Journal of Oral Rehabilitation, 1991, Volume 18, pages 507—512
Effects of phosphoric acid and tannic acid on dentine collagen Y. O K A M O T O * t , J . D . H E E L E Y * , I . L .
DOGON*t
H . S H I N T A N I t * Forsyth Dental Center atid f Harvard School of Dental Medicitie, Boston. MA. U.S.A.. and ^Hiroshima University School of Dentistry, Hiroshima, Japan.
Summary We examined the effects of phosphoric acid, the most common enamel etchant in composite resin therapy, on dentine collagen. Dentine collagen pretreated with 7M phosphoric acid was shown to be more susceptible to trypsin digestion than untreated collagen. This susceptibility increased with increasing duration of exposure to the acid. The results indicate that phosphoric acid induces a conformational change in dentine collagen (denaturation or perturbation) similar to that observed with 0-39M HCl, which has a similar pH value (0-65). However, phosphoric acid-pretreated dentine collagen, when treated with tannic acid for 2h, became as resistant to tryptic digestion as intact dentine collagen. The present results suggest that tannic acid may work as a dentine conditioner in composite resin therapy, in view of the fact that phosphoric acid etchant is applied, either deliberately or inadvertently, to dentine, and would thus induce denaturation or perturbation of collagen. Introduction Acid etching of enamel has been widely accepted in dental practice as a means of increasing the bond strength between resin and tooth. Phosphoric acid is the most common enamel etchant, although it has been reported to have inflammatory properties (Stanley, Going & Chauncey, 1975; Eriksen & Leidal, 1979). Dentine consists primarily of type-1 collagen fibres that are calcified by deposition of apatite crystals within and around the collagen fibrillar network (Glimcher, 1981). Our previous study showed that phosphoric acid decreased the stability of type-1 collagen derived from bovine Achilles tendon (Okamoto, 1985), suggesting that phosphoric acid might have promoted the perturbation or denaturation of this collagen. Collagen from both dentine and tendon probably has the same qualitative distribution of intermolecular and intramolecular cross-links (Tanzer, 1976). It therefore seemed appropriate to study the effects of phosphoric acid on dentine collagen. Since changes in the structure of collagen may well be reflected by the extent of its susceptibility to trypsin (Neuman & Tytell, 1950), we examined the effects of phosphoric acid pretreatment on the susceptibility of dentine collagen to tryptic digestion. There is considerable interest in the biological function of plant tannins, which are able to inactivate both viruses and extracellular enzymes (White, 1956). Tannic acid is an interesting tanning substance which also has effects similar to those reported for Correspondence: Dr Y. Okamoto, Hiroshima University School of Dentistry, 1-2-3 Kasumi, Minami-ku, Hiroshima. 734 Japan.
507
508
Y. Okamoto et al.
tannins. We therefore investigated the effect of tannic acid on phosphoric acidpretrcated dentine collagen, on the premise that tannic acid may protect dentine collagen from the action of trypsin. Materials and methods Preparation of dentine collagen Unerupted incisors were obtained from the jaws of freshly killed, approximately 18month-old calves. The cheese-like enamel was scraped off the extracted teeth and they were washed with ice-cold saline solution. Coronal dentine was then powdered by means of a stainless steel pestle and mortar under liquid nitrogen. The resulting 20—40 mesh dentine powder was subsequently demineralized with 0-5 M E D T A in 0-05 M Tris-HCl, pH 7-5, at 4°C until the Ca content, as determined by atomic absorption spectrophotomctry (Perkin-Elmer 3030)*, reached a constant low level of ^0-03%. The