Hybrid layer

© Ramaiah University of Applied Sciences
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Faculty of Dental Sciences©M. S. Ramaiah University of Applied Sciences
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Faculty of Dental Sciences
Presented by : Dr.Arbiya Anjum S
HYBRID LAYER

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Contents
• Introduction
• History
• Compositional & structural aspects
of enamel & dentin
• Hybrid Concept
• Goals of hybridization
• Resin infiltration
• Formation of hybrid layer
• Shag carpet appearance
• Tubule wall Hybridization
• Lateral wall Hybridization
• Etching And Its Effects On Hybrid
Layer
• Zones Of Hybrid Layer
• Concept of reverse hybrid layer
• Ghost Hybrid layer
• Marginal Hybrid layer
• Mechanical and physical properties
• Hybrid Layer Degradation
• Literature Review
• Conclusion
• References

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Introduction
Hybrid Layer : Resin Reinforced Zone Or Resin Infiltrated Layer
The zone where the adhesive resin of the dentin bonding agent
micromechanically interlocks within the inter tubular dentin and surrounding
collagen fibres

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HISTORY
1955-
Buonocore
developed
acid etching
with
phosphoric
acid
1960-1970’s –
1st and 2nd
Generation
bonding agents
1982-
Nakabayashi
proposed the
concept of hybrid
layer
•Early 1990’s-
Fusayama-
concept of total
etching and
bonding
1992-Kanca-
concept of wet
bonding

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Compositional & structural aspects of enamel & dentin
• The inorganic content of mature enamel is 95%by wt% & 86% by vol%; the primary component is
hydroxyapatite
• The remainder consists of water & organic material
• Unlike enamel, dentin contains higher % of water and organic material(type I collagen) &70 wt % of
hydroxyapatite
Mjor IA, Fegerskov o, eds. Human oral embryology & histology,1986

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Traditional Enamel bonding concept – Resin
tags mechanically locks into etched enamel
Hybrid Concept – Resin tags are supplemented
by thin resin reinforced tissue increases
bonding stability
Masuhara et al (1963) - Chemical analysis , tags themselves were pure resin, but at the end
of the tags there was a thin zone, where the resin had impregnated the interprismatic
material and formed a new material that was part tooth and part resin "hybrid layer”(highly
acid resistant) – excellent bond stability
Hybrid Layer as a Dentin-Bonding Mechanism , Nohito Nakahayashi , 1991

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1.Dentinal peptides (including collagen) must not be denatured when the dentin is
decalcified, if the acid is too aggressive, it may expose collagen below the hybrid layer
leaving a zone of weak dentin that is susceptible to long term degradation
2. Bonding resin must include monomers with both hydrophilic and hydrophobic groups
3. Catalyst must allow polymerization in the presence of oxygen and water
Characteristic required for formation of Hybrid layer
Hybrid Layer as a Dentin-Bonding Mechanism , Nohito Nakahayashi , 1991

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10Contemporary Esthetics Dentistry - 2012
Goals of hybridization
1.Sealing of dentinal tubules and intertubular dentin – lesser bacterial invasion into
tubules
2.Bonding restoration to dentin – less postoperative sensitivity
3.Minimally invasive preparations

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Hybridization (Nakabayashi 1982)- The process by which a structure formed in
dental hard tissues by demineralization of the surface and subsurface, followed by
infiltration of monomers and subsequent polymerization
Three specific ultra morphological features
• Shag carpet appearance
• Tubule wall Hybridization
• Lateral wall Hybridization
Resin Infiltration

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Hybridization is initiated by application of acid to dentin surface
Acid reacts with caoh of the intertubular dentin
Continues to a depth of 10 micrometre
Concurrently acid penetrates the dentinal tubules to a depth of
100 micrometre
Acid is washed away microscopic vacancies surrounding the
collagenous structure
Now its ready to receive the dentin bonding agent
Contemporary Esthetics Dentistry - 2012
Hybrid layer formation

