Strategies to prevent hydrolytic degradation of the hybrid layer-A review

Abstract

Objective: Endogenous dentin collagenolytic enzymes, matrix metalloproteinases (MMPs) and cysteine cathepsins, are responsible for the time-dependent hydrolysis of collagen matrix of hybrid layers. As collagen matrix integrity is essential for the preservation of long-term dentin bond strength, inhibition of endogenous dentin proteases is necessary for durable resin-bonded restorations.

Methods: Several tentative approaches to prevent enzyme function have been proposed. Some of them have already demonstrated clinical efficacy, while others need to be researched further before clinical protocols can be proposed. This review will examine both the principles and outcomes of techniques to prevent collagen hydrolysis in dentin-resin interfaces.

Results: Chlorhexidine, a general inhibitor of MMPs and cysteine cathepsins, is the most tested method. In general, these experiments have shown that enzyme inhibition is a promising approach to improve hybrid layer preservation and bond strength durability. Other enzyme inhibitors, e.g. enzyme-inhibiting monomers, may be considered promising alternatives that would allow more simple clinical application than chlorhexidine. Cross-linking collagen and/or dentin matrix-bound enzymes could render hybrid layer organic matrices resistant to degradation. Alternatively, complete removal of water from the hybrid layer with ethanol wet bonding or biomimetic remineralization should eliminate hydrolysis of both collagen and resin components.

Significance: Understanding the function of the enzymes responsible for the hydrolysis of hybrid layer collagen has prompted several innovative approaches to retain hybrid layer integrity and strong dentin bonding. The ultimate goal, prevention of collagen matrix degradation with clinically applicable techniques and commercially available materials may be achievable in several ways.

Keywords: Chlorhexidine; Cysteine cathepsins; Degradation; Dentin bonding; Durability; Enzyme inhibition; Enzymes; Hybrid layer; Matrix metalloproteinases; Review.

Figure 1

Transmission electron microscope (TEM) image of undemineralized, unstained human tooth showing the dentin-adhesive interface created with 2-step E&R adhesive (Scotchbond 1XT, 3M ESPE). A) On the top of the mineralized dentin (md) is the hybrid layer (hl), where the exposed dentin collagen mesh is infiltrated with adhesive monomers, creating a mechanical interlock between dentin-bound collagen and polymerized adhesive. On top of the hybrid layer, adhesive (a) forms a chemical bond with the restorative resin composite (c). Adhesive resin tags (rt) penetrate into dentinal tubules, sealing them and providing additional retention. B) Higher magnification image from the area marked with dashed box in A, with collagen matrix readily seen in the hybrid layer, even in unstained sections. (Images courtesy of BDs Pekka Mehtälä and Dr. Saulo Geraldeli).

Figure 2

Schematic of the complex of CHX with cysteine cathepsins B, showing CHX (stick model) completely engulfed in the cathepsin B active site cavity from S2 to S2′: Cys29 and His199 are the active site residues (light yellow). The amino acid residues Tyr75, Pro76, and Glu245 (red) are at the subsite S2; Gly74 (red) and Cys29 (light yellow) residues are at the subsite S1, and the His199 (light yellow) residue is at the subsite S1′. The enzyme secondary structure elements are shown as cyan blue schematics (arrows for extended strands and cylinders for helices); the carbon atoms of CHX are indicated by light green sticks. (Reproduced from Scaffa et al. 2012 [52], with permission)

Figure 3

A: SEM image of the fracture occurring at the bottom of the hybrid layer. Dentinal tubules are mostly exposed, with few dentinal tubules containing remaining resin tags. Partially degraded collagen at the bottom of the hybrid layer gives can be seen (asterisk). B: SEM image of the fracture cohesive failure localized in the middle of the hybrid layer. Dentinal tubules are completely filled by resin tags (black arrow), and intertubular dentin is covered by adhesive (asterisk). C: Schematic presentation of resin-bonded acid-etched dentin covered with resin composite. The acid-etched tubules no longer contain peritubular dentin, making the tubules twice their normal diameter. Resin tags extend down from the adhesive layer. The tags are hybridized with the surrounding demineralized dentin as they pass through the hybrid layer. There is no such hybridization of the resin tags as it passes into mineralized dentin. As poorly infiltrated hybrid layers age, the collagen fibrils degrade and disappear. In such hybrid layers, water replaces the collagen. The spaces in the composite are due to hydrolysis of nanofillers of silica from the resin composite. These, too become filled with water. (Figures A and B reproduced from Carrilho et al. 2007 [31], with permission.)

