Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Jul 10.
Published in final edited form as: J Oral Sci. 2012 Sep;54(3):213–217. doi: 10.2334/josnusd.54.213

Cytotoxicity evaluation of methacrylate-based resins for clinical endodontics in vitro

Esteban G Garza 1, Aniket Wadajkar 2, Chul Ahn 3, Qiang Zhu 4, Lynn A Opperman 5, Larry L Bellinger 5, Kytai T Nguyen 2, Takashi Komabayashi 6
PMCID: PMC4090924  NIHMSID: NIHMS596749  PMID: 23047031

Abstract

This study examines the cytotoxicity of Super-Bond C&B (SB-C&B), Super-Bond RC Sealer (SB-RC), MetaSEAL (Meta), and AH Plus Sealer (AH+). Freshly mixed and set materials (100 mg) were prepared in vitro and placed in cell culture medium (1 mL) for the working time and for 6 h, respectively. L929 cells seeded into 96-well plates at 5,000 cells/well were incubated with the eluted medium (200 μL) for 24 h. Cells cultured with medium alone served as the control. Cytotoxicity was evaluated by MTS assay and analyzed with ANOVA. In the freshly mixed group, the average ± SD (%) for cell viability were 66.0 ± 13.6, 55.5 ± 15.6, 10.6 ± 0.7, and 8.9 ± 2.2 for SB-C&B, SB-RC, Meta, and AH+, respectively. In the set group, the average ± SD (%) for cell viability were 100 ± 21.9, 81.8 ± 38.5, 24.9 ± 7.9, and 23.6 ± 10.0 for SB-C&B, SB-RC, Meta, and AH+, respectively. SB-C&B and SB-RC are less cytotoxic than are Meta and AH+.

Keywords: cytotoxicity, methacrylate, resin, sealer, endodontics

Introduction

Over 22 million root canal treatments are performed each year in the United States (1). Sealers in combination with a solid or semi-solid core material are used to fill voids and to seal canals during obturation. The apical seal, like the coronal seal, is critical for long-term clinical success (2). Some studies report the apical seals of resin-based sealers to be superior to those of other sealers (3,4). Thus, various resin sealers have been commercialized for endodontic applications (5-8).

Among resin sealers, the epoxy resin sealer, AH Plus (AH+), is conventionally used (9). Recently, two methacrylate resin sealers have been introduced. One is the methacrylate/sulfinate salt (MA/SS)-based resin sealer, MetaSEAL (Meta). The other is methyl methacrylate/tributylborane (MMA/TBB)-based resin sealer, Super-Bond RC Sealer (SB-RC), which was developed to address Super-Bond C&B (SB-C&B) resin cement's drawbacks: short working time, low radiopacity, and difficult removal of the material from the canal (10). The polymer components of SB-RC are poly(methyl methacrylate) (PMMA) and zirconium oxide, while that of SB-C&B is PMMA.

Materials for endodontic applications must not only provide good seals, but also exhibit excellent biocompatibility, because cytotoxicity of tissues is of concern when materials are involved with human tissue (11). Although biocompatibility is one of the factors that influence the clinician's choice of sealers in root canal therapy (12,13), cytotoxicity studies of methacrylate-based resins in fresh and set conditions are limited.

The purpose of this study therefore was to examine the cytotoxicity of SB-C&B, SB-RC, Meta, and AH+. L929 cells were used due to their sensitivity to toxic products, which explains their frequent use in the evaluation of root canal sealers (14).

Materials and Methods

Materials

The resin cement and three root canal sealers tested in this study were 1) Super-Bond C&B (Sun Medical Co., Shiga, Japan) (in the United States, C&B Metabond [Parkell, Edgewood, NY]), 2) Super-Bond RC Sealer (Sun Medical, Japan), 3) MetaSEAL (Parkell, Edgewood, NY) (In Japan, Hybrid Root SEAL [Sun Medical Co., Shiga, Japan]), and 4) AH Plus (Dentsply DeTrey, Konstanz, Germany). Lot numbers, working lengths, setting times, and classifications are summarized in Table 1.

Table 1. Materials used in this study.

