Universidade de São Paulo

2012

Effect of dental tissue conditioners and matrix

metalloproteinase inhibitors on type I collagen

microstructure analyzed by Fourier transform

infrared spectroscopy JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS,

MALDEN, v. 100B, n. 4, pp. 1009-1016, MAY, 2012http://www.producao.usp.br/handle/BDPI/42588

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Departamento de Dentística - FO/ODD Artigos e Materiais de Revistas Científicas - FO/ODD

Effect of dental tissue conditioners and matrix metalloproteinaseinhibitors on type I collagen microstructure analyzed by Fouriertransform infrared spectroscopy

Sergio B. Botta,1 Patricia A. Ana,2 Moises O. Santos,3 Denise M. Zezell,3 Adriana B. Matos1

1Operative Dentistry Department, School of Dentistry, University of Sao Paulo, Sao Paulo, SP, Brazil2Biomedical Engineering – Center of Engineering, Modeling and Applied Social Sciences, Federal University of ABC,

Santo Andre, SP, Brazil3Center for Lasers and Applications, Energetic and Nuclear Research Institute, Sao Paulo, SP, Brazil

Received 17 March 2011; revised 8 November 2011; accepted 3 December 2011

Published online 30 January 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.32666

Abstract: This study aimed to evaluate the chemical interac-

tion of collagen with some substances usually applied in

dental treatments to increase the durability of adhesive resto-

rations to dentin. Initially, the similarity between human den-

tin collagen and type I collagen obtained from commercial

bovine membranes of Achilles deep tendon was compared

by the Attenuated Total Reflectance technique of Fourier

Transform Infrared (ATR-FTIR) spectroscopy. Finally, the

effects of application of 35% phosphoric acid, 0.1M ethylene-

diaminetetraacetic acid (EDTA), 2% chlorhexidine, and 6.5%

proanthocyanidin solution on microstructure of collagen and

in the integrity of its triple helix were also evaluated by ATR-

FTIR. It was observed that the commercial type I collagen can

be used as an efficient substitute for demineralized human

dentin in studies that use spectroscopy analysis. The 35%

phosphoric acid significantly altered the organic content of

amides, proline and hydroxyproline of type I collagen. The

surface treatment with 0.1M EDTA, 2% chlorhexidine, or 6.5%

proanthocyanidin did not promote deleterious structural

changes to the collagen triple helix. The application of 6.5%

proanthocyanidin on collagen promoted hydrogen bond

formation. VC 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B:

Appl Biomater 100B: 1009–1016, 2012.

Key Words: collagen, Fourier Transform Infrared spectro-

scopy, membrane, matrix metalloproteinase, proanthocyanidin

How to cite this article: Botta SB, Ana PA, Santos MO, Zezell DM, Matos AB. 2012. Effect of dental tissue conditioners and matrixmetalloproteinase inhibitors on type I collagen microstructure analyzed by Fourier transform infrared spectroscopy. J BiomedMater Res Part B 2012:100B:1009–1016.

INTRODUCTION

The routine use of esthetic resin composite restorations hasencouraged their constant technical improvement. Perform-ing these restorations is operator dependent, and for theirretention in the cavity, it is fundamental to obtain microme-chanical bonding between the adhesive systems and dentalhard tissues (enamel and dentin).

The dentin substrate is a hydrated tissue, which is com-posed (in volume) of approximately 50% mineral, 20% offluids, and 30% of organic material.1 The organic material iscomposed mainly of 85 vol % type I collagen and theremainder is a mixture of noncollagenous proteins.2

During the restorative procedure, acid conditioners areapplied on dentin, acting on the dentin smear layer. Thesmear layer may be defined as a layer deposited on theentire dentin surface that has been instrumented with anytype of cutting or wearing instrument. The smear layer

seals the dentin tubule openings and can interfere in theformation of the hybrid layer. Thus, the use of acid condi-tioners has been proposed with the objective of removingand/or treating this smear layer and favor the completechemical and mechanical adhesion of the adhesive systemwith the etched dentin tissue,3 promoting a reduction offailures in composite restorations.

