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Chlorhexidine Arrests Subclinical Degradation of Dentin Hybrid Layers in vivo

¹ Department of Orthodontics and Pediatric Dentistry, University of São Paulo State, Araraquara Dental School, São Paulo, Brazil;
² Department of Oral Biology & Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA, USA;
³ Institute of Dentistry, University of Helsinki, Finlandm and Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital (HUCH); and
⁴ Pediatric Dentistry and Orthodontics, University of Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road, Pokfulam, Hong Kong SAR, China;

View larger version (128K): [in a new window]   Figure 1. Radiographs and TEM micrographs of the primary molars that were retrieved from the first clinical subject. C, resin composite; A, adhesive; H, hybrid layer; D, dentin. Class I restorations with circumferential enamel cavosurface margins were placed with Single Bond as the dentin adhesive. In the control group (a,c,e), the adhesive was applied to phosphoric-acid-etched dentin. In the experimental group (b,d,f), 2% chlorhexidine was applied to the phosphoric-acid-etched dentin before the use of Single Bond. (a) Bitewing radiograph showing the Class I restoration in tooth 55 (control, circled). (b) Bitewing radiograph showing the Class I restoration in tooth 65 (experimental, circled). (c) Undemineralized, unstained, silver-impregnated section from the control tooth showing extensive, black silver deposits that almost completely obscured the hybrid layer. P, polyalkenoic acid copolymer. (d). The same type of section from the experimental tooth, showing sparsely distributed silver deposits (pointer) within the hybrid layer. Arrows: polyalkenoic acid copolymer within the dentinal tubules and resin composite. (e) Demineralized section from the control tooth that was stained with phosphotungstic acid and uranyl acetate. The hybrid layer was partially degenerated and missing, with empty regions occupied by epoxy resin (asterisk). These empty regions corresponded to the regions of extensive silver deposits depicted in Fig. 1c. (f) The same type of section from the experimental tooth, showing an intact normal hybrid layer.

View larger version (136K): [in a new window]   Figure 2. A horizontal pattern of degradation within the hybrid layer, as illustrated by the radiographs and TEMs of primary molars that were retrieved from the second clinical subject (Control group, a, c, and e; Experimental chlorhexidine group, b, d, and f). C, resin composite; A, adhesive; H, hybrid layer; D, dentin. (a) Bitewing radiograph showing the Class I restoration in tooth 85 (control, circled). (b) Bitewing radiograph showing the Class I restoration in tooth 75 (experimental, circled). (c) Undemineralized, unstained, silver-impregnated section from the control tooth, showing that the top 1.5–2 µm (asterisk) of the hybrid layer (between open arrows) was almost completely impregnated with silver deposits. (d) The same type of section from the experimental tooth, showing sparsely distributed silver deposits (pointer) that occurred predominantly at the base of the hybrid layer (between open arrows). (e) Demineralized section from the control tooth that was stained with phosphotungstic acid and uranyl acetate. Partial degradation of the collagen fibrils along the top 1–1.5 µm (asterisk) into non-banded, microfibrillar strands (i.e., gelatin; high magnification not shown) of the hybrid layer (between open arrowheads) corresponded to the region of heavy silver deposits depicted in Fig. 2c. (f) The same type of section from the experimental tooth, showing the absence of degradation from the hybrid layer (between open arrowheads) when chlorhexidine was used as an MMP inhibitor.

View larger version (129K): [in a new window]   Figure 3. An abrupt, vertical pattern of degradation within the hybrid layer, as illustrated by the radiographs and TEMs of primary molars that were retrieved from the third clinical subject (Control group, a, c, and e; Experimental chlorhexidine group, b, d, and f). C, resin composite; A, adhesive; H, hybrid layer; D, dentin. (a) Bitewing radiograph showing the Class I restoration in tooth 85 (control, circled). (b) Bitewing radiograph showing the Class I restoration in tooth 75 (experimental, circled). (c) Undemineralized, unstained, silver-impregnated section from the control tooth. The hybrid layer exhibited a pale electron density (gray), with regions containing silver deposits (pointer). In addition, there were electronlucent (white) zones within the hybrid layer that did not contain silver deposits (open arrow). Since silver impregnation was performed prior to epoxy resin infiltration, these white zones probably contained materials that were more highly penetrable by the electron beam than the original resin-infiltrated collagen fibrils. (d) The same type of section from the experimental tooth, showing similar silver deposits (pointer) within the hybrid layer, but with the lack of those electronlucent regions depicted in Fig. 3c. (e) Demineralized section from the control tooth that was stained with uranyl acetate and lead citrate. Vertical regions (asterisk) were found abruptly within the hybrid layer in which the bulk of the collagen fibrils have degraded, leaving behind strands of loosely arranged microfibrils (i.e., partially degraded gelatin; see Fig. 4e). Resin tags that were surrounded by islands of intact collagen fibrils (open arrowhead) remained within these abrupt regions of degradation. (f) The same type of section from the experimental tooth, showing the absence of degradation from the hybrid layer (between open arrowheads) with chlorhexidine as an MMP inhibitor.

View larger version (139K): [in a new window]   Figure 4. Increasing severity of degradation within hybrid layers. (a–e) A series of TEM micrographs, taken from demineralized, stained sections of the other parts of the control teeth of the three clinical subjects, showing an increasing severity in the degradation that occurred within the hybrid layers after 6 mos of intra-oral functioning. In (e), most of the collagen fibrils within the hybrid layer have degraded, and only loose strands of microfibrils remain (arrow). (f) An undemineralized, unstained, silver-impregnated section depicting, from a different part of the control tooth, the electronlucent (white), silver-free zone within the hybrid layer (open arrow). These white zones probably corresponded to the regions in Fig. 4e, in which the collagen fibrils have degraded into gelatin strands. Further degradation of the denatured gelatin into polypeptides and amino acid residues could have resulted in the leaching of these smaller molecules out of the hybrid layer, with the resultant empty spaces being infiltrated by the silver deposits.