Material name Super-Bond C&B Super-Bond RC Sealer MetaSEAL AH Plus
Abbreviation SB-C&B SB-RC Meta AH+
Manufacturer Sun Medical/Parkell Sun Medical/Parkell Sun Medical/Parkell Dentsply DeTrey
Type of resin Methacrylate resin Methacrylate resin Methacrylate resin Epoxy resin
Polymerization initiator Tributylborane (TBB) Tributylborane (TBB) Sulfinate salt (SS) N/A*
Lot # VS2 RX1 TW15 (Monomer), VG14 (Powder) 1012001095
Working time 1-2 min 6 min 30 min 4 h
Setting time 5-6 min 42 min 16 h 8 h
Clinical usage Crown and bridge Endodontic sealer Endodontic sealer Endodontic sealer
Open in a new tab
*

Polymerization initiator is not necessary for polyaddition reaction.

L929 mouse fibroblasts were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (Hyclone Laboratories Inc., Logan, UT, USA), and 1% penicillinstreptomycin (Gibco BRL, Gaithersburg, MD, USA) under standard cell culture conditions (37°C and 5% CO2).

Methods

The cytotoxicity of the four materials was tested for both fresh and set sealer samples. L929 cells were seeded into 96-well plates at 5,000 cells/well and incubated for 24 h to allow adhesion for the cell cytotoxicity assay.

Samples of freshly (immediately) mixed materials were placed into 24-well plates at approximately 0.1 g/well. The fresh materials were mixed according to the manufacturers' instructions. Then 1 mL cell culture medium was added to the wells. After the required working time (specified in Table 1), the medium was removed and added (200 μL) to the cells prior to their 24-h incubation.

To obtain the set materials, samples of the different materials (setting time specified in Table 1) were placed into a 24-well plate at approximately 0.1g/well and incubated for 24 h in the cell culture incubator to allow for setting. Then 1 mL of the cell culture medium was added to the set materials and left for 6 h. Every hour, wells were shuffled for 5 s. 200 μL of the material eluate from the different eluate groups was added to the cell culture wells.

In both the fresh and set material groups, after an incubation period of 24 h at 37°C, 200 μL of MTS reagent was added to the cell culture wells, and cell viability was evaluated according to the manufacturer's instructions. Cells cultured with the medium only served as the control group (100%). Each experiment was repeated four times.

Data analysis and statistics

An ANOVA test was conducted to determine whether there were significant differences in cell viability between the experimental and the control groups, and Student's t-tests were conducted to identify which material was significantly different from control with Dunnett's correction.

Results

When cells were cultured with the eluates of the fresh materials, the average ± SD values (%) for biocompatibility were 66.0 ± 13.6, 55.5 ± 15.6, 10.6 ± 0.7, and 8.9 ± 2.2 for SB-C&B, SB-RC, Meta, and AH+, respectively. The results for Meta (P = 0.0024) and AH+ (P = 0.0005) showed statistical significance when compared with the control (Fig. 1). The cell viability in the Meta and AH+ groups was significantly less than that in the SB-C&B and SB-RC groups (P < 0.05). There was no statistically significant difference between SB-C&B and SB-RC, or between Meta and AH+.

Fig. 1.

Fig. 1

Open in a new tab

Cell viability of L929 cells after culture with elute of fresh materials.

*Significant difference from the control, Meta and AH+ groups. **Significant difference from the control, SB-C&B and SB-RC groups.

When cells were cultured with eluate of the set materials, the average ± SD values (%) for biocompatibility were 100 ± 21.9, 81.8 ± 38.5, 24.9 ± 7.9, and 23.6 ± 10.0 for SB-C&B, SB-RC, Meta, and AH+, respectively (Fig. 2). The viability of cells cultured with SB-C&B and SB-RC eluates was significantly higher than that of cells cultured with eluates of Meta and AH+ (P < 0.05). There was no statistically significant difference between SB-C&B and SB-RC, and between Meta and AH+. The cell viability in the SB-C&B and SB-RC groups was significantly higher than that in the Meta and AH+ groups (P < 0.05).

Fig. 2.

Fig. 2

Open in a new tab

Cell viability of L929 cells after culture with elute of set materials.

*Significant difference from the control, Meta and AH+ groups. **Significant difference from the control, SB-C&B and SB-RC groups.

