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					-ILO
N. Nishiyama'*, T. Asakura2, K. Suzuki3, K. Komatsu', and K. Nemoto'
Department of Dental Materials, Nihon University School of Dentistry at Matsudo, 870-1 Sakaecho, Nishi 2, Matsudo, Chiba 271-8587, Japan; 2Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan; and 3Department of Dental Materials, Okayama University, Dental School, 2-5-1 Shikadacho, Okayama 700-8525, Japan; *corresponding author, norihiroCtumascat.nihon-u.acjp

J Dent Res 79(3): 806-811, 2000

Bond Strength of Resin to Acid-etched Dentin Studied by 13C NMR: Interaction between N-methacryloyl-w-Amino Acid Primer and Dentinal Collagen
INTRODUCTION
F or dentin adhesion, it is well-understood that the application of a hydrophilic methacrylate monomer, 2-hydroxy ethyl methacrylate (HEMA), as a primer facilitates the entanglement of the resin to the dentinal collagen fiber, thereby enhancing the bonding of the resin to the acid-etched dentin. This is due to the fact thatthe HEMA facilitates the restoration of the collagenous layer in which the collagen fiber arrangement had collapsed during an air-drying process (Sugizaki, 1991; Van Meerbeek et al., 1992; Suzuki and Nakai, 1994; Nakabayashi and Pashley, 1998). Since the methacrylate monomer in a bonding agent can diffuse into the collagenous layer which was restored by the HEMA, a hybrid layer, comprised of resin and dentinal collagen fiber, could be formed in the subsurface of the intertubular dentin, due to the polymerization of the methacrylate monomer (Nakabayashi et al., 1982). Nishiyama et al. (1996) had demonstrated the effect of the pH of the Nmethacryloyl glycine (NMGly) primer on the bond strength of the resin to acid-etched dentin. The bond strength of the resin to acid-etched dentin which was treated with the primer comprised of NMGly and NMGly-Na salt was strongly dependent on the pH value of the NMGly primer solution. When the NMGly primer whose pH was below the pKa value of 3.5, which is that of the carboxylic acid group in the NMGly molecule was applied, the bond strength was 15 MPa. However, when the NMGly primer whose pH was above the pKa value was applied, the bond strength dramatically dropped and leveled off at 3 MPa, even though a 5-p.m-thick hybrid layer was formed at the resindentin interface. These data demonstrated that the adhesion mechanism of the resin to acid-etched dentin is not solely based on a micro-mechanical interlocking associated by the entanglement of resin to dentinal collagen fiber (Nakabayashi et al., 1982; Erickson, 1989; Pashley, 1990). This suggested that the amount of undissociated NMGly species in the primer solution affected the magnitude of the bond strength of the resin to the acid-etched dentin. However, the mechanism for the bonding of the resin to dentinal collagen through the NMGly primer remains to be determined. Before a more effective adhesive primer can be developed, we must understand the adsorption behavior of the NMGly primer to the dentinal collagen. In our previous paper (Nishiyama et al., 1998), the adsorption behavior of the NMGly to dentinal collagen was analyzed by the 13C NMR technique with the observation of spin-lattice relaxation times (T,) at pH = 1.6. There, it was suggested that the amide group and/or the carboxylic acid group in the NMGly molecule formed a hydrogen bond with the carboxylic acid groups of the sidechain of the amino residue in the dentinal collagen molecule. The 13C NMR technique is a very powerful technique for investigating the details of an interaction between the primer and the dentinal collagen. This is due to the fact that the T, values are very sensitive to the various types of interactions, which are a result of a change in the mobility of the primer molecule. In this study, the adsorption behavior of the N-methacryloyl-w-amino acid (NMWA) primers with different methylene lengths as a spacer group to the dentinal collagen molecule was studied by the 13C NMR T1 technique. Details

