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  • 7/29/2019 74390015

    1/9Vol 14, No 1, 2012 11

    Collagen Cross Linking Increases Its Biodegradation

    Resistance in Wet Dentin Bonding

    Changqi Xua/Yong Wangb

    Purpose: The biodegradation of exposed dentin collagen within the adhesive/dentin (a/d) interface is one of

    the main reasons for composite restoration failures and seriously affects the durability of dental restorations. In

    the present study, the objective was to investigate whether the inclusion of the cross-linking reagent (glutaralde-

    hyde, GA) in the adhesive would increase collagen biodegradation resistance within the a/d interface.

    Materials and Methods: The model adhesive consisted of ~60 % monomers (HEMA/bis-GMA, 45/55 wt/wt) and

    ~ 40 % ethanol as a solvent. 5% GA was added to the above formulation. After the dentin surfaces were etched

    for 15 s with 35% phosphoric acid, rinsed with water and blotted dry, adhesives both with and without GA were

    applied and polymerized by visible light for 20 s. These a/d specimens were immersed in the biodegradation

    solution (prepared by adding 160 mg collagenase in 1 liter of TESCA buffer solution) for up to 30 days after pro-

    ceeding with the sectioning/fracture to expose the a/d interfaces. The specimens were analyzed using SEM and

    micro-Raman spectroscopy.

    Results: SEM results indicated that for the adhesive without GA, there were many voids and a loss of collagen

    fibrils in the a/d interface after being challenged by the biodegradation solution. The Raman spectra collected

    from the interface showed that the amide I of collagen at 1667 cm-1 obviously decreased, indicating a removal

    of collagen fibrils during the degradation process. For the adhesive containing GA, the collagen fibrils within the

    interface did not degrade at all, which was also confirmed by the Raman results.

    Conclusion: The results corroborate the previous findings that by using the current adhesive system and wet

    bonding, the collagen fibrils in the a/d interface are largely unprotected and easily undergo biodegradation. Di-

    rectly including cross-linking agents in the adhesive could protect collagen fibrils from degradation in situ within

    the a/d interface.

    Keywords: dentin, bonding, collagen cross linking, Raman.

    J Adhes Dent 2012; 14: 1118. Submitted for publication: 22.06.10; accepted for publication: 01.09.10.

    doi: 10.3290/j.jad.a21494

    Generally, the longevity of ceramic or resin-compositedental restorations is reported to be 6 to 10 years.8Although many factors are responsible for shortening

    the longevity, recurrent or secondary caries7,13,17 is

    thought to be one of main reasons leading to the failure

    of dental restorations. Caries is defined as the decom-position process of hydroxylapatite and dentin collagen

    due to the influence of organic acids generated by oral

    bacteria, such as Streptococcus mutans.4,14 Recently,

    collagenase (matrix metalloproteinases, MMPs)7,10,21

    activated by acids has been proven to be involved in the

    degradation process of dentin collagen, whereas oral

    bacteria do not directly destroy dentin collagen.7 Pash-

    ley et al10

    have reported that the degradation of colla-gen in the demineralized dentin is due to host-derived

    MMPs that are induced by acid etching and released

    slowly over time.

    Several methods have been developed to control sec-

    ondary caries and improve the durability of dental restora-

    tions, for instance, utilizing the fluoride ion release26 from

    dental composites to prevent caries, since F- can improve

    apatites ability to resist acid attacks. Another method is

    to use chelating agents such as MMP inhibitors to stop

    the activation of collagenase surrounding carious lesions.

    For example, it was reported that chlorhexidine could be

    used as an antimicrobial agent for disinfection and pres-

    a Research Fellow, University of Missouri-Kansas City, School of Dentistry,

    Kansas City, MO, USA. Performed experiments, contributed to data analy-

    sis, co-wrote mansucript.

    b Associate Professor, University of Missouri-Kansas City, School of Dentistry,

    Kansas City, MO, USA. Experimental design, wrote manuscript.

