Abstract
Knowledge of human gingival cell responses to dental monomers is critical for the development of new dental materials. Testing standards have been developed to provide guidelines to evaluate biological functionality of dental materials and devices. However, one shortcoming of the traditional testing platforms is that they do not recapitulate the multi-layered configuration of gingiva, and thus cannot evaluate the layer-specific cellular responses. An oral mucosa-chip with two cell layers was previously developed as an alternative platform to assess the oral mucosa responses to dental biomaterials. The mucosa-chip consists of an apical keratinocyte layer attached to a fibroblast-embedded collagen hydrogel through interconnecting pores in a three-microchannel network. Here, cell responses in the mucosa-chip were evaluated against 2-hydroxyethyl methacrylate (HEMA), a common monomer used in restorative and aesthetic dentistry. The response of mucosal cell viability was evaluated by exposing the chip to HEMA of concentrations ranging from 1.56 to 25 mM and compared to cells in conventional well-plate monoculture. The co-cultured cells were then stained and imaged with epifluorescence and confocal microscopy to determine the layer-specific responses to the treatment. Mucosa-chips were demonstrated to be more sensitive to assess HEMA-altered cell viability than well-plate cultures, especially at lower doses (1.56 and 6.25 mM). The findings suggest that the mucosa-chip is a promising alternative to traditional platforms or assays to test a variety of biomaterials by offering a multi-layered tissue geometry, accessible layer-specific information, and higher sensitivity in detecting cellular responses.
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
References
I. About, J. Camps, T.A. Mitsiadis, M.J. Bottero, W. Butler, J.C. Franquin, Influence of resinous monomers on the differentiation in vitro of human pulp cells into odontoblasts. J. Biomed. Mater. Res. 63(4), 418–423 (2002). https://doi.org/10.1002/jbm.10253
D.R. Bienek, S.A. Frukhtbeyn, A.A. Giuseppetti, U.C. Okeke, R.M. Pires, J.M. Antonucci, D. Skrtic, Ionic dimethacrylates for antimicrobial and remineralizing dental composites. Ann. Dent. Oral Disor. 1, 108 (2018a)
D.R. Bienek, S.A. Frukhtbeyn, A.A. Giuseppetti, U.C. Okeke, D. Skrtic, Antimicrobial monomers for polymeric dental restoratives: Cytotoxicity and physicochemical properties. J Funct Biomat 9(1), 20 (2018b). https://doi.org/10.3390/jfb9010020
U. Bilitewski, M. Genrich, S. Kadow, G. Mersal, Biochemical analysis with microfluidic systems. Anal. Bioanal. Chem. 377(3), 556–569 (2003). https://doi.org/10.1007/s00216-003-2179-4
S. Bouillaguet, M. Virgillito, W. Jc, B. Ciucchi, J. Holz, The influence of dentine permeability on cytotoxicity of four dentine bonding systems, in vitro. J. Oral Rehabil. 25, 45–51 (1998). https://doi.org/10.1046/j.1365-2842.1998.00205.x
I.P. Caldas, G.G. Alves, I.B. Barbosa, P. Scelza, F. de Noronha, M.Z. Scelza, In vitro cytotoxicity of dental adhesives: A systematic review. Dent. Mater. 35(2), 195–205 (2019). https://doi.org/10.1016/j.dental.2018.11.028
K.-H. Cho, S.-K. Yu, M.-H. Lee, D.-S. Lee, H.-J. Kim, Histological assessment of the palatal mucosa and greater palatine artery with reference to subepithelial connective tissue grafting. Anat. cell biol. 46(3), 171–176 (2013). https://doi.org/10.5115/acb.2013.46.3.171
M. Falconi, G. Teti, M. Zago, S. Pelotti, L. Breschi, G. Mazzotti, Effects of HEMA on type I collagen protein in human gingival fibroblasts. Cell Biol. Toxicol. 23(5), 313–322 (2007). https://doi.org/10.1007/s10565-006-0148-3