Discussion

In our study, AH plus was found to be the most cytotoxic of the sealers. AH plus is a two-component system consisting of two pastes. The epoxide paste contains diepoxide. The amine paste contains 1-adamantane amine, T N,N′-dibenzyl-5-oxa-nonandiamine-1,9, and TCD-diamine. When the two pastes are mixed, the poly addition reaction starts immediately. The amines polymerize with the diepoxide to co-polymers. The un-polymerized monomers which were eluted to the medium during setting cause the cytotoxic effect. AH 26 releases formaldehyde and exhibits cytotoxicity (15-20). Comparing with AH 26 only a minimum release of formaldehyde from AH plus was observed (16). AH 26 and AH plus both demonstrated cytotoxicity in rat cerebral astrocyte cell culture (21). AH plus was more cytotoxic than AH 26 in human cervical carcinoma cells and mouse skin fibroblasts (22). AH Plus has shown to be cytotoxic in the first three days and does not reach non-cytotoxicity until 3 to 5 weeks (23). The results of the present experiment are in agreement with the previous studies. However, studies have shown AH plus exhibiting close to no cytotoxicity (24,25). These differences may due to the differences in experimental conditions and methods.

Meta was found to be very cytotoxic, especially when fresh. These results have been found in similar studies on the cytotoxic effects of Meta (23,26). Meta's monomer components include 4-methacryloxyethyl trimellitate anhydride (4-META) and hydroxyethyl methacrylate (HEMA). Based on 50% inhibition of cell growth, the relative cytotoxicity of the monomers MMA: 4-META: HEMA was calculated as 1:6:7 (27,28). It is thought that HEMA diffuses through dentine to cause cellular damage (29,30). HEMA also induces cell growth inhibition and cycle perturbation as well as glutathione depletion and reactive oxygen species production (31).

HEMA has been reported to inhibit intracellular tyrosine phosphorylation in L929 cells (32). These are reasonable explanations for our cytotoxicity results for Meta.

The main monomer component of SB-C&B and SB-RC is MMA, which has a low potential for pulp irritation compared with other poly-functional methacrylate monomers (27,33). The polymerization initiator of SB-C&B and SB-RC is TBB. Tronstad et al. studied pulp responses to composite resin (Concise) and MMATBB-based resin (Polycap) in deep Class V cavities in monkeys after 8 days (34). In composite resin, 30% of responses were slight; 50%, moderate; and 20%, severe. In MMA/TBB-based resin, 75% of responses were slight; 25%, moderate; and 0%, severe. Overall, the MMA/TBB-based resin had lower cytotoxicity than did the composite resin. No severe response to the MMA/TBB-based resin was reported.

The success of MMA/TBB-based resin in endodontic applications can be attributed to several material properties. MMA is the least cytotoxic among the monomers used in dentistry (27,35,36). Also, the residual MMA is low after setting and decreases with time because of the presence of TBB (37). Finally, the polymerization at the dentin interface enables reliable sealing of the interface due to the presence of TBB (38).

Several studies have indicated that cytotoxic effects in cell culture can be caused by released monomers (39,40). The curing of resin-based cement is usually not complete, so unconverted monomers can be released from the resin into an adjacent aqueous phase and can diffuse through dentin to the pulp space. In short, before setting, the cytotoxicity of the monomer itself will affect the pulp, while after setting, the residual effect of the monomer will depend on amount and elution kinetics. In this regard, in addition to the low cytotoxicity of the MMA in SB-C&B and SB-RC, the TBB initiator significantly reduced the residual MMA during the course of the resin's setting (37). Our data are consistent with those of a similar study that found minimal cytotoxic effects with SB-RC (41). Although the results of our in vitro study are significant and offer good clinical information, it is necessary to confirm the in vitro results with future in vivo studies.

This study demonstrates that the MMA/TBB resins SB-C&B and SB-RC are less cytotoxic than are Meta and AH+. While excellent sealing of the canal has been reported with MMA/TBB resins (10,17), SB-C&B is not suitable for root canal use due to its poor handling properties. Therefore, among all tested materials, SB-RC's low cytotoxicity and excellent sealing of the canal at the resin-dentin interface recommend it for clinical use.