    Correspondence: Dr. Yong Wang, University of Missouri-Kansas City, School

    of Dentistry, 650 E. 25th St., Kansas City, MO 64108, USA. Tel: +1-816-235-

    2043, Fax: +1-816-235-5524. e-mail: [email protected]

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    Xu et al

    times with distilled water, with each rinse lasting one

    minute. The rest of the specimens in the buffer solution

    were used as controls. The specimens were classified

    into four groups: A), adhesive (without GA)/dentin in-

    terface specimens (without biodegradation challenge);B), adhesive (without GA)/dentin interface specimens

    (with biodegradation challenge); C), adhesive (with

    GA)/dentin interface specimens (without biodegrada-

    tion challenge); D), adhesive (with GA)/dentin interface

    specimens (biodegradation challenge).

    Micro-Raman Spectroscopy

    As described above, separate adhesive/dentin slabs

    (including those that were stored in biodegradation solu-

    tion and those that were not) were prepared for inves-

    tigation using micro-Raman spectroscopy. Since the

    micro-Raman spectroscopic technique is nondestructive,

    these same specimens were available for analysis usingSEM. A LabRam HR 800 Raman spectrometer (Horiba

    Jobin Yvon; Paris, France) using monochromatic radia-

    tion emitted by a He-Ne laser (wavelength 632.8 nm)

    was used to collect Raman spectra. It was equipped with

    a confocal microscope (Olympus BX41), a piezoelectric

    XYZ stage with a minimum step width of 50 nm, and an

    air-cooled CCD detector of 1024 x 256 pixels. The follow-

    ing parameters were used: 600 grating, 140-m confocal

    hole, and 100-m slit width. Spectra were Raman-shift

    frequency calibrated using known lines of silicon.

    Micro-Raman spectra were acquired by focusing the

    laser beam through a 100X Olympus objective to an ap-

    proximately 1 m beam diameter onto the a/d speci-

    mens. Spectral maping was performed at positions cor-

    responding to 1-m intervals across the a/d interfaces

    using the computer-controlled stage. Two consecutive

    scans of spectra (with 60 s accumulation time each)were obtained from each site. An imaging system and

    high-resolution monitor enabled visual identification of

    the position at which the Raman spectra were obtained.

    No post processing of the data was performed.

    Scanning Electron Microscopy

    Following micro-Raman analysis, the specimens de-

    scribed above were prepared for SEM examination.

    After dehydration in different ethanol solutions (33%,

    67%, 85%, 95%, 100%), the prepared specimens were

    mounted on aluminum stubs and sputter coated with

    ca 20 nm of gold-palladium. The specimens were then

    examined at a variety of magnifications and tilt anglesin a Philips XL30 ESEM-FEG (Philips Electron Optics;

    Hillsboro, OR, USA) at 10 or 15 kV.

    RESULTS

    Figure 1 depicts representative SEM micrographs of

    the dentin interfaces with model adhesives with and

    without GA. The specimens were cross sectioned and

    slightly polished to create flat surfaces. In Figs 1A and

    1C, the micrographs were taken from sectioned speci-

    mens not exposed to the biodegradation solution; the

    Fig 1 RepresentativeSEM micrographs of

    the dentin interfaces

    with model adhesives

    with and without glu-

    taraldehyde (GA).

    The specimens were

    cross sectioned and

    slightly polished to

    create flat surfaces

    before immersion in

    the biodegradation

    solution. A) adhesive

    without GA, before

    degradation; B) ad-hesive without GA,

    after degradation;

    C) adhesive with GA,

    before degradation;

    D) adhesive with GA,

    after degradation; Ad:

    adhesive; D: dentin.

    Before Degradation

    WithoutGA

    With

    GA

    After Degradation

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    Xu et al

    14 The Journal of Adhesive Dentistry

    micrographs from the specimens exposed to the biodeg-

    radation solution are shown in Figs 1B and 1D. Before

    biodegradation, the appearance of the interface with

    adhesive containing GA appears similar to the interface

    in the adhesive without GA. The margin lines for theinterfaces can be recognized in both specimens. After

    biodegradation, there is almost no change in appear-

    ance of the interface with the adhesive containing GA,

    but the interfacial margin line disappeared. In addition,

    there are gaps and/or cracks at the a/d interface when

    GA was not included in the formulation.