ABSTRACT
The application of the hydrophilic methacrylate primer to acid-etched dentin increases the bond strength of the resin to the acid-etched dentin. However, the mechanism for the bonding of the resin to dentinal collagen through the primer remains to be determined. Before a more effective adhesive primer can be developed, we must understand the adsorption behavior of the primer to the dentinal collagen. The purpose of this study was to determine how 5 derivatives of N-methacryloylw-amino acid (NMwA) primers enhance the bonding of the resin to acid-etched dentin. The interaction between the NMwA primers and dentinal collagen was studied by the '3C NMR technique, including the observation of spin-lattice relaxation times, T1. When the dentinal collagen was dispersed into the NMwA solution, the T values of the two carbonyl carbons attributed to the amide and the carboxylic acid in the NMwA molecule decreased dramatically. This result was due to the interaction between the amide group and the carboxylic acid group in the NMwA molecule and the dentinal collagen molecule. The T1 values of these carbonyl carbons decreased when the number of methylene groups in the NMwA molecule increased. The interaction became stronger as the number of methylene groups in the NMwA molecule was increased. Further, the bond strength of the resin to the acid-etched dentin primed with NMwA increased with a decrease in the T, value of the amide carbonyl carbon. The strength of the interaction of the NMwA primer to the dentinal collagen molecule, determined by the '3C NMR technique, showed a direct correlation with the bond strength of the resin to acid-etched dentin that was treated with the NMwA primer.

KEY WORDS: adhesion mechanism of dentin,
dentin primer, N-methacryloyl-c-amino acid, dentinal collagen, 13C NMR.

Received July 8, 1998; Last Revision July 27, 1999; Accepted September 3, 1999

806

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Interaction between NM.A and Dentinal Collagen Studied by 13C NMR

807

of the interaction between the NMwA and the dentinal collagen were investigated by the change in the T1 value of the carbonyl carbons attributed to the amide and the carboxylic acid groups, based on our previous paper as described above. The adhesion mechanism of the resin to the acid-etched dentin, which was treated with NMwA primers, is discussed.

MATERIALS & METHODS
Preparation of the NMwA Primer Solution
Five species of NMwA-NMGly (glycine), NMPAla (P-alanine), NMBu (y-amino butyric acid), NMVa (8-amino valeric acid), and NMCa (e-amino caproic acid) were synthesized with the condensation of methacryloyl chloride (Tokyo Kasei Chem. Indust., Tokyo, Japan) to the amino group which corresponded to the oamino acid (Tokyo Kasei) in the presence of NaOH (Nishiyama et al., 1994). Fig. 1 shows the chemical formulae of the 5 NMwA derivatives with different methylene lengths as a spacer group. Five mol% of the NMwA compounds were dissolved in water, and these solutions were used as dentin primer. Because of its low solubility, NMCa solution was prepared by the dissolution of 5 mol% NMCa in 30% (w/w) aqueous ethanol solution.

polyethylene ring (intemal diameter, 3.8 mm; depth, 2.0 mm) was fixed on the surface by sticky wax. After being primed with NM)A solution for 30 sec, the surface was air-dried for 15 sec, and the surface was then coated with a bonding agent (Chemically cured type, Clearfil New Bond®', Kuraray, Osaka, Japan). After being air-dried, a composite resin (Chemically cured type, Clearfil SC-IL3, Kuraray) was immediately injected into the cavity of the polyethylene mold. This preparation was left to harden at room temperature for 15 min. The polyethylene ring mold was then removed. The specimen was stored in water at 37°C. After 24 hrs, the specimen was then thermocycled in water between 4°C and 60°C for 20,000 cycles. The immersion time in each bath was 1 min. Next, the specimen was positioned on the tensile testing machine (DCS-2000, Shimadzu, Kyoto, Japan). The tensile bond strength of the composite resin to the dentin was measured at a cross-head speed of 2.0 mm/min. Ten specimens were used for each experiment.

Statistical Analyses
A one-way factorial analysis of variance (ANOVA) was used individually to analyze each factor in terms of the effects of the application of the thermocycle on the bond strength of the resin to the etched dentin pre-treated with the NMwA primer, and the effects of the methylene chain length in the NMwA molecule on the bond strength of the resin to the acid-etched dentin pre-treated with the NMwA derivatives. Scheffe's Comparison Test was used to evaluate statistical differences in the bond strength among the subgroup at p = 0.50%.