Acknowledgments

This paper was supported in part by NIH KL2 RR024983(TK) and UL1 RR024982. Dr. Komabayashi is a consultant for Sun Medical.

Footnotes

The publisher regrets that in the above-mentioned article, Table 1 was omitted.

References

  • 1.American Dental Association. Survey of dental services rendered and distribution of dentists in the United States by region and state. ADA Survey Center; Chicago: 2005-6. [Google Scholar]
  • 2.Shipper G, Ørstavik D, Teixeira FB, Trope M. An evaluation of microbial leakage in roots filled with a thermoplastic synthetic polymer-based root canal filling material (Resilon) J Endod. 2004;30:342–347. doi: 10.1097/00004770-200405000-00009. [DOI] [PubMed] [Google Scholar]
  • 3.Limkangwalmongkol S, Burtscher P, Abbott PV, Sandler AB, Bishop BM. A comparative study of the apical leakage of four root canal sealers and laterally condensed gutta-percha. J Endod. 1991;17:495–499. doi: 10.1016/S0099-2399(06)81797-8. [DOI] [PubMed] [Google Scholar]
  • 4.Oguntebi BR, Shen C. Effect of different sealers on thermoplasticized Gutta-percha root canal obturations. J Endod. 1992;18:363–366. doi: 10.1016/s0099-2399(06)81219-7. [DOI] [PubMed] [Google Scholar]
  • 5.Cobankara FK, Orucoglu H, Sengun A, Belli S. The quantitative evaluation of apical sealing of four endodontic sealers. J Endod. 2006;32:66–68. doi: 10.1016/j.joen.2005.10.019. [DOI] [PubMed] [Google Scholar]
  • 6.Belli S, Ozcan E, Derinbay O, Eldeniz AU. A comparative evaluation of sealing ability of a new, self-etching, dual-curable sealer: hybrid root SEAL (MetaSEAL) Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2008;106:e45–52. doi: 10.1016/j.tripleo.2008.07.027. [DOI] [PubMed] [Google Scholar]
  • 7.Stoll R, Thull P, Hobeck C, Yüksel S, JablonskiMomeni A, Roggendorf MJ, Frankenberger R. Adhesion of self-adhesive root canal sealers on gutta-percha and Resilon. J Endod. 2010;36:890–893. doi: 10.1016/j.joen.2010.01.004. [DOI] [PubMed] [Google Scholar]
  • 8.Yamanaka Y, Shigetani Y, Yoshiba K, Yoshiba N, Okiji T. Immunohistochemical analysis of subcutaneous tissue reactions to methacrylate resin-based root canal sealers. Int Endod J. 2011;44:669–675. doi: 10.1111/j.1365-2591.2011.01874.x. [DOI] [PubMed] [Google Scholar]
  • 9.Pameijer CH, Zmener O. Resin materials for root canal obturation. Dent Clin North Am. 2010;54:325–344. doi: 10.1016/j.cden.2009.12.004. [DOI] [PubMed] [Google Scholar]
  • 10.Imai Y, Komabayashi T. Properties of a new injectable type of root canal filling resin with adhesiveness to dentin. J Endod. 2003;29:20–23. doi: 10.1097/00004770-200301000-00006. [DOI] [PubMed] [Google Scholar]
  • 11.Bertram JS. The molecular biology of cancer. Mol Aspects Med. 2000;21:167–223. doi: 10.1016/s0098-2997(00)00007-8. [DOI] [PubMed] [Google Scholar]
  • 12.Geurtsen W, Leyhausen G. Biological aspects of root canal filling materials – histocompatibility, cytotoxicity, and mutagenicity. Clin Oral Investig. 1997;1:5–11. doi: 10.1007/s007840050002. [DOI] [PubMed] [Google Scholar]
  • 13.Dahl JE. Toxicity of endodontic filling materials. Endodontic Topics. 2005;12:39–43. [Google Scholar]
  • 14.Pissiotis E, Spångberg LS. Toxicity of Pulpispad using four different cell types. Int Endod J. 1991;24:249–257. doi: 10.1111/j.1365-2591.1991.tb01150.x. [DOI] [PubMed] [Google Scholar]