    To minimize the effect that cutting/polishing may have

    on the interfacial morphology, the specimens were frozen-

    fractured to expose the interfaces before immersion in

    the biodegradation solution. Figure 2 depicts representa-

    tive SEM micrographs of the adhesive/dentin fractured

    interface specimens from four groups. The interfaces are

    not readily recognizable as compared to those in Fig 1.However, without the effect from smear layers caused

    by polishing, the collagen fibrils are visible in the dentin

    interfaces with both model adhesives with and without

    GA before biodegradation (Figs 2A, and 2C). After biodeg-

    radation, for adhesive without GA, there are many voids

    in the a/d interface (marked by circles), and the collagen

    fibrils were lost (Fig 2B). Nevertheless, for the adhesive

    containing GA, there was almost no change in morphol-

    ogy, and the collagen fibrils did not degrade at all after

    immersion in the biodegradation solution (Fig 2D). By

    comparing the micrographs from Figs 1 and 2, it is evident

    that fracturing provides more detailed structural informa-

    tion, although the surfaces are not flat. However, cutting

    and polishing diminishes the morphological differences

    between specimens.

    To study the chemical compositional changes, the cor-

    responding Raman mapping spectra of the above a/dinterfaces with and without degradation were obtained.

    Representative Raman spectra from pure dentin, inter-

    face, and pure adhesive are shown in Fig 3. The intense

    peaks related to the adhesive occur at 1720 cm-1 (car-

    bonyl), 1609 cm-1 (phenyl C-C), 1453 cm-1 (CH2 def), and

    1113 cm-1 (C-O-C). These peaks are related to methacry-

    late monomers in the model adhesives.23 In the dentin

    spectrum, the peaks related to collagen occur at 1667

    cm-1 (amide I), and 1245 cm-1 (amide III); the peak related

    to minerals occurs at 961 cm-1 (i1 PO43-).25 The spectrum

    of the interface shows the contribution from the adhesive

    and dentin. Some peaks of dentin (especially collagen)

    are overlapped with the peaks of model adhesive; how-ever, the amide I peak at 1667 cm-1 for collagen is not

    covered and is still distinguishable from the interface

    spectrum.

    Representative micro-Raman mapping spectra of the

    dentin interfaces with model adhesives without and with

    GA are shown in Fig 4, which were collected before and

    after biodegradation. Figure 4A represents a series of

    mapping spectra acquired at 1-m intervals across the

    dentin interface with an adhesive (without GA) before

    biodegradation. In the first two spectra, the relative in-

    tensity of the 961 cm-1 (PO43-) peak associated with the

    mineral component suggested that the second spectrum

    Fig 2 Representative

    SEM micrographs of

    the dentin interfaces

    with model adhe-

    sives with and with-

    out glutaraldehyde

    (GA). The specimens

    were frozen fractured

    to expose the inter-

    faces before putting

    in the biodegradation

    solution. A) adhesive

    without GA, before

    degradation; B) ad-

    hesive without GA,after degradation;

    C) adhesive with GA,

    before degradation;

    D) adhesive with GA,

    after degradation; Ad:

    adhesive; HL: hybrid

    layer; T: resin tag.

    Before Degradation

    Withou

    tGA

    With

    GA

    After Degradation

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    Xu et al

    represented the bottom of the demineralized dentin. The

    last two spectra were acquired from an adhesive. Vibra-

    tional peaks associated with the adhesive and collagen

    component of dentin were noted from the third to eighth

    spectra, indicating that the model adhesive monomers

    had penetrated into the interface and that the thickness

    of the interfacial zone of adhesive/dentin was approxi-

    mately 6 to 7 m. After biodegradation, the amide I peakintensity at 1667 cm-1 for collagen obviously decreased

    across the whole interfacial zone, indicating damage/

    removal of collagen fibrils during the degradation process

    (Fig 4B). However, for the adhesive containing GA, there

    was almost no change in the mapping spectra of the inter-

    face before and after degradation (Figs 4C and 4D), which

    indicated that the composition of the a/d interface did not

    change after being challenged by the biodegradation solu-

    tion. The collagen in the interface did not degrade, which

    conformed to the SEM results.