C.M. Franca, A. Tahayeri, N.S. Rodrigues, S. Ferdosian, R.M. Puppin Rontani, G. Sereda, et al., The tooth on-a-chip: A microphysiologic model system mimicking the biologic interface of the tooth with biomaterials. Lab Chip 20(2), 405–413 (2020). https://doi.org/10.1039/c9lc00915a
S. Gröger, J. Michel, J. Meyle, Establishment and characterization of immortalized human gingival keratinocyte cell lines. J. Periodontal Res. 43(6), 604–614 (2008). https://doi.org/10.1111/j.1600-0765.2007.01019.x
C.T. Hanks, R.G. Craig, M.L. Diehl, D.H. Pashley, Cytotoxicity of dental composites and other materials in a new in vitro device. J. Oral. Pathol. 17(8), 396–403 (1988). https://doi.org/10.1111/j.1600-0714.1988.tb01304.x
C.T. Hanks, S.E. Strawn, J.C. Wataha, R.G. Craig, Cytotoxic effects of resin components on cultured mammalian fibroblasts. J. Dent. Res. 70(11), 1450–1455 (1991). https://doi.org/10.1177/00220345910700111201
H.C. Hildebrand, L. Hakkinen, C.B. Wiebe, H.S. Larjava, Characterization of organotypic keratinocyte cultures on de-epithelialized bovine tongue mucosa. Histol Histopathol 17(1), 151–163 (2002). https://doi.org/10.14670/hh-17.151
P. Hu, S.A. Rooholghodos, L.H. Pham, K.L. Ly, X. Luo, Interfacial Electrofabrication of freestanding biopolymer membranes with distal electrodes. Langmuir 36(37), 11034–11043 (2020). https://doi.org/10.1021/acs.langmuir.0c01894
R.P. Illeperuma, Y.J. Park, J.M. Kim, J.Y. Bae, Z.M. Che, H.K. Son, et al., Immortalized gingival fibroblasts as a cytotoxicity test model for dental materials. J. Mater. Sci. Mater. Med. 23(3), 753–762 (2012). https://doi.org/10.1007/s10856-011-4473-6
K. Izumi, H. Kato, S. Feinberg, Tissue Engineered Oral Mucosa, in Stem Cell Biology and Tissue Engineering in Dental Sciences, vol 53 (Academic Press, Elsevier, Cambridge, 2015), pp. 721–731. https://doi.org/10.1016/B978-0-12-397157-9.00077-1
G. Kaufman, D. Skrtic, Morphological and kinetic study of oral keratinocytes assembly on reconstituted basement membrane: Effect of TEGDMA. Arch. Oral Biol. 104, 103–111 (2019). https://doi.org/10.1016/j.archoralbio.2019.05.019
S. Krifka, G. Spagnuolo, G. Schmalz, H. Schweikl, A review of adaptive mechanisms in cell responses towards oxidative stress caused by dental resin monomers. Biomaterials 34(19), 4555–4563 (2013). https://doi.org/10.1016/j.biomaterials.2013.03.019
R.H.W. Lam, X. Cui, W. Guo, T. Thorsen, High-throughput dental biofilm growth analysis for multiparametric microenvironmental biochemical conditions using microfluidics. Lab Chip 16(9), 1652–1662 (2016). https://doi.org/10.1039/C6LC00072J
I. Lignos, R. Maceiczyk, A.J. deMello, Microfluidic technology: Uncovering the mechanisms of Nanocrystal nucleation and growth. Acc. Chem. Res. 50(5), 1248–1257 (2017). https://doi.org/10.1021/acs.accounts.7b00088
L. Loan Khanh, N. Thanh Truc, N. Tan Dat, N. Thi Phuong Nghi, V. van Toi, N. Thi Thu Hoai, et al., Gelatin-stabilized composites of silver nanoparticles and curcumin: Characterization, antibacterial and antioxidant study. Sci. Technol. Adv. Mater. 20(1), 276–290 (2019). https://doi.org/10.1080/14686996.2019.1585131
Ly, K., Raub, C. B., & Luo, X. (2020a). Tuning the porosity of biofabricated chitosan membranes in microfluidics with co-assembled nanoparticles as template. Mater. Adv., 34-44. doi:https://doi.org/10.1039/D0MA00073F
K. Ly, S. Rooholghodos, C. Rahimi, B. Rahimi, D. R. Bienek, G. Kaufman, C. B. Raub, X. Luo, Oral mucosa-chip as an alternative platform to evaluate the impacts of dental monomers. Paper presented at The 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences (2020b), pp. 953–954
K. Moharamzadeh, Oral mucosa tissue engineering, in Biomaterials for Oral and Dental Tissue Engineering. (Woodhead Publishing/Elsevier, Cambridge, 2017), vol 14 pp. 223–244. https://doi.org/10.1016/B978-0-08-100961-1.00014-1
