative histological analysis of human pulp following direct pulp capping with Propolis, mineral trioxide aggregate and Dycal. Aust Dent J 2010; 55:59-64.  Back to cited text no. 19
    
20.
Ahangari Z, Alborzi S, Yadegari Z, Dehghani F, Ahangari L, Naseri M. The effect of Propolis as a biological storage media on periodontal ligament cell survival in avulsed tooth: an in vitro study. Cell J 2013; 15:244-249.  Back to cited text no. 20
    
21.
Keiser K, Johnson CC, Tipton DA. Cytotoxicity of mineral trioxide aggregate using human periodontal ligament fibroblasts. J Endod 2000; 26:288-291.  Back to cited text no. 21
    
22.
Vajrabhaya LO, Korsuwannawong S, Jantarat J, Korre S. Biocompatibility of furcal perforation repair materials using cell culture technique: Ketac Molar versus ProRoot MTA. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006; 102:e48-e50.  Back to cited text no. 22
    
23.
Koulaozidou EA, Papazisis KT, Economides NA, Beltes P, Kortsaris AH. Antiproliferative effect of mineral trioxide aggregate zinc oxide-eugenol cement and glass-ionomer cement against three fibroblastic cell lines. J Endod 2005; 31:44-46.  Back to cited text no. 23
    
24.
Ragnarsson B, Carr G, Daniel JC. Isolation and growth of periodontal ligament cells in vitro. J Dent Res 1985; 64:1026-1030.  Back to cited text no. 24
    
25.
Camilleri J, Montesin FE, Silvio LD, Pitt Ford TR. The chemical construction and biocompatibility of accelerated Portland cement for endodontic use. Int Endod J 2005; 38:834-842.  Back to cited text no. 25
    
26.
Scweikl H, Schmalz G, Gottke C. Toxicity parameters for cytotoxicity testing of dental materials in two different mammalian cell lines. Eur J Oral Sci 1996; 104:292-299.  Back to cited text no. 26
    
27.
Telli C, Serper A, Dogan LA, Gu D. Evaluation of the cytotoxicity of calcium phosphate root canal sealer by MTT assay. J Endod 1999; 25:811-813.  Back to cited text no. 27
    
28.
Osorio RM, Hefti A, Vertucci FJ, Shawley AL. Cytotoxicity of endodontic materials. J Endod 1998; 24:91-96.  Back to cited text no. 28
    
29.
Camargo SEA, Camargo CHR, Hiller KA, Rode SM, Schweikl H, Schmalz G. Cytotoxicity and genotoxicity of pulp capping materials in two cell lines. Int Endod J 2009; 42:227-237.  Back to cited text no. 29
    
30.
Kueng W, Silber E, Eppenberger U. Quantification of cells cultured on 96-well plates. Anal Biochem 1989; 182:16-19.  Back to cited text no. 30
    
31.
Ko H, Yang W, Park K, Kim K. Cytotoxicity of mineral trioxide aggregate (MTA) and bone morphogenitic protein 2 (BMP-2) and response of rat pulp to MTA and BMP-2. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109:e103-e108.  Back to cited text no. 31
    
32.
De Deus G, Ximenes R, Filho ED, Pltkowski EC, Filho TC. Cytotoxicity of MTA and Portland cement on human ECV 304 endothelial cells. Int Endod J 2005; 38:604-609.  Back to cited text no. 32
    
33.
Karimjee CK, Koka S, Rallis DM, Gound TG. Cellular toxicity of mineral trioxide aggregate mixed with alterative delivery vehicle. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006; 102:e115-e120.  Back to cited text no. 33
    
34.
De Deus G, Canabarro A, Alves G, Linbares A, Senne MI, Granjeiro JM. Optimal cytocompatibility of bioceramic nanoparticulate cement in primary human mesenchymal cells. J Endod 2009; 35:1387-1390.  Back to cited text no. 34
    
35.
Jacob A, Parolia A, Pau A, Amalraj FD. The effect of Malaysian propolis and Brazilian red Propolis on connective tissue fibroblast in the wound healing process. BMC Comp Alter Med 2015; 15:294.  Back to cited text no. 35
    
36.
De Funari CS, De Oliveira FV, Mathor MB. Analysis of Propolis from Baccharis dracunculifolia DC. (Compositae) and its effects on mouse fibroblasts. J Ethnopharmacol 2007; 111:206-212.  Back to cited text no. 36
    
37.
Bretz WA, Chiego DJ, Marcucci MC, Cunha I, Custaodio A, Schneider LG. Preliminary report on the effect of propolis on wound healing in the dental pulp. Z Naturforsch 1998; 53:1045-1048.  Back to cited text no. 37
    
38.
Jahromi MZ, Ranjbarian P, Shiravi S. Cytotoxicity evaluation of Iranian Propolis and calcium hydroxide on dental pulp fibroblasts. J Dent Res Dent Clin Dent Prospects 2014; 8:130-133.  Back to cited text no. 38
    
39.
Mahal NK, Singh N, Thomas AM, Kakkar N. Effect of three different storage media on survival of periodontal ligament cells using collagenase-dispase assay. Int Endod J 2013; 46:365-370.  Back to cited text no. 39
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1]


This article has been cited by
1 The analysis of the effects of propolis products as food supplements on the viability of baby hamster kidney (BHK-21) and murine macrophage (RAW 264.7) cells by spectrophotometric MTT assay
Serol KORKMAZ, Ayse PARMAKSIZ, Ahmet SAIT, Burcu Irem OMURTAG KORKMAZ
Journal of Advances in VetBio Science and Techniques. 2022;
[Pubmed] | [DOI]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Materials and me...
Results
Discussion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed2353    
    Printed137    
    Emailed0    
    PDF Downloaded177    
    Comments [Add]    
    Cited by others 1    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]