www.elsevier.com/locate/jmbbm Available online at www.sciencedirect.com Research Paper In vitro load-induced dentin collagen-stabilization against MMPs degradation Manuel Toledano a,n , Fa ´ tima S. Aguilera a , Monica Yamauti b , Marı´a Estrella Ruiz-Requena c , Raquel Osorio a a Department of Dental Materials, School of Dentistry, University of Granada, E-18071 Granada, Spain b Department of Restorative Dentistry, Piracicaba Dental School – University of Campinas, Areião, Piracicaba – SP 13414-903, Brazil c Department of Biochemistry and Molecular Biology, School of Medicine, Av. de Madrid s/n, University of Granada, E-18071 Granada, Spain article info Article history: Received 25 January 2013 Received in revised form 27 May 2013 Accepted 6 June 2013 Available online 20 June 2013 Keywords: Dentin Load cycling Collagen degradation Resin infiltration Longevity Restoration Adhesion abstract Introduction: Teeth are continuously subjected to stresses during mastication, swallowing and parafunctional habits, producing a significant reduction of the bonding efficacy in adhesive restorations. The purpose of this study was to evaluate the metalloproteinases (MMPs)-mediated dentin collagen degradation of hybrid layers created by using different demineralization processes, previous resin infiltration, and in vitro mechanical loading. Methods: Human dentin beams (0.75 Â 0.75 Â 5.0 mm) were subjected to different treat- ments: (1) untreated dentin; (2) demineralization by 37% phosphoric acid (PA) or by 0.5% M ethylenediaminetetraacetic acid (EDTA); (3) demineralization by PA, followed by applica- tion of Adper ™ Single Bond (SB); (4) demineralization by EDTA, followed by application of SB. In half of the specimens, mechanical loadings (100,000 cycles, 2 Hz, 49 N) were applied to dentin beams. Specimens were stored in artificial saliva. C-terminal telopeptide (ICTP), determinations (which indicates the amount of collagen degradation) (radioimmunoassay) were performed after 24 h, 1 week and 4 weeks. Results: Load cycling decreased collagen degradation when dentin was untreated or PA- demineralized and EDTA-treated. ICTP values increased when both PA-demineralized and EDTA-treated and infiltrated with SB dentin beams were loaded, except in samples that were subjected to EDTA treatment and SB infiltration after 4 w of storage, which showed similar values of collagenolytic activity than the non loaded specimens. Load cycling preserved the initial (24 h) ICTP determination at any time point, in all groups of the study, except in PA-demineralized and SB infiltrated dentin which showed an increased of collagen degradation values, over time. This same trend was observed in all groups without loading. Interpretation: Mechanical loading enhances collagen's resistance to enzymatic degradation in natural and demineralized dentin. Mild acids (EDTA) lead to a lower volume of demineralized/unprotected collagen to be cleaved by MMPs. Load cycling produced an increase of collagen degradation when PA-demineralized dentin and EDTA-treated dentin 1751-6161/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jmbbm.2013.06.002 n Correspondence to: Facultad de Odontología, Campus de Cartuja s/n, University of Granada, E-18071 Granada, Spain. Tel.: +34 958 243788; fax: +34 958 240908. E-mail address: toledano@ugr.es (M. Toledano). journal of the mechanical behavior of biomedical materials27 (2013) 10–18