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• Tubule Wall Hybridization:
• Extension of the hybrid layer into tubule wall area. Hermetically sealing the pulp dentinal complex
against microleakage, Especially protective when bond fails at top or bottom of the hybrid layer
• The resin tags keep tubules sealed as they break off at the level of hybrid layer
• Ensures a leakage free seal of tubules
• Lateral Tubule Hybridization :
• Formation of tiny hybrid layer into the walls of lateral tubule branches. This micro version of hybrid layer
typically surrounds a central core of resin called Micro Resin Tag
• Shag Carpet Appearance
• Appears when dentin surface after being acid etched is actively scrubbed with an acidic primer
solution
• The combined mechanical and chemical action of rubbing the acid etched dentin with an acidic
primer dissolves additional mineral salts while fluffing and separating the entangled collagen at the
surface
Sorarira et al(2006): The hybrid layer analysis revealed
different hybridization patterns, suggesting that the
attachment seems to be influenced by many factors and a
standardization of dentinal substrate is impossible

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Etching And Its Effects On Hybrid Layer

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Self etch adhesive system
• Retain or modify the smear layer
• Due to the low acidity of self etch adhesives as compared to etchant used in total-etch
systems, the smear layer is not completely removed which then interferes with resin tags
penetration resulting in shorter resin tags and thinner Hybrid layer formation affecting the
long term bond strength
Total-etch adhesive systems
• Smear layer is completely removed
• Due to high acidity of etchant used and remaining debris is rinsed off. Thicker hybrid layer
and long and dense resin tags are formed which results in better long term bond strengths

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Albaladejo 2010 : Hybrid layers of etch-and-rinse versus self-etching adhesive systems:
The formed hybrid layer obtained with the two-step selfetching adhesives and the etch-
and-rinse systems were continuous and uniform in thickness
Droplets within the all-in-one adhesive layer may occur as a result of water absorption
from dentin through osmosis, and may interfere with proper resin polymerization
Resin tags obtained with SEB and the etch-and-rinse adhesive systems showed lateral
branches, which is a sign of proper resin infiltration

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17Sturdavent 7th edition,2019

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Perdiago et al (1996)
Demineralized dentin has three distinct zones
Top layer : Residual smear layer or denatured collagen
and silica particles
Middle layer : Collagen fibrils separated by electro lucent
spaces of 10- 20 nm
Bottom layer : Gradual transition to underlying unaltered
dentin, with partially demineralized zone containing HA
crystals enveloped by resin/abrupt transition called
Hiatus
Zones Of Hybrid Layer

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• C Prati et al reported the formation of a "reverse" hybrid layer by application of NaOCI
after acid etching
• This procedure not only removes the exposed collagen but also solubilizes the fibrils down
into the underlying mineralized matrix to create sub micron porosities within the mineral
phase
• Cylindrical channels previously occupied by collagen fibrils are now available for resin
infiltration within the mineralized matrix
• Such dentin surfaces have been proposed as mineralized, hydrophilic alternatives to
collagen rich, hydrophobic acid- demineralized dentin, possessing increased stiffness and
bond strength
REVERSE HYBRID LAYER
Effect of removal of surface collagen fibrils on resin–dentin bonding , C. Prati,* e al ,Dental Materials 15 (1999) 323–331

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Diameter – 0.5 – 1
micrometre
Effect of removal of surface collagen fibrils on resin–dentin bonding , C. Prati,* e al ,Dental Materials 15 (1999) 323–331

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• Hybridiod region
• Aluminum oxide air abrasion (white arrows) resulted in partial removal of the original hybrid
layer (HL), followed by the formation of a new ghost-like hybrid layer (HL2). The adhesive
layer interface (ALi) was also modified, allowing for the incorporation of aluminum oxide
powder even after cleaning
Ghost Hybrid Layer

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• The quality of the hybrid layer created in dentin at the external margin of resin composite restorations which
determine the longevity of such restorations, for it is very thin and exhibits porosities and grooves, indicating
that it is not completely infiltrated with resin
• The thickness of the marginal hybrid layer measured under SEM was 1.5-2.5 μm thick in Scotchbond MP and
Scotchbond 1 but varied from 0.0 to 12.0 μm in Clearfil Liner Bond 2V when observed after NaOCl post polishing
procedures
• Collagen fibrils are not completely infiltrated by adhesive resin at the CEJ level along the Cavo surface margin of
the restorations
• The presence of noninfiltrated collagen along the external margins may reduce the sealing capability of bonding
systems, leaving many open spaces and gaps, which may jeopardize the durability of the bond over time
Marginal Hybrid Layer
Marginal Hybrid Layer in Class V Restorations, C Prati, Operative Dentistry, 2000, 25, 228-233