13C NMR Measurement of NMwA in the Presence of Dentinal Collagen
Immediately after extraction, the crown dentin of bovine teeth was cross-sectioned by a diamond cutter under a stream of water. The sliced dentin disks, after being frozen by liquid nitrogen, were reduced to powder by means of a ball agate mill (Type P-5, Fritsch, Idar-Oberstein, Germany). The dentin particles with diameters less than 75 1Lm were separated by means of a testing sieve (200 mesh) and demineralized by means of 40% phosphoric acid for 15 min at 0°C. We did this to avoid a structural change of the dentinal collagen from a triple helix to a random coil structure-by the neutralization heat which was associated by the demineralization of the hydroxyapatite with the acid. The insoluble dentinal collagen was decanted with de-ionized water for removal of any soluble dentinal component. This process was repeated until the pH value of the supematant solution increased to 6. The insoluble dentinal collagen fibers which were aggregated among themselves were reduced to powder by means of an agate mortar, and then dried at 20°C. Dentinal collagen powder (70 mg) was dispersed into 0.600 g of deuterium oxide (99.8 atom%, CEA, Paris, France) whose pH value had been previously adjusted to 1.0 by means of hydrochloric acid. Into this suspension, 6.73 x 10-5 mol of NMwA was then dissolved. The spin-lattice relaxation times, T1, of the carbons attributed to the NMoA molecule were observed in an EX-270 spectrometer (JEOL, Tokyo, Japan) operating at 67.80 MHz at 25°C (Nishiyama et al., 1998). The (180'-T-90'-5T1)n pulse sequence was used, where the accumulation and the delay times were 400 scans and 20 milliseconds, respectively. The NMR observation was conducted twice for each experiment. Both T1 values of the carbonyl carbons were then averaged.

RESULTS
or Without Dentinal Collagen Fig. 2 shows the 13C NMR spectra of the carbonyl region of the
5 species of NMwA in the absence or presence of dentinal collagen at pH = 1.6. When 70 mg of dentinal collagen was dispersed into 0.600 g of deuterium oxide with pH = 1.0, the pH value of the solution increased to 1.6. At this pH value, the carboxylic acid groups of the side-chain of the amino acid residue in the dentinal collagen molecule and the carboxylic acids of the NMwA molecules are considered to be in the undissociated state (-COOH). This was due to the pKa values of

13C NMR Spectra of NMwA With

OH3 CH2=C-C-N-( CH2)nr COOH
OH
Formula of N-methacryloyl-w-amino acid

(NMwA)

Adhesion Test
The surface of a bovine tooth was ground and polished, under a stream of water, by means of no. 100 and 600 silicon carbide papers. Thereafter, this fresh dentin surface was etched with 40% phosphoric acid for 30 sec and then rinsed under a stream of water. The acid-etched dentin surface was then air-dried for 15 sec. A

n=1, NMglycine; n=2, NMg-alanine; n=3, NMy-butylic acid, n=4, NM6-aminovaleric acid; n=5, NMc-caproic acid Figure 1. Formulae of N-methacryloyl-w-amino acids (NMwA) with various methylene chain lengths. The NMwA have an amide and a carboxylic acid group as functional groups.