    The evidence of GA cross linking dentin collagen is

    shown in Fig 5. Representative Raman spectra in the

    region of 1000 to 1050 cm-1 were selected from the in-

    terfaces of the above four groups. The peak around 1001

    cm-1 is associated with phenylalanine of collagen,22 and

    the peak around 1031 cm-1 is assigned to the pyridinium

    ring vibration.15 It has been found that a trivalent amino

    acid based on a 3-hydroxypyridinium ring is a prominent

    cross-linking residue in collagen.15 The relative ratio of

    1031/1001 can be used to measure the cross-linking

    reaction between GA and collagen.15 The higher the ratio

    is, the greater the extent of collagen cross linking. As

    compared to the spectra from specimens without GA, an

    increase in peak intensity at 1031 cm-1 was observed in

    the spectra from the interfaces with adhesive contain-

    ing GA (Fig 5). This peak intensity was the same before

    and after biodegradation. The results indicated the GA

    included in the adhesive cross linked demineralized den-

    tin collagen.

    Fig 3 Representative Raman spectra from the dentin, adhe-

    sive and interface.

    Raman

    Intensity

    AmideI

    CH2

    CH2

    AmideIII

    C-O-C

    C-C

    inPhenyl

    Dentin

    Interface

    Adhesive

    1800 1600 1400 1200 1000 800

    Raman Shift/cm-1

    1PO4

    3-

    Fig 4 Representa-

    t ive micro-Raman

    mapping spectra

    of the dentin inter-

    faces with model

    adhesives without (A

    and B) and with GA

    (C and D), collected

    before and after bio-

    degradation.

    After degradationBefore degradation

    PO43-

    PO43-

    1600 1400 1200 1000 800 1600 1400 1200 1000 800

    1600 1400 1200 1000 800 1600 1400 1200 1000 800

    A

    C

    B

    D

    44

    22

    -4-4

    -2-2

    00

    X(

    m)

    X(

    m)

    Amide IAmide I

    DentinDentin

    InterfaceInterface

    Adhesive wo GAAdhesive wo GA

    Raman Shift/cm-1

    Raman Shift/cm-1

    Raman Shift/cm-1

    Raman Shift/cm-1

    PO43-4

    2

    -4

    -2

    0

    X(

    m)

    Amide I

    Dentin

    Interface

    Adhesive w GA

    PO43-4

    2

    -4

    -2

    0

    X(

    m)

    Amide I

    Dentin

    Interface

    Adhesive w GA

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    Xu et al

    16 The Journal of Adhesive Dentistry

    The effects of adding GA to the adhesive on the degree

    of conversion and penetration of adhesive in the interface

    were evaluated. Representative spectra of polymerized

    adhesives with and without GA are shown in Fig 6. The

    peak appearing at 1637 cm-1 is associated with the C=C

    of methylacrylate, and the peak at 1608 cm-1 is related

    to C-C in phenyl of the adhesive monomer.9,27 The adhe-

    sive degree of conversion can be calculated based on

    the intensity ratio of 1637/1608,9,27 which did not show

    any difference between the adhesives with and without

    GA. This indicates that adding GA to an adhesive does

    not induce a negative effect on the degree of conver-

    sion. However, adding GA may slightly affect the adhesive

    penetration into the a/d interface. To determine the dif-

    ferences in adhesive penetration, the relative intensity

    ratios of 1113 cm-1 (C-O-C, adhesive)/1667 cm-1 (amide I,

    collagen) were calculated using the spectral subtraction

    technique.23 The ratio of 1113/1667 shows a gradual de-

    cline for both adhesives with and without GA as a function

    of position (Fig 7). The ratio for the adhesive with GA is

    slightly lower than that for the adhesive without GA, while

    the difference is not significant (p > 0.05).

    DISCUSSION

    Current dentin bonding strategies rely on micromechani-

    cal retention between collagen and infiltrated resin in the

    demineralized dentin layer. The strength of interlocking

    via monomers/resin penetration and entanglement of

    exposed collagen fibrils depend on the quality and lon-

    gevity of both resin and collagen phases. Since it has to

    be formed in the presence of water (wet bonding), there

    is substantial evidence to suggest that the quality of this

    layer is very poor.5,11,12,18-20,23-25 Instead of serving as a

    stable connection between the bulk adhesive and subja-cent intact dentin, the layer has been called the weakest

    link in the a/d bond.18 Results from both in vitro and in

    vivo studies have indicated that the poor quality of infil-

    trated resin (due to inadequate monomer/polymer con-

    version, phase separation, hydrolysis) and unprotected/

    exposed collagen fibrils (inducing degradation) are two

    major factors inhibiting the formation of a durable a/d

    bond when using current adhesive systems.