insoluble residue was collected and washed thoroughly with ice-cold distilled water. The demineralized dentine powder was then extracted with three daily changes of 0-1 M acetic acid at 4°C for 3 days followed by three daily changes of 0-1 M disodium phosphate at 4°C. Thereafter, the residue was thoroughly washed with icecold distilled water. The partially purified dentine collagen was stored at —25°C. Treatment of dentine collagen with phosphoric acid Samples of dentine collagen (5mg ml ') were suspended in 7 M phosphoric acid (approximately 40% w/w) at 20°C, maintained with gentle shaking for 1, 10, 20 or 30 min and then filtered. As an experimental control for the effects of low pH alone, 0 39 M HCI was employed; the pH of both acids was approximately 0-65. The residues were thoroughly washed with ice-cold distilled water (20ml ml ' of acid used; repeated five times) and lyophilized. Dentine collagen that had been washed with water and then lyophilized served as the control. Treatment of dentine collagen with tannic acid Phosphoric acid-treated dentine collagen (5mg m p ' ) was suspended in 1% tannic acidt in 0-1 M phosphate buffer, pH 7-5, at 20°C for 5, 10, 30, 60 or 120min, or for 24 h. Samples were then thoroughly washed with ice-cold distilled water (20ml m P ' of acid used; repeated five times) and lyophilized. Digestion of dentine collagen with trypsin Samples of phosphoric acid- and/or tannic acid-treated dentine collagen (2mg m P ' ) were incubated with trypsin (TPCK treated)t in phosphate buffer, pH 8-0, at 37°C for 16h with gentle shaking, and then heated to 60°C for 30min (Kuboki et al, 1981). The enzyme/substrate weight ratio was 1:100. After centrifugation at 12tK)0xg for 15min, the supernatants were removed, lyophilized and aliquots were then hydrolysed in vacuo with 6 M HCI at IIOX for 20 h. Hydroxyproline was determined in the hydrolysates according to the method of Woessner (1961). The amount of collagen was estimated from the hydroxyproline content, assuming 102 residues/1000 total residues (Volpin & Veis, 1973). *Norwalk. Connecticut. U,S,A. t Sigma Chemical Co.. St Louis, Missouri, U,S,A.
Effects of phosphoric acid and tannic acid
509
Results
The effect of phosphoric acid or HCl on dentine collagen is shown in Table 1. Untreated dentine collagen was solubilized to a small extent (3%) by trypsin, while pre-exposure to phosphoric acid increased the solubility of collagen considerably. The amount of collagen solubilized by trypsin increased with increasing duration of pre-exposure to this acid. The amount of solubilized dentine collagen increased to 30%. 70% and 80% after treatment with phosphoric acid for 1, 10 or 20 min, respectively. In addition, dentine collagen pretreated with 0-39 M HCl exhibited similar susceptibility to trypsin. By contrast, when exposure to phosphoric acid was followed by tannic acid treatment, tryptic digestion of dentine collagen was reduced. This reduction became more
Table 1. Solubilization of dentine collagen pretreated with phosphoric acid or HCl by tryptic digestion Collagen solubiiizcd (%) Time of treatment
Control
Phosphoric acid
HCl
3-0 ±0-4*
0 1 10 20 30
33-1 :t3-4 66-6: 79-3 :t6-2 85-7 :t7-7
34-6 64-7 77-8 84-2
±2-6
±5-9 ±5-6 ±6-4
Values arc expressed as mean values ± S.D. of five experiments.
100
0
10 20 30 Time of phosphoric acid treatment (min)
Fig 1. The effects of phosphoric acid and tannic acid on the solubilization of dentine collagen by trypsin. Phosphoric acid- and tannic acid-treated dentine collagen was digested with trypsin at 37°C for 16 h. The data arc expressed as the average percentage of collagen solubilized by trypsin in five separate experiments: T, = treatment with tannic acid for t min, (• • ) = T,, ( • A) = T5,
(•
• ) = T,o. (A
A) = T3o, (•
• ) = T^,. (o
o) = T|2o.
510
y. Okamoto et al.