Etching procedure causes exposure of the collagenfibrils, facilitating hybrid layer formation. However, at thesame time, if these exposed fibers are not completely envel-oped by the resinous monomer, they may be dissolved andwill be the site where degradation of the bond interfacebegins.4

Another important factor to consider is that exposure ofthe extracellular matrix in collagenous tissues related to en-dogenous and exogenous stimuli, such as the use of acids,may lead to disturbances in the regulatory mechanisms of

Correspondence to: A. B. Matos; e-mail: bona@usp.br

Contract grant sponsor: Fundacao de Amparo �a Pesquisa do Estado de Sao Paulo (FAPESP); contract grant numbers: 1995/5651-0, 2006/05684-1,

2006/06746-0

Contract grant sponsor: Conselho Nacional de Desenvolvimento Cientıfico e Tecnol�ogico (CNPq); contract grant number: 143395/2009-2

Contract grant sponsor: CEPID; contract grant number: 05/51689-2

VC 2012 WILEY PERIODICALS, INC. 1009

extracellular matrix metalloproteinase (MMP) activity, capa-ble of participating in exposed collagen degradation at thebond interface between the restorative material and dentinduring the adhesive procedure.5–7 Activation of the MMPsmay favor the proteolytic degradation of dentin tissue,8

compromising bond durability7 due to the reduction in themechanical strength of the dentin collagen fibrils.9 This deg-radation results in diminishing the bond strength betweendentin tissue and the adhesive system, and may lead to pre-mature loss of restorations.

These facts have motivated the use of chemical agentsduring the adhesive procedure, which may restrict the dele-terious effects produced by acid-containing tissue condi-tioners, or inhibit the action of MMPs. The reduction in col-lagen fiber exposure, resulting in less MMPs activation, canbe obtained by replacing the application of 35% orthophos-phoric acid for 15 s (classical bonding protocol) with theapplication of a chelating agent [0.1M ethylenediaminetetra-acetic acid (EDTA), for 60 s], whose preclinical evaluationshowed similar bond strength results in the comparisonbetween the two tissue conditioning agents.10

Another front on which one could act would be theapplication of substances known to be MMPs inhibitors,such as 2% chlorhexidine digluconate for 60 s, a widelyindicated clinical protocol and with evidence of increasingthe durability of restorations.11,12 The use of agents promot-ing crosslinking between collagen fibrils, such as proantho-cyanidin, has recently been reported in the literature as astep to be performed before the adhesive procedure, in anattempt to prevent degradation of the bond interface.13,14

Proanthocyanidin interacts with collagen in four differentways: by covalent interaction with proteins,15 ionic interac-tions,16 hydrogen bridge formation,17 or hydrophobic inter-actions.18 All of these different interactions keep the colla-gen protein structure intact, even after it has been cleavedby an enzyme.19

To evaluate whether the application of the above-men-tioned substances would in fact be efficient in improving arestorative protocol, it is imperative to consider the chemi-cal interactions between such products and collagen (mainorganic constituent of dentin) in addition to investigatingwhether the products applied to human dentin do not pro-mote alterations in the tridimensional structure of the colla-gen fibers. Studies have shown that collagen fibrils sub-jected to disturbances in the links of their polymeric chainor that have been denatured, show a reduction in their re-sistance to the action of proteolytic enzymes and a reduc-tion in their mechanical strength.9 For this in vitro analysis,ideally, the use of human dentin would be necessary. How-ever, the difficulties inherent to this substrate, such as dif-ferences in mineralization, dentinal tubule dimensions, andthe difficulty of obtaining teeth, make it necessary to usesubstitutes during chemical analyses, which suggests theuse of type I collagen membranes,20 and which has not yetbeen reported in the literature.

Thus, this study has two distinct objectives: (1) to verifythe chemical similarity of spongy samples of type I collagen,obtained from bovine Achilles deep tendon, and samples of

demineralized human dentin, to determine if these two dif-ferent substrates behaved in similarly under an analysis byAttenuated Total Reflectance technique of Fourier TransformInfrared (ATR-FTIR) spectroscopy and (2) to evaluate theinteraction of some substances usually applied before dentalrestorations on tridimensional structure of type I collagenfibrils.