    Numerous efforts have been made to improve the qual-

    ity of infiltrated resin by introducing new materials or tech-

    niques. Only recently, improving the stability of collagen

    fibrils by cross linking has been attempted.1,2 In these

    Fig 5 Representa-

    tive Raman spec-

    tra in the region of

    1000 to 1050 cm-1,

    selected from the in-

    terfaces of the above

    four groups.

    Fig 6 Representative spectra of polymerized adhesives with

    and without GA, showing the information on the degree of con-

    version.

    C-C in Phenyl

    C = C

    Adhesive without GA

    Adhesive with GA

    1637c

    m-1

    1608cm-1

    Without GA With GA0.9320.009 0.9360.008

    1680 1660 1640 1620 1600 1580 1560 1540

    Ram

    anIntensity

    Raman Shift/cm-1

    1+&+2&+&+2

    JOXWDUDOGHK\GH

    FROODJHQ

    S\ULGLQLXPULQJ

    1050 1040 1030 1020 1010 1000

    Without GA before degradation

    Without GA after degradation

    With GA before degradation

    With GA after degradation

    RamanIntensity

    Raman Shift/cm-1

    1031.2cm-1

    1029.3cm-1

    1001.3cm-1

    &+

    &+2

    &+&+2

    &+&+21

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    Xu et al

    studies, dentin collagen is cross linked by immersing in

    the cross-linking solution for various time periods (up to

    72 h).1,2 Although the application of cross linkers did im-

    prove mechanical strength and stability of dentin collagen,

    to date there has been no feasible way to clinically deliver

    cross-linking agents to the dentin bonding surface, since

    effective cross-linking induction usually requires a long in-

    cubation period in the cross-linking solution (> 1 h). In the

    previous studies, after acid etcing, the demineralized den-

    tin surfaces were immersed in the respective cross-linking

    solutions for 1 h,1,16 which makes this approach clinically

    unfeasible. In this study, we investigated the potential of

    adding a cross-linking reagent (glutaraldehyde, GA) to the

    adhesive for collagen cross linking. Our results indicated

    that the cross-linking agent included in the model adhesivewould not only cross link demineralized dentin collagen in

    situ, but also increase collagen biodegradation resistance

    within the adhesive/dentin interface formed under wet con-

    ditions. The null hypothesis was thus rejected.

    In the control group (adhesive without GA), collagen

    fibrils in the a/d interface were obviously degraded or

    removed after immersion in the biodegradation solution

    (Figs 2B and 4B). Based on the Raman spectra, collagen

    in mineralized dentin did not degrade after the biodeg-

    radation challenge, since it was protected by minerals

    (data not shown). It is very likely that collagen fibrils in

    the demineralized dentin layer are not entirely protected

    or sealed by adhesive resin, which is consistent withour previous study.23 In that study, we used a novel mi-

    croscopic staining technique to characterize the ideal or

    optimum hybrid layer as compared with the a/d interface

    prepared by the wet-bonding technique. The ideal resin-

    collagen hybrid structure was prepared under controlled,

    optimum conditions. Using a histomorphological staining

    technique, any collagen that is not encased in adhesive

    resin is available for reaction with the Goldners trichrome

    stain.20,23 The results indicate that the section of the

    ideal resin-collagen hybrid specimen does not pick up any

    stains, but that the a/d interface always picks up stains,

    indicating that the adhesive does not encapsulate the

    collagen fibrils throughout the width of the demineralized

    dentin. Thus, it is almost impossible to form an optimum

    hybrid structure under wet bonding conditions. The results

    of this study further confirmed that collagen fibrils in the

    a/d interface were not protected by resin. After being chal-

    lenged by the biodegradation solution, the unprotected,

    exposed collagen fibrils were digested by collagenase.

    In the past, the difficulty in recognizing the fact that the

    collagen is not protected or sealed by infiltrated resin un-

    der wet bonding conditions might be partly due to the SEM

    techniques used. The most popular SEM techniques for

    determining the quality of the interface have relied on mor-

    phological characterization of the polished a/d specimens.