40
0
0-5 1 2 24 Time of tannic acid treatment (h)
Fig 2. Relationship between the duration of tannic acid treatment and the solubilization of dentine collagen. Collagen was treated with phosphoric acid for 1 min, and then treated with tannic acid for 5 min to 24 h.
marked with increasing duration of exposure to tannic acid, as shown in Figs 1 and 2. Although more than 85% of dentine collagen that had been pretreated with 7 M phosphoric acid for 30 min was solubilized by tryptic digestion, the amount of dentine eollagen solubilized, when treated with tannie aeid for 5 or 30 min after such phosphoric acid pretreatment, deereased to about 75% and 20%, respectively (Fig. 1). In addition, when treated with tannic acid for 5 or 30 min after pretreatment with 7 M phosphoric acid for 1 min, the amount of dentine eollagen that was solubilized decreased to about 28% and 12%, respectively (Fig. 2). After tannic acid treatment for 2 h, dentine collagen reeovered its normal resistance to tryptie digestion (Fig. 2). Treatment of samples with HCI followed by tannic acid exposure resulted in a similar reduction of solubilized collagen by tryptic digestion (data not shown). Discussion
Untreated dentine collagen is surprisingly resistant to trypsin treatment, as reported by Carmichael, Dodd and Veis (1977). However, the susceptibility of dentine eollagen to trypsin increased with increasing duration of exposure to 7 M phosphoric acid or 0-39M HCI. It is well established that denatured or perturbed eollagen exhibits decreased resistanee to non-specific proteolytic enzymatie digestion (Piez, 1967). The results of the present study therefore indieate that acid treatment, at the eoncentrations used, induces denaturation or perturbation of dentine eollagen. In view of the results obtained with 0-39M HCI, which has a similar pH to that of 7 M phosphoric acid, it seems likely that the phosphorie acid effeets are due to its low pH rather than its high phosphate eontent. In any ease, dentine eollagen that has been freshly exposed by phosphoric acid may well exhibit surfaee demineralization, following the removal of the smear layer. It would thus appear that a conformational change such as denaturation or perturbation may also occur to some extent in collagen exposed superficially in vivo, even though mineralized dentine eollagen would probably be less affected than demineralized
Effects of phosphoric acid and tannic acid
511
collagen, since hydroxyapatite released during acid etching may have a buffering effect. When the integrity of dentine is compromised by demineralization and collagen denaturation, a reduction in the strength ofthe dentine may occur (Causton & Johnson, 1979). It seems clear that the integrity of collagen in the surface layer of dentine is compromised by 7M phosphoric acid treatment. The amount of collagen solubilized by trypsin, when pretreated with phosphoric acid, decreased with increasing duration of exposure to tannie acid, which is a well-known tanning agent. Furthermore, phosphoric acid-pretreated dentine collagen was shown to regain its normal resistance to tryptie digestion after exposure to tannie acid for 2h. The mechanism whereby tannic acid protects dentine collagen from the action of trypsin is the subject of some speeulation at present. The acid may act as an astringent, changing the tertiary conformation of collagen and rendering it resistant to trypsin. It is also possible that this acid is able to inactivate trypsin directly, or to create unfavourable conditions for the action of trypsin by ehelating the catalytic cations (White, 1956). In addition, since tannic acid is unlikely to form stable intermolecular crosslinks in collagen under the physiological conditions employed in the study, it may form reversible, readily dissociated cross-links, and thus insolubilize dentine collagen, so preventing digestion by trypsin. Although the mechanism whereby tannic acid protects dentine collagen from the action of trypsin is unclear, it would appear to improve the resistance of dentine collagen etched with phosphoric acid to enzymatic digestion. Further studies using mineralized dentine collagen are in progress to investigate the potential benefits of tannic acid as a dentine conditioner in composite resin therapy. Acknowledgements