K. Moharamzadeh, I.M. Brook, R. Van Noort, A.M. Scutt, M.H. Thornhill, Tissue-engineered oral mucosa: A review of the scientific literature. J. Dent. Res. 86(2), 115–124 (2007). https://doi.org/10.1177/154405910708600203
L. Niu, H. Zhang, Y. Liu, Y. Wang, A. Li, R. Liu, et al., Microfluidic Chip for Odontoblasts in vitro. ACS Biomater. Sci. Eng. 5(9), 4844–4851 (2019). https://doi.org/10.1021/acsbiomaterials.9b00743
C. Rahimi, B. Rahimi, D. Padova, S.A. Rooholghodos, D.R. Bienek, X. Luo, et al., Oral mucosa-on-a-chip to assess layer-specific responses to bacteria and dental materials. Biomicrofluidics 12(5), 054106 (2018). https://doi.org/10.1063/1.5048938
G. Schmalz, S. Krifka, H. Schweikl, Toll-like receptors, LPS, and dental monomers. Adv. Dent. Res. 23(3), 302–306 (2011). https://doi.org/10.1177/0022034511405391
H. Schweikl, G. Spagnuolo, G. Schmalz, Genetic and cellular toxicology of dental resin monomers. J. Dent. Res. 85, 870–877 (2006a). https://doi.org/10.1177/154405910608501001
H. Schweikl, G. Spagnuolo, G. Schmalz, Genetic and cellular toxicology of dental resin monomers. J. Dent. Res. 85(10), 870–877 (2006b). https://doi.org/10.1177/154405910608501001
J. Szczepanska, T. Poplawski, E. Synowiec, E. Pawlowska, C.J. Chojnacki, J. Chojnacki, J. Blasiak, 2-hydroxylethyl methacrylate (HEMA), a tooth restoration component, exerts its genotoxic effects in human gingival fibroblasts trough methacrylic acid, an immediate product of its degradation. Mol. Biol. Rep. 39(2), 1561–1574 (2012). https://doi.org/10.1007/s11033-011-0895-y
G. Teti, G. Mazzotti, M. Zago, M. Ortolani, L. Breschi, S. Pelotti, et al., HEMA down-regulates procollagen alpha1 type I in human gingival fibroblasts. J. Biomed. Mater. Res. A 90(1), 256–262 (2009). https://doi.org/10.1002/jbm.a.32082
G. Teti, G. Orsini, V. Salvator, S. Focaroli, M.C. Mazzotti, A. Ruggeri, M. Mattioli-Belmonte, M. Falconi, HEMA but not TEGDMA induces autophagy in human gingival fibroblasts. Front. Physiol. 6(275) (2015). https://doi.org/10.3389/fphys.2015.00275
W.M.W. Tra, J.W. van Neck, S.E.R. Hovius, G.J.V.M. van Osch, S. Perez-Amodio, Characterization of a three-dimensional mucosal equivalent: Similarities and differences with native Oral mucosa. Cells Tissues Organs 195(3), 185–196 (2012). https://doi.org/10.1159/000324918
H.A. Tran, K.L. Ly, K.E. Fox, P.A. Tran, T.H. Nguyen, Immobilization of antimicrobial silver and antioxidant flavonoid as a coating for wound dressing materials. Int. J. Nanomedicine 14, 9929–9939 (2019). https://doi.org/10.2147/ijn.S230214
Vardar-Sengul, S., Arora, S., Baylas, H., & Mercola, D. (2009). Expression Profile of Human Gingival Fibroblasts Induced by Interleukin-1β Reveals Central Role of Nuclear Factor-Kappa B in Stabilizing Human Gingival Fibroblasts During Inflammation. 80(5), 833–849. doi:https://doi.org/10.1902/jop.2009.080483
J. Wu, Z. He, Q. Chen, J.-M. Lin, Biochemical analysis on microfluidic chips. TrAC Trends Anal. Chem. 80, 213–231 (2016). https://doi.org/10.1016/j.trac.2016.03.013
Acknowledgements
This effort was supported in part by the American Dental Association (ADA), ADA Foundation, the National Institute of Dental and Craniofacial Research (R01-DE26122-02), and the National Institute of General Medical Science (1R15GM129766-01). Authors gratefully acknowledge donation of HEMA from Esstech, Essington, PA.
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Ly, K.L., Rooholghodos, S.A., Rahimi, C. et al. An Oral-mucosa-on-a-chip sensitively evaluates cell responses to dental monomers. Biomed Microdevices 23, 7 (2021). https://doi.org/10.1007/s10544-021-00543-6
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DOI: https://doi.org/10.1007/s10544-021-00543-6