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23Marginal Hybrid Layer in Class V Restorations, C Prati, Operative Dentistry, 2000, 25, 228-233

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Sano, H., Takatsu, T., Ciucchi, B., Russell, C.M. and Pashley, D.H., 1995. Tensile properties of
resin-infiltrated demineralized human dentin. Journal of dental research, 74(4), pp.1093-1102.
Mechanical and Physical properties of Hybrid layer

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• Endogenous proteases are actually hydrolases, require unbound water to cleave collagen peptides,
dentin bonding, it is very difficult, if not impossible, to fully envelope the deepest portion of the
demineralized collagen fibrils within the HL with resin
• The gaps between the exposed collagen fibrils are filled with water, enabling the activation of the
endogenous enzymes and collagen degradation, leading to plasticization of the adhesive resin, and
mechanical strain on the exposed fibrils
• Inhibition of collagen degradation also results with the improved overall HL Integrity, lower increase of Nano
leakage and improved bond strength durability Loss of collagen allows increased water flow in and under the
hybrid layer, which may accelerate resin plasticization
• Several bonding strategies -
 Directly by collagenolytic enzyme inhibition / Increasing collagen resistance to degradation
 Indirectly by eliminating water between the exposed collagen fibrils
Hybrid Layer Degradation

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Mechanisms of degradation of the hybrid layer inadhesive
dentistry and therapeutic agents toimprove bond durability—A
literature review – Frassetto 2016
• Resin–dentin bond degradation is a complex process, involving the hydrolysis of both the
resin and the collagen fibril phases contained within the hybrid layer
• Collagen fibers become vulnerable to mechanical and hydraulic fatigue, as well as
degradation by host-derived proteases with collagenolytic activity (matrix
metalloproteinases and cysteine cathepsins)
• Inhibition of the collagenolytic activity and the use of cross-linking agents are the two main
strategies to increase the resistance of the hybrid layer to enzymatic degradation

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• By a chelating mechanism, since the activity of the MMPs independent on the metal ions
that can be chelated
• Chlorhexidine (CHX) - Apart from its antimicrobial properties, CHX was shown to inhibit
the activity of the MMPs and cysteine cathepsins
• As low as 0.2% solutions of CHX increase the longevity of the HL
• Attempts to incorporate it into the components of dental adhesive systems with the
intention of shortening chair time, with promising result, but CHX binding was shown to
be a reversible mechanism, and probably due to leaching, the resin–dentin interfaces
treated with CHX were shown to be unstable after 18months
Inhibition of the enzymatic activity

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Ethylenediaminetetraacetic acid (EDTA)-
• Chelating agent
• EDTA removes the Ca2+from the collagen matrices, and binds the Zn2+ions from the catalytic
site of the MMPs .However, several downsides of this agent have been reported, such as long
application time and reversibility caused by water solubility
Quaternary ammonium compounds
• Benzalkonium chloride (BAC), which is a mixture of alkyl benzyl-dimethylammonium
chlorides of various alkyl chains that strongly binds to demineralized dentin and that was
shown to have an immediate inhibiting effect comparable to that of CHX with promising
results in bond strength preservation over time
• 12-methacryloyloxydodecylpyridinium bromide (MDPB), incorporated into a commercially
available adhesive system showing a good ability to inhibit the MMPs activity

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• Galardin - a reduction in the degradation of the HL after one year
• SB-3CT, a specific MMP-2 and -9 inhibitor showed a positive influence on HL
Preservation
• Specific inhibitors for cysteine cathepsins, such as E-64 and odanacatib express
their inhibiting effect through binding to the enzyme at the cleavage site, mimicking
the substrate , but the data on effect on dentin bond durability is very limited s