The authors are grateful to Dr S. Kashket for providing laboratory facilities. This study was supported in part by PHS grant RR-()54(S3 from the National Institute of Health. References C'AKMKHAHt. D..J.. DoDD, CM. & VKI.S. A. (1977) The solubilization of bone and dentin collagens by pepsin. Effect of cross-linkages and tion-collagcti components. Biochimua et Bioplnsica Acta, 491. 177. CAUSTON. B.E. & JOHNSON. N . W . (1979) Changes in the dentine of huirian teeth following extraction and their implication for in-vitro studies of adhesion to tootli substanee. Archives of Oral Biology, 24. 229. ERtKSEN. H.M. & LHIDAL. T.I, (1979) Monkey pulpal respotise to composite resin restorations in cavities treated with various cleansing agents. Scandinavian Journal of Dental Research. 87. 3(19. GLiMCHtK. M..I. (1981) On the form and function of bone: from molecules to organs. Wolffs law revisited. In: The Chemistry and Biology of Mineralized Connective Tissues (Ed. A. Vcis), p. 617. Elsevier & North Holland. New York. KuBOKt, Y.. TsuzAKi. M.. Liu. C.F. & MECHANIC G.L. (1981) Location of the intcrmolceular crosslinks in bovine dentin collagen, solubilization with trypsin and isolation of cross-link peptides containing dihydroxylysinonorleucinc and pyridinoline. Biochemical and Biophysical Research Communications, 102. 119. NHUMAN. R.E. & TYTHLL. A.A. (1930) Action of protcolytic enzymes on collagen. Proceedings ofthe Society for Experimental Biology and Medicine, 13, 409. OKAMOTO. Y. (1985) Influence of composite resin on organic matrix in dentine. Quintessence, 4. 1097. PiEz, K.A. (1967) Soluble collagen and the components resulting from its denaturation. In: Treatise on Collagen I (Ed. G.N. Ramachandran). p. 207. Academic Press. New York. STANLEY, H.R.. GOING. R.E. & CHAUNCEY, H . H . (1975) Human pulp response to acid pretreatment
512
y. Okamoto et al.
of dentin and to composite restoration. Journal of the American Dental Association. 91, 817. M.L. (1976) Crosslinking. In: Biochemistry of Collagen (Eds G.N. Ramaehandran & A.H. Reddi), p. 137. Plenum Press, New York. VoLPiN, D. & VEIS, A. (1973) Cyanogen bromide peptides from insoluble skin and dentin bovine eollagens. Biochemistry, 12, 1452. WHITE, T . (1956) The scope of vegetable tannin ehemistry. In: Symposium on the Chemistry of Vegetable Tannins (Ed. Society of Leather Trades' Chemists), p. 7. Croydon, London. WoESSNER, J.F. (1961) The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Archives of Biochemistry and Biophysics., 93, 440. TANZER,
Effects of phosphoric acid and tannic acid on dentine collagen Y. O K A M O T O * t , J . D . H E E L E Y * , I . L .
DOGON*t
H . S H I N T A N I t * Forsyth Dental Center atid f Harvard School of Dental Medicitie, Boston. MA. U.S.A.. and ^Hiroshima University School of Dentistry, Hiroshima, Japan.
Summary We examined the effects of phosphoric acid, the most common enamel etchant in composite resin therapy, on dentine collagen. Dentine collagen pretreated with 7M phosphoric acid was shown to be more susceptible to trypsin digestion than untreated collagen. This susceptibility increased with increasing duration of exposure to the acid. The results indicate that phosphoric acid induces a conformational change in dentine collagen (denaturation or perturbation) similar to that observed with 0-39M HCl, which has a similar pH value (0-65). However, phosphoric acid-pretreated dentine collagen, when treated with tannic acid for 2h, became as resistant to tryptic digestion as intact dentine collagen. The present results suggest that tannic acid may work as a dentine conditioner in composite resin therapy, in view of the fact that phosphoric acid etchant is applied, either deliberately or inadvertently, to dentine, and would thus induce denaturation or perturbation of collagen. Introduction Acid etching of enamel has been widely accepted in dental practice as a means of increasing the bond strength between resin and tooth. Phosphoric acid is the most common enamel etchant, although it has been reported to have inflammatory properties (Stanley, Going & Chauncey, 1975; Eriksen & Leidal, 1979). Dentine consists primarily of type-1 collagen fibres that are calcified by deposition of apatite crystals within and around the collagen fibrillar network (Glimcher, 1981). Our previous study showed that phosphoric acid decreased the stability of type-1 collagen derived from bovine Achilles tendon (Okamoto, 1985), suggesting that phosphoric acid might have promoted the perturbation or denaturation of this collagen. Collagen from both dentine and tendon probably has the same qualitative distribution of intermolecular and intramolecular cross-links (Tanzer, 1976). It therefore seemed appropriate to study the effects of phosphoric acid on dentine collagen. Since changes in the structure of collagen may well be reflected by the extent of its susceptibility to trypsin (Neuman & Tytell, 1950), we examined the effects of phosphoric acid pretreatment on the susceptibility of dentine collagen to tryptic digestion. There is considerable interest in the biological function of plant tannins, which are able to inactivate both viruses and extracellular enzymes (White, 1956). Tannic acid is an interesting tanning substance which also has effects similar to those reported for Correspondence: Dr Y. Okamoto, Hiroshima University School of Dentistry, 1-2-3 Kasumi, Minami-ku, Hiroshima. 734 Japan.