MATERIALS AND METHODS

Experimental designThis in vitro study was divided into two experimentalphases. In the first one, it was evaluated whether collagenmembranes obtained from bovine Achilles deep tendon(type I collagen) could substitute demineralized human den-tin samples in chemical analyses done by ATR-FTIR. For thispurpose, three samples of human demineralized dentin andthree samples of type I collagen were analyzed by ATR-FTIR; the intensities and positioning of the main absorptionbands was evaluated, to verify similarity between the sam-ples. In the second experimental phase, the chemical inter-actions provided by phosphoric acid, chlorhexidine andproanthocyanidin on structure and integrity of the collagentriple helix were evaluated by ATR-FTIR.

Phase 1: collagen similarity from human dentinand bovine Achilles tendonThis study was approved by the Research Ethics Committeeof the School of Dentistry, University of Sao Paulo, ProtocolNo. 09/08. Three recently extracted impacted human thirdmolar teeth, with absence of caries lesions and structuralanomalies were used. After extraction, the collected teethwere washed with soap and water and then cleaned by pro-phylaxis with pumice stone and water. The teeth werestored in distilled water under refrigeration (þ4�C) for amaximum period of 30 days.

From each tooth, one 5 mm � 5 mm � 2 mm humandentin slice was obtained by sectioning the occlusal surfacewith a diamond disc with water cooling. The samplesobtained (n ¼ 3) were submitted to an ultrasonic bath for10 min, and after this were immersed in a 0.5M EDTA solu-tion at pH 7.4, at 37�C for 07 days21 until the completedemineralization, which was confirmed by X-ray images.Next, the samples were copiously washed with Milli-Q waterfor 30 min.

For comparison of dentin with bovine collagen, spongesof bioabsorbable membranes of type I collagen from bovineAchilles deep tendon (Technodry Liofilizados M�edicos, MG,Brazil) were cut with the aid of a sterile biopsy punch withan internal area of 5 mm. Thus, three discs of type I colla-gen, measuring 5 mm in diameter and 2 mm thick wereobtained.

The compositional analysis of all samples was performedusing the ATR-FTIR (Thermo Nicolet Smart Orbit) with aDTGS detector using a diamond crystal, coupled to a FTIRspectrometer (6700 ThermoNicolet). The ATR-FTIR spectraof each sample were obtained with 4.0 cm�1 resolution,with 80 scans in the range of 4000–500 cm�1, and wererecorded using the OMNIC Spectra Software. Infrared bands

1010 BOTTA ET AL. TYPE I COLLAGEN MICROSTRUCTURE AND MATRIX METALLOPROTEINASE INHIBITORS

considered for this study 1630 cm�1 (amide I), 1551 cm�1

(amide II), 1237 cm�1 (amide III), and 1454 cm�1 (prolineand hydroxyproline). The center band positions and bandswidths were obtained and the areas under the consideredbands were calculated and normalized by amide I band forsemiquantitative comparison between groups.

The record and conversion of absorption spectra wereperformed by means of the specific program of the spec-trometer (OMNIC 8.0, ThermoNicolet, Madison, WI). Thestatistical analysis was performed using the OriginPro 8software, and Student’s t-test, considering the level of statis-tical significance of a < 5%.

Power analysis (SPSS SamplePower – IBM) was per-formed to estimate the test power considering the samplesize used (n ¼ 3) in each experimental group, the one-wayanalysis of variance test and assuming a difference betweenthe means of a ¼ 0.05 and b ¼ 0.95. The analysis indicateda power of 0.999 for dentin collagen and 0.997 for bovinetendon collagen.

Phase 2: effects of tissue conditioning substances andMMP inhibitors on the microstructure of collagenFor this analysis, 25 membranes of type I collagen wereprepared as previously described, and were randomly dis-tributed into five experimental groups (n ¼ 5), as describedin Table I. For the treatments, each membrane wasimmersed in 5 mL of the corresponding solution.