Figure 4

The effect of 2% CHX pretreatment on the distribution of failure modes (in percentage) as observed with SEM in vitro (A) and in vivo (B). External inhibitor indicates the absence or presence of a protease inhibitor cocktail used in incubation medium (artificial saliva, AS). A) In immediate testing, no differences between the fracture modes were detected. After 6-month incubation, statistically significant increase in failures located at the bottom of the hybrid layer were seen in control group, but not in CHX group. External inhibitors in AS significantly reduced the failures at the bottom of the hybrid layer in controls, indicating partial elimination of endogenic enzyme function; respective effect in CHX-treated samples was non-significant. (Data from Carrilho et al. 2007 [31]) B) Failure modes in restorations tested immediately or after 14 months (14 m) in service in vivo. After 14 months, cohesive failures in the hybrid layer and dentin increased in controls, while in CHX-pretreated group failures in the hybrid layer decreased and no cohesive failures in dentin were observed. (Data from Carrilho et al. 2007 [32]).

Figure 5

Cellular metabolism (SDH enzyme production detected by MTT assay) of odontoblast-like cells on the pulpal side of dentin disks following different concentrations of EDC solutions applied on the occlusal side of 0.4 mm-thick dentin discs. Columns are mean absorbance (570 nm) and error bars are standard-deviations, n=12. Columns connected by the horizontal line do not differ statistically.

Figure 6

Comparison of the loss of bond strength between enamel and dentin in vitro after 6 month water storage within the same study [15]. Bond strengths in enamel after the storage did not show statistically significant difference with any adhesive when compared to the intial bond strengths. In dentin, significant decrease (p<0.05) was observed with all adhesives except Protect Bond and Optibond FL.

Figure 7

A–E show adjacent confocal laser scanning microscopy (CLSM) and transmission electron microscopy images obtained from dentin bonds made with the self-etching dental adhesive, Adper Prompt-L-Pop. After bonding, the bonded tooth was sectioned vertically into 1 mm thick slabs and incubated in control media (Figs. A and B) or in biomimetic remineralization medium for 6 months. The specimen in Fig. A was immersed in 0.1% rhodamine B overnight. The red fluorescent tracer diffused into water-rich, resin-poor spaces within the hybrid layer (the area between the opposite arrows). In the absence of remineralizing reagents, no mineralization of the water-filled spaces occurred. The adjacent TEM shows no remineralization of the hybrid layer (area between opposing arrows). Fig. C is a CLSM image of the same resin-dentin bond that was allowed to remineralize for 6 months with biomimetic reagents. Note that the hybrid layer (area between opposing arrows) is less fluorescent and more grey indicating that much of the water was replaced by apatite mineral. This is better shown in the adjacent TEM images showing mineralization (asterisk region) of the bottom of the hybrid layer in 2 months and most of the hybrid layer in 6 months (from Kim et al. 2010 [166], with permission).

Figure 8

A: The effect of biomineralization on the dynamic mechanical behavior of hybrid layers (between opposing white arrowheads). Specimens were examined in the hydrated condition after 2 months of biomineralization. R: hydrophilic adhesive resin; T: resin-filled dentinal tubule; M: underlying mineralized dentin. The color scale shows the complex modulus of elasticity from 0–30 GPa. Much of the hybrid layer contains blue and green colors corresponding to complex moduli of 10 to 15 GPa. B: The right image is a TEM micrograph from the same specimen showing that much of the lower half of the hybrid layer (area between opposing white arrowheads) is filled with mineralized collagen (dark appearance). The top of the hybrid layer is electron transparent and not mineralized because the collagen fibrils were enveloped by adhesive resin.

Figure 9

A: Control resin-bonded dentin that was subjected to 2 months of immersion in a simulated body fluid without biomimetic mineralizing reagents. Note that the overlying adhesive resin (R) has a complex modulus of 2.5 GPa, as does most of the hybrid layer (area between opposing white arrowheads) indicating that the hybrid layers remained unmineralized and relatively soft (complex modulus of 2.5 GPa). B: TEM micrographs from the same resin-bonded dentin specimen that show areas where the collagen within the hybrid layer has been solubilized by endogenous dentin protease activity (asterisks).