    Using the polishing preparation technique, the existence of

    smooth, acid-resistant interfacial layers has been consist-ently reported for most adhesive systems. Our previous

    studies have indicated that polishing the a/d interface

    during specimen preparation for SEM can adversely af-

    fect and even obscure the morphological detail of the a/d

    specimens, which actually possess a porous interfacial

    structure. The quality of the a/d interface can be easily

    overestimated due to polishing.24 In the present study,

    there is almost no change in appearance of the polished

    interfaces before and after challenge by the biodegrada-

    tion solution, except the after degradation, there are some

    cracks at the a/d interface when GA is not included in the

    formulation (Fig 1). It once again shows that the polishing

    techqnique could mask the morphological detail and shieldthe interface from attack by the collagenase.

    The artifacts just described can be avoided by using

    the fracture techqnique. Before the degradation chal-

    lenge, dentin collagen fibrils could be clearly observed in

    the fractured interfaces (Figs 2A and 2C). After degrada-

    tion, the changes in interfaces were able to be monitored

    easily. It showed that collagen fibrils were degraded in or

    had disappeared from the interface with adhesive (with-

    out GA) (Fig 2B), but were still visible in the interface

    with adhesive (containing GA) after the biodegradation

    challenge (Fig 2D). These results are confirmed by the

    confocal Raman mapping results (Fig 4). For the adhesive

    Fig 7 Raman intensity ratios of

    1113/1667 as a function of spa-

    tial position across the dentin inter-

    faces with adhesive containing and

    adhesive not containing GA.

    Collagen

    AmideIat1667

    C-O-Cat1113

    Raman Shift/cm-1

    RamanIntensity

    Adhesive

    1700 1600 1100

    1,2

    1

    0,8

    0,6

    0,4

    0,2

    01 2 3 4 5 6 7

    Depth/um

    Rationof1113/1667

    Adhesive without GA

    Adhesive with GA

    Penetration of BIS-GMA

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    Xu et al

    18 The Journal of Adhesive Dentistry

    containing GA, there was almost no change in the amide

    I peak intensity for collagen before and after degrada-

    tion, indicating the preservation of collagen fibrils dur-

    ing the biodegradation process (Fig 4). However, for the

    adhesive without GA, there was a dramatic decrease in

    the collagen amide I peak after biodegradation (Fig 4),

    which indicated that collagen fibrils were removed after

    being challenged by the collagenase solution. The confo-cal Raman technique has many advantages. It not only

    provides information on chemical compositional changes,

    but also can detect information from the sub-surface of

    the specimen due to its confocal setup. SEM is a surficial,

    morphological technique. As discussed above, its results

    are very sensitive to the surface preparation techniques.

    Unlike SEM, the surface influence or interference from the

    preparation techniques can be eliminated in the confocal

    Raman studies by adjusting the focusing positions.

    CONCLUSION

    In summary, the results of this study corroborate theprevious findings that when using current adhesive

    systems and wet bonding, the collagen fibrils in the a/d

    interface are largely unprotected and easily undergo

    biodegradation. Collagen cross linking shows promise

    as a way to improve and preserve the durability of bond-

    ing. Based on the Raman results, GA included in the

    adhesive uniformly cross linked the demineralized col-

    lagen (Fig 5). Cross-linked collagen fibrils survived in astrong biodegradation solution. Directly including cross-

    linking agents in the adhesive could be a good, clinically

    practicable method of protecting collagen fibrils from

    degradation in situ within the a/d interface, as well as

    improving the durability of adhesively luted dental res-torations. Although GA has been used in Gluma desen-

    sitizer clinically, due to the toxicity concerns about GA,

    the delivery of more biocompatible cross-linking agents

    should be studied in the future.

    ACKNOWLEDGMENTS

    This investigation was supported in part by USPHS Research

    Grants DE 015281 and DE 021023 from the National Institute of

    Dental and Craniofacial Research, National Institutes of Health,

    Bethesda, MD 20892, USA. The authors would like to acknowl-

    edge the SEM technical support of Dr. Vladimer Dusevich and the

    secretarial support of John Fife from UMKC School of Dentistry.

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    Clinical relevance: Directly including cross-linking

    agents in the adhesive could protect collagen fibrils

    from degradation in situ within the adhesive/dentin

    interface.

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