The treatment times of each solution are different, aim-ing to simulate the clinical procedure to be performedbefore a restorative procedure. In this way, the acid phos-phoric was applied for 15 s, necessary to etch dentin beforethe application of an etch-and-rinse adhesive system; theapplication time for distilled water and 2% chlorhexidinedigluconate was 60 s, accordingly to the literature11; theapplication time for EDTA was 60 s, accordingly to the liter-ature10; and the application time selected for the proantho-cyanidin solution was 60 min, accordingly to theliterature.13,14

After the immersion time had elapsed, each collagenmembrane was carefully removed with a sterile clinical for-ceps, which was changed for each sample. After the immer-sions, the membranes of groups G1 and G4 (immersed inwater and 2% chlorhexidine, respectively) were only lightlydried with filter paper (Whatman No. 6, Whatman Interna-tional, England), to avoid dehydration and to reproduce thatwhich occurs in prerestorative clinical applications. The

membranes of groups G2, G3, and G5 (immersed in 35%phosphoric acid, 0.1M EDTA, and 6.5% proanthocyanidin so-lution, respectively) were abundantly washed with Milli-Qwater (Millipore, Bedford) for 1 min and lightly dried withfilter paper. All samples were kept in humid environment toavoid dehydration.

The specimens were analyzed by ATR-FTIR technique, aspreviously described. From each spectrum obtained, themain absorption bands in infrared were identified, and asemiquantitative comparison of the intensity of the bandswas made by means of comparison with the spectrumobtained of the membranes immersed in water (positivecontrol). Calculation of the area under the bands of interestwas made after the baseline tracing. Normalization wasdone by dividing the areas under the bands of the treatedsamples considered by the areas of the membranes of thepositive control group.

To evaluate the integrity of the collagen triple helix, thepeak absorbance ratios of 1235 cm�1/1450 cm�1 were con-sidered, according to Tonhi and Plepis.22 Therefore, if thisratio was close to 0.5, the integrity of the collagen triplehelix is compromised; if this ratio was closer to 1, theintegrity of amide III and CAH bond of the pyrrolidine ringof the type I collagen triple helix is maintained.23

RESULTS

Similarity of human dentin collagen to bovine Achillestendon collagenFigure 1 shows the infrared spectra of demineralized dentinand type I collagen from bovine Achiles tendon (mem-brane). In both spectra, bands characteristic of collagenwere identified, corresponding to the groupings amide A(3309 cm�1) and amide B (2930 cm�1). In addition, thetwo spectra presented the three main bands of the collagenfingerprint: in 1630 cm�1, typical of amide I (due to car-bonyl stretching – CAH), in 1551 cm�1, related to amide II(due to the vibrations in the plane of NAH bond and CANstretching), and in 1232 cm�1, corresponding to the vibra-tions in the plane of amide III (due to CAN stretching andNAH deformation). The peaks identified in 1454 cm�1 andin the region between 1417 cm�1 and 1360 cm�1 corre-spond to the stereochemistry of the pyrrolidine rings of pro-line and hydroxyproline, in the same way as the peaksfound in the region between 3100 and 3400 cm�1 occurdue to OAH and NAH stretching of amide A.

TABLE I. Substances Evaluated with Regard to Collagen Integrity

ExperimentalGroup Substance Brand Name

ApplicationTime

G1 Distilled water n.a 1 minG2 35% ortophosphoric acid 3M ESPE Dental Products St. Paul, MN 15 sG3 Ethylenediaminetetraacetic

acid � 0.1M EDTAF�ormula e Acao Produtos Odontol�ogicos,

Sao Paulo, SP, Brazil1 min

G4 2% chlorhexidine digluconate FGM Produtos Odontol�ogicos, Joinvile, SC, Brazil 1 minG5 6.5% proanthocyanidin MegaNatural-BP � Polyphenolics, Madera, CA 60 min

n.a., not applicable.

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Figure 2 illustrates the region of the collagen fingerprint.It can be noted that the two samples present the bands inthe same position, no new bands or the disappearance ofbands were evident. It was also observed that the dentinsamples showed the peaks 938 cm�1 (PO2

�), 1005 cm�1

(vibration CAH), 1029 cm�1 (vibrations CAO and CAC),1074 cm�1 (PO2

�), and 1157 cm�1 (vibration CAO) withgreater intensity when compared with the membranes. It isimportant to note the presence of phosphate ions binding todemineralized dentin (bands 1239, 1201, 1075, 1031, and960 cm�1), which is important to the reaction withchlorhexidine.

The semiquantitative comparison of the spectra obtainedshowed that the relative quantity of amide I is similar forthe two types of samples. Nevertheless, it was observedthat the quantities of amide II, amide III, and proline arehigher in the membrane samples when compared with thedentin samples (Table II).