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Nishiyama et al.
NMCa

J Dent Res 79(3) 2000

-COOH

-COOH
-CONH-

^-CONH-

NMVa

NMBu

NM.AJa

NMGIy

I

a

1

I

I

I

a

I

I

I

I

I

I

I

I

I

I

I

I

I

I

1180

175

170

180

175 174D ppm from TMS

degree of change in the mobility of the NMwA molecule. The T1 values of the 2 carbonyl carbons assigned to the amide and the carboxylic acid in the 5 species of NMwA molecules were determined with or without dentinal collagen (Table 1). When the dentinal collagen was dispersed into the NMoA solutions, the T1 values of the 2 carbonyl carbons decreased. These observations were attributed to the fact that the dentinal collagen molecule affected the mobility of the amide and carboxylic acid groups in the NMwA molecule. Fig. 3 shows the ratio of the T1 value of the previously discussed 2 carbonyl carbons in the presence of dentinal collagen to the result obtained in the absence of dentinal collagen. In the 5 NMwA compounds, the T1 ratio value tended to decrease with the increase in the number of methylene groups in the NMwA molecule. This indicated that the interaction among the amide and the carboxylic acid and the dentinal collagen molecule was generally stronger when the number of methylene groups increased. of Acid-etched Dentin Treated with NMvA Primer
on the Tensile Bond Strength

Effect of Thermocycle

(A)

(B)

Figure 2. Expanded 13C NMR spectra of the carbonyl region of 5 NMbA derivatives in the absence or presence of dentinal collagen. (A) NMR spectra of the 5 species of NMwA only. (B) NMR spectra of the 5 species of NMwA in the presence of dentinal collagen.
these carboxylic acids of the amino acid residue and that of the 5 species of NMwA: ranging from 3.9 to 4.3 (Stryer, 1975) or from 3.5 to 4.9 (Suzuki et al., 1997), respectively. The 13C NMR peaks of the carbonyl carbons attributed to the amide and the carboxylic acid in the NMwA molecule are shown in Figs. 2A and 2B. Here, the 13C NMR peaks attributed to the dentinal collagen were not observed in the NMR spectrum (2B), even though the dentinal collagen was dispersed in the NMNA solution. This suggested that the mobility of the carbons attributed to the dentinal collagen molecules was limited, because the triplehelix structure of the dentinal collagen was maintained. The addition of dentinal collagen resulted in very broad spectral peaks (Fig. 2B), of which the half-width became wider than those observed in the corresponding peaks when dentinal collagen was absent. This change was due to the decrease in the mobility of the NMoA molecule. The strength of the interaction between the 5 species of NMwA and the dentinal collagen appears to be determined by the

Table 2 shows the tensile bond strength of the resin to acidetched dentin that was treated with the NMwA primers before and after the thermocycle. The statistical analysis of the bond strength of the resin to the acid-etched dentin pre-treated with the NMwA with different methylene lengths is summarized in Table 3. When the NMwA primers were applied to the acid-etched dentin that had been air-dried, the bond strength increased from 5 MPa to a range of 9 to 16 MPa. The thermocycle caused a slight decrease in the bond strength of the resin to the acidetched dentin treated with the NMwA primers compared with those obtained before the thermocycling treatment. The methylene number-dependency of the bond strength, however, was maintained in either case.

DISCUSSION
The surface of the acid-etched dentin treated with HEMA primer was analyzed by Fourier transform infrared spectroscopy (Suzuki et al., 1994) and Fourier transform