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Removal of the unbound/residual water within Hybrid Layer
1.Ethanol wet-bonding brings ethanol-solvated resin closer to collagen than is possible during water wet-
bonding
• Pashley et al - altered the wet-bonding technique by replacing rinse-water with ethanol, so that the dentin
was saturated by ethanol rather than water
• Tay et al - excellent resin–dentin bonding by applying bis-GMA, the least soluble dimethacrylate, to ethanol-
saturated dentin
• Applying ethanol-solvated adhesives to ethanol-saturated dentin avoids any risk of phase separations ,
reduce the presence of residual water in the resin–dentin bonds
• Jee et al - used molecular dynamic simulations to recreate the three layers of bound water in collagen
matrices - first and second layers of tightly-bound water in collagen could not be replaced by ethanol.
However, half of the bound water in the outermost layer of bound water could be replaced by ethanol

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2. Development Of SE Primer Adhesive
• 10-methacryloyloxydecanethylene phosphoric acid (mdp) added in higher concentrations than were
used before for wet-bonding adhesives (i.E. 20–25 vol%)
• After “self-etching” for 10 s, one dries the primed dentin and then seals it with a solvent-free adhesive
before light-curing.
• Hybrid layers of only 1 m thick and contain much of the smear layer including the smear plugs that
prevent dentinal fluid from wetting the surface during bonding procedures. In general, these bonds
show good durability, but are still prone to loss of bond strength with time
3.Dimethyl Sulfoxide (DMSO) -
• as a primer or a solvent, low surface energy and can be used as a solvent to facilitate radical
polymerization reactions such as are used in dental adhesion
• It also possesses the polarity needed to break down water’s self-association and forms stable
complexes with water to create “hydrophobic water”
• Increase bothin immediate and long-term bond strength. Improved penetration may also contribute to
the durability of the bond strength, but DMSO may also have MMP-inhibitory effects

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• Al-Ammar et al., 2009; Macedo et al., 2009; Bedran-Russo et al., 2009, 2010 - Extensive efforts have been made
in the past few years to induce cross-linking in acid-demineralized dentin collagen
• Glutaraldehyde, genipin, proanthrocyanidin, and carbodiimide
• Proanthocyanidine inhibits MMP-2 and MMP-9. (Matchett et al, 2005)
• Cathepsin-K can also be regulated by molecules like sulphated GAGs (Glycosaminoglycans)
• The major drawback in the use of cross-linking agents to inactivate MMPs and cysteine cathepsins is that a
water-rich, resin-sparse collagen matrix with poor mechanical properties is retained within the hybrid layer.
Hence additional strategies are required so as to improve the mechanical properties of the denuded collagen
matrices from resin -dentin bond degradation.
MMP and Cathepsin Silencing via the use of Cross-linking Agents

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Biomimetic remineralization
• Biomimetic mineralization is one of the most reliable approaches which makes use of nanotechnology
principles to actually mimic what occurs in biomineralization (Tay and Pashley, 2008)
• It removes water from resin deficient regions of hybrid layer and replaces it with apatite crystallites which
are extremely small so as to fill the spaces for collagen matrices which have been reshaped for
remineralization of resin -dentin bonds
• For etch-and-rinse adhesives, apatite crystallites were present in both extrafibrillar and intrafibrillar spaces
after biomimetic mineralization. One of the hurdles for biomimetic remineralization of the poorly infiltrated
hybrid layer is that there has to be no residual hydroxyapatite crystallites in the demineralized collagen
network to serve as nucleation site for further remineralization.

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Literature Review
SEM evaluation of the hybrid layer after cavity
preparation with Er:YAG laser - Barceleiro et al
• Thickness of the hybrid layer formed using Scotchbond Multi-Purpose Plus, Single Bond 2, Prime & Bond
2.1 and Xeno III on a dentin surface prepared with a diamond bur in a high speed handpiece or prepared
with an Er:YAG laser used with two parameters of pulse energy (200 and 400 mJ) and frequency (4 and 6
Hz)
• Results -4 tested dentin adhesive systems produced a 2.90 +/- 1.71 microm hybrid layer in dentin prepared
with a diamond bur and hybrid layer was regular and routinely found. i.e. laser groups, the dentin adhesive
systems produced hybrid layers ranging from 0.41 +/- 1.00 microm to 2.06 +/- 2.49 microm, which were
very irregular and not routinely found
• Conclusion : Er:YAG laser, with the parameters used in this experiment, has a negative influence on the
formation of a hybrid layer and cavity preparation methods influence formation of the hybrid layer