Effects of tissue conditioning substances and MMPinhibitors on the microstructure of collagenFigure 3 shows the mean infrared spectra of type I colla-gen samples submitted to the different treatments pro-posed. In all the spectra, maintenance of the main bandscharacteristic of collagen is observed in 1630 cm�1 (amideI), in 1551 cm�1 (amide II), in 1232 cm�1 (amide III), andin 1450 cm�1 (pyrrolidine rings of proline andhydroxyproline).

Figure 4 shows the infrared spectra of the fingerprint ofcollagen for the tissue conditioner substances, and Figure 5shows the infrared spectra obtained in the region of the fin-gerprint of collagen for the MMP inhibitor substances. It ispossible to observe that the main absorption peaks of colla-gen were preserved in all samples.

In Figure 4, it is observed that the application of phos-phoric acid for 15 s promoted a significant decrease in therelative intensity of bands corresponding to amides I, II, andIII and in the 1405 cm�1 band. In addition, it is presented

an increase on relative intensities of absorption peaks ofphosphate (bands 1239, 1201, 1075, 1031, and 960 cm�1),which indicates the incorporation of phosphate ions fromphosphoric acid treatment to the collagen structure. Consid-ering the application of 0.1M EDTA solution, it was not pro-moted the appearance, disappearance of displacement ofabsorption bands.

In Figure 5, it is noted that the 2% chlorhexidine digluc-onate application did not result in the disappearance or dis-placement of new absorption bands. However, it is impor-tant to note a slight increase on relative intensity ofabsorption bands 1200 cm�1 (collagen and P¼¼O band),1080 cm�1 (phosphate vibration), 1033 cm�1 (collagen),1015 cm�1 (polysaccharides), 960 cm�1 (phosphate), 938cm�1 and 917 cm�1 (phosphodiester stretching bands);also, it is possible to note the formation of 1045 cm�1 band(CAO stretching and CAOH of carbohydrates).

The 6.5% proanthocyanidin application resulted inthe appearance of an absorption band located in 1520cm�1, corresponding to the vibrational mode of C¼¼N andaromatic tannin ring C¼¼C stretching. In addition, anincrease could be seen in the intensities of bands 1375cm�1 (corresponding to the vibrational mode of CAOstretching, deformation CAH, NAH, or CAOH of polyphe-nols), 1284 cm�1 (corresponding to the vibrational modeof amide III and pyrolidine ring stretching CAO of the fla-vonoid), 1204 cm�1 (corresponding to the vibrationalmode of amide III or vibrations CAOAC and CO of poly-saccharides or stretching CAOH of polyphenols) and1064 cm�1 (corresponding to the vibrational mode ofCAO of ribose and deformation CAH of aromatic ring ofpolyphenols composites). Thus, it was observed thatthere were no alterations in the absorption bands rela-tive to amides I, II, and III.

The analysis of integrity of collagen triple helix showedthat all tested substances did not promote collagen denatu-ration, because the means of the absorbance ratios in 1235cm�1 (amide III) and 1450 cm�1 (pyrrolidine ring) wereclose to the unit (Table III).

FIGURE 2. Spectral region between 1800 and 800 cm�1 of the means

of demineralized dentin and collagen membrane samples.

FIGURE 1. Absorption spectrum in infrared region between 4000 and

500 cm�1 of the collagen present in demineralized human dentin and

in the collagen membranes analyzed by ATR-FTIR, with identification

of the peaks of greater intensity.

1012 BOTTA ET AL. TYPE I COLLAGEN MICROSTRUCTURE AND MATRIX METALLOPROTEINASE INHIBITORS

DISCUSSION

For the study of chemical changes produced in the collagenstructure, the ATR-FTIR technique is highly indicated. Theinfrared spectrum is characteristic of every molecule, andcertain groups of atoms give rise to bands that occur closeto one and on the same frequency, irrespective of the struc-ture of the molecule. It is precisely the presence of thesecharacteristic bands of groups that allow one to obtain use-ful structural information.24 For different chemical groups,the wavelengths absorbed and the natural frequency of thevibrations are unique and depend on the existent bond type(C¼¼C, CAH, C¼¼O, NAH, and OAH).25 The portion of great-est use for the characterization of organic compounds is inthe range between 4000 and 400 cm�1 in the mid infrared.