J Dent Res 79(3) 2000

Interaction between NMoA and Dentinal Collagen Studied by 13C NMR
1.0

809

Raman spectroscopy (Xu et al., 1997). Suzuki et al. determined the amount of HEMA adsorbed onto the collagen. They noted that the adsorbed amount of HEMA was dependent on the HEMA concentration in the aqueous solution, and related to the bond strength of the resin to the acid-etched dentin primed with the HEMA. Xu et al. suggested that the carbonyl group of the ester in the HEMA molecule might interact with the dentinal collagen molecule. In this study, the interaction between the NMwA primers and the dentinal collagen molecule in an aqueous solution was studied by the 13C NMR technique, including the observation of spin-lattice relaxation times, T1. The T, value of the carbonyl carbons attributed to the amide group and the carboxylic acid group in the presence of dentinal collagen decreased. This result was achieved due to the formation of a hydrogen bonding from the amide group and the carboxylic acid group in the NMwA molecule to the carboxylic acids of the side-chain in the amino acid residues in the dentinal collagen molecule (Nishiyama et al., 1998). However, the degree of the decrease in the T1 value of the 2 carbonyl carbons was strongly dependent upon the number of methylene groups in the NMwA molecule. The reduction in the T, value of the carbonyl carbon attributed to the amide group in the NMwA molecule was greater than the T1 value of the carbonyl carbon attributed to the carboxylic acid group (Table l). The observed greater reduction in the T1 values of the carbonyl carbon of the amide group in the NMwA molecule in the presence of collagen suggested that the amide group may form a stronger interaction with the dentinal collagen molecule than may the carboxylic acid group in the NMwA molecule. To obtain the correlation between the strength of the interaction of the NMwA primer to the dentinal collagen and the bond strength of the resin to acid-etched dentin, we determined the bond strength of the resin to the acid-etched dentin treated with NMwA. When the NMwA primers were applied to the acid-etched dentin that had been air-dried, the bond strength increased from 5 MPa to a range of 9 to 16 MPa. This increase in the bond strength may be attributed to the formation of a hybrid layer comprised of resin and dentinal collagen fibers (Suzuki et al., 1997). This result was achieved due to the fact that the NMwA primer restored the collapsed collagenous layer during the air-drying process (Nishiyama et

o :-COOH
0

o

-CONH-

0 0

0.4 0.2

-

5 1 3 4 6 0 2 Number of methylene in the NMwA molecule
Figure 3. Relation of the number of methylene groups in the NMoA molecule and the ratio of the T1 value of the 2 carbonyl carbons of the amide and carboxylic acid. The T1 ratio is a ratio of the T, value with dentinal collagen to the T, value without dentinal collogen. T1 ratio =
al., 1994, 1995; Suzuki et al., 1998), and provided a microspace inside the collagenous layer where the bonding agent could diffuse. However, despite the 4-pm-thick hybrid layer that was formed at the interface with each NMwA primer, the bond strength varied independently of the number of methylene groups in the NMwA molecule (Suzuki et al., 1997). The thickness of the hybrid layer may not be directly related to the degree of the bond strength of the resin to acid-etched dentin. The thermocycle caused a slight decrease in the bond strength of the resin to the acid-etched dentin treated with the NMwA primers compared with those obtained before the thermocycling treatment.

Table 1. T, value (in sec) of the Carbons of the NMwA Molecule in the Absence or Presence of Dentinal Collagen Dentinal Collagen
NMGly
-a

Table 2. The Bond Strength of the Resin to the Acid-etched Dentin Treated with NMoA Primer Before and After Thermocycle

-CONH33.01 18.32 29.07 14.29 30.16 10.07 26.73 7.53 24.37
8.54

-COOH 23.44 14.19 23.18 15.83 22.90 13.57

Before Thermocycle

After Thermocycle
1.62
7.53 10.72 12.77 13.70 13.00

+

NMI3Ala
NMBu
NMVa

+ + + +

Non-treated Treated with NMwA
NMGly

5.00 9.15

(2.20)a
(1.73) (3.50) (2.79) (1.50)a (2.20)

(0.79)a
(4.03) (3.17) (3.68) (3.20)p (3.70)

NM,BAla
NMBu
NMVa NMCa

11.84
12.12 16.40 13.90

22.84
13.07 23.69 13.05

NMCa
a

- = NM,A only. + = NMwA in the presence of dentinal collagen.

Significant difference corresponded and after thermocycle (p = 0.50%).

to the bond

strength before

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Nishiyama

et a/.

J Dent Res 79(3) 2000
20
Va
o :1Before thermnocycle U After thermocycle

Table 3. The Statistical Analysis of the Bond Strength of Resin to Acid etched Dentin Primed with the NM(,)A Before and After Thermocycle
NMGly NMI3Ala
NMGly
NMBu

NMVa

NMCa

NMIt,Ala
NMBu NMVa NMCa

N N S

S

Before Thermocycle St, N' N N S N S S S N N S
-

0O m
c

Bu Ca

\

S N N
S

15 F

I

PAIa

c

(D

0)1

-0
S N N N
0) C 0)

Aher Thermocycle
NMGly
N
N S S S
N N N

NMI,Ala
NMBu NMVa NMCa

S N
N N

S N N
N

I-

5r

N = No significant difference corresponded to the bond strength of the resin primed with the NMWA derivatives with different methylene chain lengths at this level (p = 0.50%). S = Significant difference at this level.