507
508
Y. Okamoto et al.
tannins. We therefore investigated the effect of tannic acid on phosphoric acidpretrcated dentine collagen, on the premise that tannic acid may protect dentine collagen from the action of trypsin. Materials and methods Preparation of dentine collagen Unerupted incisors were obtained from the jaws of freshly killed, approximately 18month-old calves. The cheese-like enamel was scraped off the extracted teeth and they were washed with ice-cold saline solution. Coronal dentine was then powdered by means of a stainless steel pestle and mortar under liquid nitrogen. The resulting 20—40 mesh dentine powder was subsequently demineralized with 0-5 M E D T A in 0-05 M Tris-HCl, pH 7-5, at 4°C until the Ca content, as determined by atomic absorption spectrophotomctry (Perkin-Elmer 3030)*, reached a constant low level of ^0-03%. The insoluble residue was collected and washed thoroughly with ice-cold distilled water. The demineralized dentine powder was then extracted with three daily changes of 0-1 M acetic acid at 4°C for 3 days followed by three daily changes of 0-1 M disodium phosphate at 4°C. Thereafter, the residue was thoroughly washed with icecold distilled water. The partially purified dentine collagen was stored at —25°C. Treatment of dentine collagen with phosphoric acid Samples of dentine collagen (5mg ml ') were suspended in 7 M phosphoric acid (approximately 40% w/w) at 20°C, maintained with gentle shaking for 1, 10, 20 or 30 min and then filtered. As an experimental control for the effects of low pH alone, 0 39 M HCI was employed; the pH of both acids was approximately 0-65. The residues were thoroughly washed with ice-cold distilled water (20ml ml ' of acid used; repeated five times) and lyophilized. Dentine collagen that had been washed with water and then lyophilized served as the control. Treatment of dentine collagen with tannic acid Phosphoric acid-treated dentine collagen (5mg m p ' ) was suspended in 1% tannic acidt in 0-1 M phosphate buffer, pH 7-5, at 20°C for 5, 10, 30, 60 or 120min, or for 24 h. Samples were then thoroughly washed with ice-cold distilled water (20ml m P ' of acid used; repeated five times) and lyophilized. Digestion of dentine collagen with trypsin Samples of phosphoric acid- and/or tannic acid-treated dentine collagen (2mg m P ' ) were incubated with trypsin (TPCK treated)t in phosphate buffer, pH 8-0, at 37°C for 16h with gentle shaking, and then heated to 60°C for 30min (Kuboki et al, 1981). The enzyme/substrate weight ratio was 1:100. After centrifugation at 12tK)0xg for 15min, the supernatants were removed, lyophilized and aliquots were then hydrolysed in vacuo with 6 M HCI at IIOX for 20 h. Hydroxyproline was determined in the hydrolysates according to the method of Woessner (1961). The amount of collagen was estimated from the hydroxyproline content, assuming 102 residues/1000 total residues (Volpin & Veis, 1973). *Norwalk. Connecticut. U,S,A. t Sigma Chemical Co.. St Louis, Missouri, U,S,A.