Collagen is the most abundant class of proteins in thehuman body and represents about 30% of its total protein.There are over 20 types of collagens, being the most abun-dant in the human body, types I, II, and III.26 Possible sour-ces of type I collagen are the bovine Achilles deep tendons,rat tail, pericardium, and skin of animals including fish.

For studies of chemical and/or mechanical effects of sev-eral substances in dentin substract, most studies recom-mend an excessive number of teeth per experimental group,taking into account that human teeth present differences inmineralization, dentinal tubule dimensions, age, and donorethnicity.1 Although literature shows several studies employ-ing infrared spectroscopy analysis of dentin, there is no con-sensus about the number of speciments that should beused. In this way, considering some difficulties in obtainanceof sound human tooth, it is necessary to find an optimal

substitute for demineralized dentin for infrared spectros-copy analysis. Thus, the option of evaluating collagen mem-branes produced from bovine Achilles deep tendons (com-pletely composed of type I collagen) has become important.

In the comparison of the absorption bands in the infra-red spectra obtained from the bovine collagen and demine-ralized human dentin, it is possible to identify their similar-ity with regard to the presence and position of the mainabsorption bands. In both spectra obtained, it is observedthe presence of the absorption bands corresponding to thefingerprint region of collagen, composed by amide I (1600–1660 cm�1), amide II (1500–1550 cm�1), amide III (1320–1220 cm�1), and pyrrolidine rings (1450 cm�1). Therefore,although differences were detected in the relative intensitiesof the absorption bands (Table II) and concerning thehigher presence of phosphate ions binding to demineralizeddentin collagen, these differences did not interfere in thechemical properties of these substrates. For ATR-FTIR analy-sis, dentin can be substituted by commercial membrane.

It is imperative to remember that every collagen typeconsists of three polypeptide chains, composed fundamen-tally by Gly–proline–hydroxyproline structured in a-like he-lix,27 and some minor characteristics distinguish the colla-gen chains each other, such as the terminal domains.Triplets of glycine, praline, and hydroxyproline are the mostcommon and the most stabilizing tripeptide sequence in col-lagens.27 The intramolecular hydrogen bonds stabilize thetriple helix, with one direct hydrogen bond between the gly-cine NAH and the carbonyl of the proline residue in an ad-jacent chain.

FIGURE 3. Absorption spectrum in the infrared region of collagen

membranes immersed in the test substances of this study. (A) Amide I

band (1630 cm�1); (B) amide II band (1551 cm�1); (C) pyrrolidine ring

(1450 cm�1); (D) amide III band (1232 cm�1); (E) band with reference to

phosphate ion (1080 cm�1). Water, hydrated collagen membrane; PRS,

6.5% proanthocyanidin-rich solution; CHX, 2% chlorhexidine; EDTA,

0.1M ethylenediaminetetraacetic acid; PA, 35% phosphoric acid.

FIGURE 4. Spectral region between 1800 and 800 cm�1 of the means

of samples from phosphoric acid, EDTA, and water groups of this

study. PA, 35% phosphoric acid; water, hydrated collagen membrane;

EDTA, 0.1M ethylenediaminetetraacetic acid.

TABLE II. Areas Under the Bands of Interest Obtained in the Dentin and Collagen Membrane Spectra (Mean 6 SD)

Amide I Amide II/Amide I Amide III/Amide I Proline/Amide I

Dentin 15.640 6 2.40a 0.336 6 0.05b 0.081 6 0.01d 0.095 6 0.01f

Membrane 15.659 6 1.51a 0.478 6 0.03c 0.114 6 0.00e 0.116 6 0.01g

Different letters in each column represent statistically different means in accordance with the Student’s t-test.

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Considering the analysis by FTIR, the intensities of am-ide I and II are related to the helical structure of collagen,and the intensity of amide I could be used for characterizingthe secondary structure of proteins.28,29

The amides I and II are the main infrared absorptionbands of the peptide group in collagen. Both bands absorbin different regions considering if they participate or not inhydrogen bonding interactions. Hydrogen bonds affect thevibration frequencies of the participating atoms. The pres-ence of hydrogen bonds is an indication of the polypeptidechains assuming regular secondary structures. In fact bothamides I and II bands absorb in slightly different regions ifthey are in a-like helix or if they are in b-sheets.30 Thus, theamide bands can be used to probe the structure of the col-lagen.29 The ones that are most sensitive are the amides I,II, and III. Amide I band is predominantly a C¼¼O stretchingmode, the amide III results from a mixture of CAN stretch-ing and NAH in plane bending, and a CAC stretching andthe amide II band is also used to distinguish between a-likehelix, b-sheets and random coil conformations ofbiomolecules.