.

0

0.2

0.4

4 showxs the rclationiship hctwscci thic axveriaged Ti ratuos ol tihc calhonyl carhon attributed to the amilide gr-oUp and the mican hond strcriiths ol thc Iesili to thc acid-ctched dentini pie-treated \xihi the NM(,)A primner. In the 5 NM(w)A dci Lati\ es. tic 2 bond streinethis befot-c aLid alter the Th thici-ilocyclc \\55-c pioroitionadl to tic / ratio withi the corielaittion coctlicicints, R' = ().771 and R- I).91 1. ItspezctixclV 'I'lie Streigtth ol' the intelaction hetxecen the NM(,)A pkinret aidi tlIe dentinal collagen rnolccnlC exilibihed a dii cct correlation xx ithi the bond strel-th ola thie iesin to the acidetchic(d dctini tricated itih the NMi(A pritcmr I'lic NMcaA mole0Cilc x IlicIhI iteracted w ith the dentinal Collagen mOICCulIC iiiay pionotc tIe h1ybridi/ation ol'tlie resini to dentinal collagen ti-et I-lUS tiiis iaciceascd the adliesiveniess at tlhc resin to the dcniiitial collalcern that wxas dcpncident upon the strcngthl ol the interaction hict\ccin the NM(\)A Molecule and the dentinal collaIgCH IIIolcClIIC. IleC quality of th lC hyrid layer that x as totlmeld on the suLbsulaice ola the intCihubulal dCentin on the dcntin adhesion xx as signil'icantly increased. Th'lese l'indiiigs a gcestid that. xxith this tecliii pic, a high-quiality hybr-id layer, \\hiichi xas alTecctcd h the strerngth ol tlc inteiaction betwecn the NM(,)A priicrit anid the Iciitinial cal lagen nioleeCIC, was lotilicd at the inter lace. F ilil thc icsults at the 1( NMIR aiialysis, thc adliesioti inicclian isnis ol tihc iesiii to the acid-etched deiitiii trcated with the NM(,)A )liiCIeS are postIulatecd as ltollows. The application ol the NM(,)A primner lacilitated the i-estolatitioll ol the cot1laecHtIs laxc1\r1lichi had1 collapsed dtii ilig thc air-drying process (Nishiyti)iaa (c/ a!., 1995). I LlrtieCi, the amiiiide group aiid oi tilC CalhO\Ylie acid glOUp ill the NMwA iiiolcculc h\dirogcn bonided xith ithe denttinaial collagen iiolecile. Sinace ilic iltCeIaction ol the NM(,)A moleCCulC plOliiOtCd thc ilhiidliiatiOil ol'tthe IC'Sill tO the dCilttill colla'i`il iiioteCuLe at thlc ittel-tlace it ciaticed the hliadincg ol the resiti to the dcritiinal collaucii tibet. aiid tlitis plovideld a noticeably highei hoilit sticithtli. I lC adhliesioi ola the resiii to ctciitiii is ani iniportaut Iliictioii

0.6 T1 ratio

0.8

1.0

Figure 4. Relation of the T, ratio of the carbonyl carbon of amide group in the NM(,,A molecule and the bond strength of the resin to the acid-etched dentin primed with NM(,)A. Gly, NMGly; [Aa, NMI,Ala; Bu, NMBu; Va, NMVa; Ca, NMCa; NM, N-methacryloyl (,)-. t1 the priimer's actioii, and in particLIlar. the priiiier lacilitates the lot1 iiatioin o a hlybrid lIyCI 1111tOh IgitS tCiIaiCtiO \ itli thlC o it j denitinal collagcn. 1 lie borid streiigthi to the detitini was itticld to be stroligly Cortrclatcd to tic streiigthi oa li is interaction atid m1Ya bhe rlated to tic ciqality oi the h) Iiyhid laxcI tlat is toirilcd.