  • 15.Spångberg LS, Barbosa SV, Lavigne GD. AH 26 releases formaldehyde. J Endod. 1993;19:596–598. doi: 10.1016/S0099-2399(06)80272-4. [DOI] [PubMed] [Google Scholar]
  • 16.Cohen BI, Pagnillo MK, Musikant BL, Deutsch AS. Formaldehyde evaluation from endodontic materials. Oral Health. 1998;88:37–39. [PubMed] [Google Scholar]
  • 17.Leonardo MR, Bezerra da Silva LA, Filho MT, Santana da Silva R. Release of formaldehyde by 4 endodontic sealers. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1999;88:221–225. doi: 10.1016/s1079-2104(99)70119-8. [DOI] [PubMed] [Google Scholar]
  • 18.Merdad K, Pascon AE, Kulkarni G, Santerre P, Friedman S. Short-term cytotoxicity assessment of components of the epiphany resin-percha obturating system by indirect and direct contact millipore filter assays. J Endod. 2007;33:24–27. doi: 10.1016/j.joen.2006.10.003. [DOI] [PubMed] [Google Scholar]
  • 19.Lodiene G, Morisbak E, Bruzell E, Ørstavik D. Toxicity evaluation of root canal sealers in vitro. Int Endod J. 2008;41:72–77. doi: 10.1111/j.1365-2591.2007.01321.x. [DOI] [PubMed] [Google Scholar]
  • 20.Donadio M, Jiang J, He J, Wang YH, Safavi KE, Zhu Q. Cytotoxicity evaluation of Activ GP and Resilon sealers in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009;107:e74–78. doi: 10.1016/j.tripleo.2009.01.041. [DOI] [PubMed] [Google Scholar]
  • 21.Huang TH, Yang JJ, Li H, Kao CT. The biocompatibility evaluation of epoxy resin-based root canal sealers in vitro. Biomaterials. 2002;23:77–83. doi: 10.1016/s0142-9612(01)00081-3. [DOI] [PubMed] [Google Scholar]
  • 22.Miletić I, Anić I, Karlović Z, Marsan T, Pezelj-Ribarić S, Osmak M. Cytotoxic effect of four root filling materials. Endod Dent Traumatol. 2000;16:287–290. doi: 10.1034/j.1600-9657.2000.016006287.x. [DOI] [PubMed] [Google Scholar]
  • 23.Pinna L, Brackett MG, Lockwood PE, Huffman BP, Mai S, Cotti E, Dettori C, Pashley DH, Tay FR. In vitro cytotoxicity evaluation of a self-adhesive, methacrylate resin-based root canal sealer. J Endod. 2008;34:1085–1088. doi: 10.1016/j.joen.2008.06.020. [DOI] [PubMed] [Google Scholar]
  • 24.Leyhausen G, Heil J, Reifferscheid G, Waldmann P, Geurtsen W. Genotoxicity and cytotoxicity of the epoxy resin-based root canal sealer AH plus. J Endod. 1999;25:109–113. doi: 10.1016/S0099-2399(99)80007-7. [DOI] [PubMed] [Google Scholar]
  • 25.Camps J, About I. Cytotoxicity testing of endodontic sealers: a new method. J Endod. 2003;29:583–586. doi: 10.1097/00004770-200309000-00010. [DOI] [PubMed] [Google Scholar]
  • 26.Ames JM, Loushine RJ, Babb BR, Bryan TE, Lockwood PE, Sui M, Roberts S, Weller RN, Pashley DH, Tay FR. Contemporary methacrylate resin-based root canal sealers exhibit different degrees of ex vivo cytotoxicity when cured in their self-cured mode. J Endod. 2009;35:225–228. doi: 10.1016/j.joen.2008.11.008. [DOI] [PubMed] [Google Scholar]
  • 27.Yoshii E. Cytotoxic effects of acrylates and methacrylates: relationships of monomer structures and cytotoxicity. J Biomed Mater Res. 1997;37:517–524. doi: 10.1002/(sici)1097-4636(19971215)37:4<517::aid-jbm10>3.0.co;2-5. [DOI] [PubMed] [Google Scholar]