If the polypeptide is a a-like helix the NAH and C¼¼Ogroups and the hydrogen bonds between them are orientedalong the helix axis. The bonds that make up the amide IIband are perpendicular to the helix axis. In b-sheets, the sit-uation is just the opposite, with the NAH and amide bandsbeing perpendicular to the chain direction, and the amide IIbands being parallel to it.30

Commercial membranes were used in a later experimen-tal phase to evaluate the chemical interaction of collagenwith 35% phosphoric acid, 0.1M EDTA, 2% chlorhexidinedigluconate, and 6.5% proanthocyanidin. It was observedthat the use of 35% phosphoric acid, even for a short periodof time (15 s), significantly altered the content of amide,proline and hydroxyproline, in addition to enabling theincorporation of phosphate ions into the collagen micro-structure (Figure 4). In spite of the short application time(15 s), this acid is capable of producing demineralization of

peritubular and intertubular dentin at a depth of between 3and 5 lm, exposing a collagen fiber network.31 This timeminimizes the deleterious effects of phosphoric acid, whichwas indeed demonstrated in the test of triple helix integrity.

From the analysis of the spectra obtained (Figure 4), itis observed that 0.1M EDTA can be a feasible alternative tothe use of phosphoric acid, considering that this substanceproduced no significant changes in the collagen structure,and did not alter the triple helix integrity. Previous studieshave suggested the substitution of phosphoric acid by 0.1MEDTA for prerestorative tissue conditioning.10,32 EDTA pro-motes less demineralization of the dentin, exposing asmaller depth of collagen fibers, producing a thinner hybridlayer when associated with an adhesive system.32

It is known that EDTA chelating action leave less freecalcium ions to bind with the MMP activation sites,33 thenkeeping the MMP inactive by the absence of free calciumions. On the other hand, pro-MMPs that are buried in thedentin are not available to cleave collagen proteins, butMMPs that came from saliva or from the dentinal tubulescan access the hybrid layer by an eventual nanoleakage and,as a consequence, can weak the dentin bond.33,34

The MMP inhibitor substances appear as an alternativefor use in association with phosphoric acid to reduce itsside effects on the collagen structure. Indeed, in this study,it was confirmed that 2% chlorhexidine digluconate doesnot produce any chemical change in the microstructure ofcollagen (Figure 5), nor does it interfere in its integrity (Ta-ble III). In addition, it is shown a slight increase on relativeintensity of some absorption bands of collagen after chlo-rhexidine application, which suggests that this substancecan interact chemically with collagen. It was reported byMisra35 that chlorhexidine reacts with phosphate presentedat hydroxyapatite and, considering the low presence ofphosphate ions binding to demineralized dentin, these ionscan be the main responsible for the interaction of chlorhexi-dine with dentin. In this study, it was not possible to dem-onstrate the presence of chlorhexidine phosphate becauseboth demineralized dentin and collagen membranes presentsignificant less quantity of phosphates ions binding than hy-droxyapatite. However, the increase on relative intensity ofsome peaks, including the phosphate ones, indicates theinteraction of this substance with collagen.

Chlorhexidine is an efficient broad spectrum antibacte-rial agent, which has been shown to be effective in the inhi-bition of at least three types of matrix MMP: MMP-2, MMP-

TABLE III. Absorbance Ratios of the Collagen Triple Helix

After Immersion in Each Tested Solution

Tested SolutionApplication

TimeIntegrity

(1235/1450 cm�1)

Water 1 min 1.01 6 0.0235% phosphoric acid 15 s 1.03 6 0.040.1M EDTA 1 min 1.01 6 0.022% chlorhexidine solution 1 min 1.04 6 0.016.5% proanthocyanidin-rich

solution60 min 1.05 6 0.02

FIGURE 5. Spectral region between 1800 and 800 cm�1 of the means

of samples from chlorhexidine, water and proanthocyanidin of this

study. Water, hydrated collagen membrane; PRS, 6.5% proanthocyani-

din-rich solution; CHX, 2% chlorhexidine.