ACKNOWLEDGMENTS
Part ol' this work was suppoited hy a grant-in-aid tor ScieintiFic Researci ir-oiii the Miinistr-y of EdLucation, SCiCICC, Sports ailic C'LiltLilC in Japaii (#t0tO6720)12) aniid hy a General Itclixictidl.l (iralnt lroii thc SuItiki Meriorial Girant ot Nilioii tJiiix\c sit\ School of l)entistrv at MaItSilco (#9S- 11()6).

REFERENCES
rickson RIL 1 989). Mechanism and clinical inpilicLitiorl. oat bondi 1triniatioii loi- i\ o (leitiii hobondiing gents .1/)m li 2:1 17-1 2. i Nakahayashi N, Pashlcy t)i ( 1()98). I lyhridlization ol deniatl hiarc tiSStICs Istic. c(l. iChlica'go IL QLlilltCSSClec Piiblishiiille ( o ctd. Nakabayashii N, Kojinia K. MasUiWLar ( 1 9S'). 11 e poalnotioll o0 acdIesion b1 al1ti haitioll ol Ilio1m011ci-S ilto tooth sti Ist CS. / iomic(/ S l ( Ahlle /Rcs 16 05-7'. Nishuivaiia N. Sl/iki K Yainarioto K. loi ic K. Neiroto K (I994). Actliesiveniess ol the prinicr u coitaliin ')-'unno ,vi(i dIcrix lis o dclctiii. *Jp .1 J li 1acr 13: 1-5. I Nishiviania N. Asakulll r. SU/Uik K. I loric K Ncinioto K ( 1)S). t1acts ot a siitiructiril clialno in Lolkttcic upoll hirlding to Coil(iitiOIlcii dclitiii h stLidiei hv ( NNMR. .1l iomcd i cr,'i I& 19:10171 1. Nishiyan1a N. SLIaLki K. \saktkl-l 1. Nakai 11 YISiladit S. Nciitioto K

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Interaction between NMwA and Dentinal Collagen Studied by 13C NMR

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(1996). The effects of pH on N-methacryloyl glycine primer on bond strength to acid-etched dentin. JBiomed Mater Res 31:379-384. Nishiyama N, Asakura T, Suzuki K, Sato T, Nemoto K (1998). Adhesion mechanisms of resin to etched dentin primed with N-methacryloyl glycine studied by 13C NMR. JBiomed MaterRes 40:458-463. Pashley DH (1990). Interactions of dentinal materials with dentin. Trans Acad Dent Mater 3:55-73. Stryer L (1975). Biochemistry. 1st rev. ed. San Francisco: W.H. Freeman and Company. Sugizaki J (1991). The effect of the various primers on the dentin adhesion of resin composite. Jpn J Conserv Dent 34:228-265. Suzuki K, Nakai H (1994). Adhesion of restorative resin to tooth substance. Treatment of acid-etched dentin by aqueous solution. Jpn JDent Mater 12:34-44.

Suzuki K, Nishiyama N, Nemoto K, Asakura T, Nakai H (1997). Effects of N-methacryloyl-w-amino acid primer pretreatment on bond strength of the resin to acid-etched dentin. J Biomed Mater Res 37:261-266. Suzuki K, Nishiyama N, Nemoto K, Torii Y, Inoue K (1998). Effects of N-methacryloyl amino acid applications on hybrid layer formation at the interface of intertubular dentin. J Dent Res 77:1881-1888. Van Meerbeek B, Inokoshi S, Braem M, Lambrechts P, Vanherle G (1992). Morphological aspects of the resin-dentin interdiffusion zone with different dentin adhesive systems. JDent Res 71:1530-1540. Xu J, Stangel I, Butler IS, Gilson DF (1997). An FT-Raman spectroscopic investigation of dentin and collagen surfaces modified by 2-hydroxyethylmethacrylate. JDent Res 76:596-601.