  • 28.Imazato S, Tarumi H, Ebi N, Ebisu S. Cytotoxic effects of composite restorations employing self-etching primers or experimental antibacterial primers. J Dent. 2000;28:61–67. doi: 10.1016/s0300-5712(99)00039-1. [DOI] [PubMed] [Google Scholar]
  • 29.Bouillaguet S, Wataha JC, Hanks CT, Ciucchi B, Holz J. In vitro cytotoxicity and dentin permeability of HEMA. J Endod. 1996;22:244–248. doi: 10.1016/s0099-2399(06)80141-x. [DOI] [PubMed] [Google Scholar]
  • 30.Hume WR, Gerzina TM. Bioavailability of components of resin-based materials which are applied to teeth. Crit Rev Oral Biol Med. 1996;7:172–179. doi: 10.1177/10454411960070020501. [DOI] [PubMed] [Google Scholar]
  • 31.Chang HH, Guo MK, Kasten FH, Chang MC, Huang GF, Wang YL, Wang RS, Jeng JH. Stimulation of glutathione depletion, ROS production and cell cycle arrest of dental pulp cells and gingival epithelial cells by HEMA. Biomaterials. 2005;26:745–753. doi: 10.1016/j.biomaterials.2004.03.021. [DOI] [PubMed] [Google Scholar]
  • 32.Kaga M, Noda M, Ferracane JL, Nakamura W, Oguchi H, Sano H. The in vitro cytotoxicity of eluates from dentin bonding resins and their effect on tyrosine phosphorylation of L929 cells. Dent Mater. 2001;17:333–339. doi: 10.1016/s0109-5641(00)00091-9. [DOI] [PubMed] [Google Scholar]
  • 33.Fujisawa S, Atsumi T. Cytotoxicities of a 4-META/MMA-TBBO resin against human pulp fibroblasts. Dent Mater J. 2004;23:106–108. doi: 10.4012/dmj.23.106. [DOI] [PubMed] [Google Scholar]
  • 34.Tronstad L, Spångberg L. Biologic tests of a methyl methacrylate composite material. Scand J Dent Res. 1974;82:93–98. doi: 10.1111/j.1600-0722.1974.tb00371.x. [DOI] [PubMed] [Google Scholar]
  • 35.Imai Y, Watanabe M, Lee HE, Kojima K, Kadoma Y. Cytotoxicity of monomers used in dental resins. Iyo Kizai Kenkyujo Hokoku. 1988;22:87–90. in Japanese. [Google Scholar]
  • 36.Takeda S, Hashimoto Y, Miura Y, Kimura Y, Nakamura M. Cytotoxicity test of dental monomers using serum-free cell culture (in vitro) Shika Zairyo Kikai. 1993;12:613–619. in Japanese. [Google Scholar]
  • 37.Hirabayashi C, Imai Y. Studies on MMA-TBB resin. I. Comparison of TBB and other initiators in the polymerization of PMMA/MMA resin. Dent Mater J. 2002;21:314–321. doi: 10.4012/dmj.21.314. [DOI] [PubMed] [Google Scholar]
  • 38.Imai Y, Kadoma Y, Kojima K, Akimoto T, Ikakura K, Ohta T. Importance of polymerization initiator systems and interfacial initiation of polymerization in adhesive bonding of resin to dentin. J Dent Res. 1991;70:1088–1091. doi: 10.1177/00220345910700071401. [DOI] [PubMed] [Google Scholar]
  • 39.Gerzina TM, Hume WR. Effect of hydro-static pressure on the diffusion of monomers through dentin in vitro. J Dent Res. 1995;74:369–373. doi: 10.1177/00220345950740011301. [DOI] [PubMed] [Google Scholar]
  • 40.Gerzina TM, Hume WR. Diffusion of monomers from bonding resin-resin composite combinations through dentine in vitro. J Dent. 1996;24:125–128. doi: 10.1016/0300-5712(95)00036-4. [DOI] [PubMed] [Google Scholar]
  • 41.Eldeniz AU, Mustafa K, Ørstavik D, Dahl JE. Cytotoxicity of new resin-, calcium hydroxideand silicone-based root canal sealers on fibroblasts derived from human gingiva and L929 cell lines. Int Endod J. 2007;40:329–337. doi: 10.1111/j.1365-2591.2007.01211.x. [DOI] [PubMed] [Google Scholar]

RESOURCES