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A New Technique for Assessing Hybrid Layer Interfacial
Micromorphology and Integrity: Two-photon Laser
Microscopy - D’Alpino 2006
Two-photon laser fluorescence microscopy technique developed to evaluate the interfacial micromorphology of
the hybrid layer in bonded restorations
Results: Subsurface fluorescent imaging using this technique enabled interfacial micromorphology to be
characterized at submicrometer resolution and provided high-contrast images. The quality of surrounding
structures and potential presence of gaps were also precisely assessed
Conclusion: Two-photon laser microscopy provided high quality, high-resolution images of the bonded interface
and surrounding areas, allowing accurate qualitative and quantitative analysis of the structure and integrity of
the hybrid layer.

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Effects of Nd:YAG Laser Irradiation on the Hybrid
Layer of Different Adhesive Systems – Ferreira et al 2009
To evaluate the microtensile bond strength (μTBS) and hybrid layer morphology of different adhesive
systems, either followed by treatment with Nd:YAG laser irradiation or not
Results: Control groups (27.81 ± 1.38) showed statistically higher values than lased groups (21.37 ± 0.99), and
CSEB control group values (31.26 ± 15.71) were statistically higher than those of SBMP (24.3 ± 10.66).
There were no significant differences between CSEB (20.34 ± 10.01) and SBMP (22.43 ± 9.82) lased groups.
Among parameters tested, 0.8 W/10 Hz showed the highest value (25.54 ± 11.74). Nd:YAG laser irradiation
caused dentin to melt under the adhesive layer of both adhesive systems tested.
Conclusion:Nd:YAG laser irradiation of the hybrid layer promoted morphological changes in dentin and
negatively influenced the bond strength of both adhesive systems.

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Dentin bonding: effect of tubule orientation on
hybrid layer formation
Schupbach P, Krejci I, Felix L: Dentin bonding: effect of tubule orientation on
hybrid-layer formation. Em J Oral Sci 1997; 105; 344-352, © Mmiksgaard, 1997

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Conclusion
Prior to hybrid layer concept dentin bonding was poor and there
was a gap between composite and tooth which is colonized by
bacteria with deleterious effects
Thus hybridization improved bond strength
Further research to stabilize the degradation of this layer is needed

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References
• Contemporary Esthetics Dentistry - 2012
• Current concepts on adhesion to dentin-j.D. Eick
• Adhesion to enamel and dentin: current status and future challenges- B van meerbeek
• Water treeing-a potential mechanism for degradation of dentin adhesives- tay fr,pashley
• Effects of dentin permeability on restorative dentistry –dent clin N am 46 2002
• Hybridization of dental hard tissues – Nakabayashi ,david pashley
• Bonding to Dentin: Smear Layer and the Process of Hybridization K. Van Landuyt, J. De Munck, E. Coutinho, M.
Peumans, P. Lambrechts, B. Van Meerbeek
• Dental adhesion review: aging and stability of the bonded interface lorenzo breschi
• Effects of water on dentin bonding thomas jacobsenl, karl-johan siiderholm
• The smear layer in endodontics – a review D. R. Violich1 & N. P. Chandler
• Marginal Hybrid Layer in Class V Restorations, C Prati, Operative Dentistry, 2000, 25, 228-233

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• SEM evaluation of the hybrid layer after cavity preparation with er:yag laser - barceleiro et al
• A new technique for assessing hybrid layer interfacial micromorphology and integrity: two-photon laser
microscopy - d’alpino 2006
• Dentin bonding performance and ability of four mma-based adhesive resins to prevent demineralization along
the hybrid layer – nurrohman et al 2012
• Mechanisms of degradation of the hybrid layer inadhesive dentistry and therapeutic agents toimprove bond
durability—a literature review – frassetto 2016
• Schupbach p, krejci i, felix l: dentin bonding: effect of tubule orientation on
• Hybrid-layer formation. Em J oral sci 1997; 105; 344-352, © mmiksgaard, 1997
• Effects of nd:yag laser irradiation on the hybrid layer of different adhesive systems – ferreira et al 2009
• Dentin bonding: sem comparison of the resin-dentin interface in primary and permanent teeth j.E. N'or,

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