1014 BOTTA ET AL. TYPE I COLLAGEN MICROSTRUCTURE AND MATRIX METALLOPROTEINASE INHIBITORS

8, and MMP-9.36. Preclinical studies have shown that theapplication of 2% chlorhexidine resulted in interfaces thatwere less susceptible to degradation after 12 months37 and14 months12 of function in the oral cavity, or after 24months38 in vitro. Therefore, according to the resultsobtained in this study, it is suggested that 2% chlorhexidinemay be used for 60 s safely in prerestorative etch-and-rinseprocedures, as it does not interfere in the quality of thebonding resins remaining after tissue conditioning.

This study also demonstrates that the 6.5% proantho-cyanidin-rich solution is a promising agent for maintainingcollagen integrity. Previous studies have shown that the car-boxylic groups of collagen fibers interact with the hydroxylgroups of the aromatic rings of proanthocyanidin, resultingin bonds of the hydrogen bridge type.17 The stability of thisinteraction with proline-rich proteins, such as collagen,when compared with other polyphenols, suggests the struc-tural specificity of proanthocyanidin.17,18 This interaction isowing to proline being an amino acid that has an atom ofoxygen from the functional carboxylic group; the proximityto an atom of nitrogen of the adjacent amino acid favors thehydrogen bridge bond type.18 Therefore, the proline-richproteins, such as type I collagen, form highly stable bondswith proanthocyanidin.

Evaluation of the integrity of the collagen triple helixwas performed by analysis of the ratio of the absorbance ofbands 1235 cm�1 (amide III) and 1450 cm�1 (stereochem-istry of the pyrrolidine rings). Although the first band issensitive to the presence of the secondary structure of colla-gen, the second is independent of the ordered structure ofcollagen.39 For type I collagen fibers, the integrity of theirsecondary structure may be verified when the value of theratio 1235/1450 cm�1 is greater than or equal to the unit.Changes in this absorption ratio indicate significant struc-tural alterations in the collagen triple helix. In this study,the value found for all substances applied to bovine collagenwas approximately 1.00; significantly higher than that whichwould be observed for denatured structures, whose valuewould be around 0.5.39 The results showed that none of thesubstances applied interfered negatively in the structuralarrangement of collagen triple helix.

In spite of the routine use of 2% chlorhexidine for 60 s,35% phosphoric acid for 15 s and of 0.1M EDTA for 60 s onthe mineralized tissue, this test of the integrity of collagenfibers had not yet been performed. It has thus been provedthat these aqueous solutions may be clinically used inpatients without harm to the structure of dentin collagenfibers, and that the 6.5% proanthocyanidin solution hasbeen shown to be a promising alternative for maintainingthe integrity of collagen over time.

The presence of native inter- and intramolecular cross-links of the triple helix collagen structure provides the basisfor the stability and strength of the collagen fibrils is re-sponsible for maintaining its mechanical properties, whichhave a great influence on the success of the dental restora-tive procedure on demineralized dentin.

The use of extrinsic collagen cross-linking agents caninduce additional formation of inter- and intramolecular

cross-links.18 Proanthocyanidin is a natural collagen cross-linker18 well known to readily precipitate proline-rich pro-teins (such as collagen) due to hydrogen and covalentbonds.20 The Proanthocyanidin have been demonstrated toincrease the ultimate tensile strength and elastic modulus ofdemineralized dentin.14,40

According to the results obtained in this study, it couldbe concluded that type I collagen fibers from human dentinor bovine Achilles deep tendon are structurally similarwhen evaluated by ATR-FTIR spectroscopy. The 35% phos-phoric acid used for surface conditioning for a short periodof time (15 s), significantly altered the organic content ofamide, proline and hydroxyproline. The surface treatmentwith 0.1M EDTA or 2% chlorhexidine did not promote dele-terious structural alterations to the collagen triple helix. Theapplication of 6.5% proanthocyanidin-rich solution do notpromote deleterious structural alterations to the collagentriple helix, although important chemical interactions weredetected with hydrogen bond formation.

ACKNOWLEDGMENTS

The authors thank Ms. Margery Galbrigth for the English revi-sion of this manuscript.

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