Effects of phosphoric acid and tannic acid
509
Results
The effect of phosphoric acid or HCl on dentine collagen is shown in Table 1. Untreated dentine collagen was solubilized to a small extent (3%) by trypsin, while pre-exposure to phosphoric acid increased the solubility of collagen considerably. The amount of collagen solubilized by trypsin increased with increasing duration of pre-exposure to this acid. The amount of solubilized dentine collagen increased to 30%. 70% and 80% after treatment with phosphoric acid for 1, 10 or 20 min, respectively. In addition, dentine collagen pretreated with 0-39 M HCl exhibited similar susceptibility to trypsin. By contrast, when exposure to phosphoric acid was followed by tannic acid treatment, tryptic digestion of dentine collagen was reduced. This reduction became more
Table 1. Solubilization of dentine collagen pretreated with phosphoric acid or HCl by tryptic digestion Collagen solubiiizcd (%) Time of treatment
Control
Phosphoric acid
HCl
3-0 ±0-4*
0 1 10 20 30
33-1 :t3-4 66-6: 79-3 :t6-2 85-7 :t7-7
34-6 64-7 77-8 84-2
±2-6
±5-9 ±5-6 ±6-4
Values arc expressed as mean values ± S.D. of five experiments.
100
0
10 20 30 Time of phosphoric acid treatment (min)
Fig 1. The effects of phosphoric acid and tannic acid on the solubilization of dentine collagen by trypsin. Phosphoric acid- and tannic acid-treated dentine collagen was digested with trypsin at 37°C for 16 h. The data arc expressed as the average percentage of collagen solubilized by trypsin in five separate experiments: T, = treatment with tannic acid for t min, (• • ) = T,, ( • A) = T5,
(•
• ) = T,o. (A
A) = T3o, (•
• ) = T^,. (o
o) = T|2o.
510
y. Okamoto et al.
40
0
0-5 1 2 24 Time of tannic acid treatment (h)
Fig 2. Relationship between the duration of tannic acid treatment and the solubilization of dentine collagen. Collagen was treated with phosphoric acid for 1 min, and then treated with tannic acid for 5 min to 24 h.
marked with increasing duration of exposure to tannic acid, as shown in Figs 1 and 2. Although more than 85% of dentine collagen that had been pretreated with 7 M phosphoric acid for 30 min was solubilized by tryptic digestion, the amount of dentine eollagen solubilized, when treated with tannie aeid for 5 or 30 min after such phosphoric acid pretreatment, deereased to about 75% and 20%, respectively (Fig. 1). In addition, when treated with tannic acid for 5 or 30 min after pretreatment with 7 M phosphoric acid for 1 min, the amount of dentine eollagen that was solubilized decreased to about 28% and 12%, respectively (Fig. 2). After tannic acid treatment for 2 h, dentine collagen reeovered its normal resistance to tryptie digestion (Fig. 2). Treatment of samples with HCI followed by tannic acid exposure resulted in a similar reduction of solubilized collagen by tryptic digestion (data not shown). Discussion
Untreated dentine collagen is surprisingly resistant to trypsin treatment, as reported by Carmichael, Dodd and Veis (1977). However, the susceptibility of dentine eollagen to trypsin increased with increasing duration of exposure to 7 M phosphoric acid or 0-39M HCI. It is well established that denatured or perturbed eollagen exhibits decreased resistanee to non-specific proteolytic enzymatie digestion (Piez, 1967). The results of the present study therefore indieate that acid treatment, at the eoncentrations used, induces denaturation or perturbation of dentine eollagen. In view of the results obtained with 0-39M HCI, which has a similar pH to that of 7 M phosphoric acid, it seems likely that the phosphorie acid effeets are due to its low pH rather than its high phosphate eontent. In any ease, dentine eollagen that has been freshly exposed by phosphoric acid may well exhibit surfaee demineralization, following the removal of the smear layer. It would thus appear that a conformational change such as denaturation or perturbation may also occur to some extent in collagen exposed superficially in vivo, even though mineralized dentine eollagen would probably be less affected than demineralized
Effects of phosphoric acid and tannic acid
511
collagen, since hydroxyapatite released during acid etching may have a buffering effect. When the integrity of dentine is compromised by demineralization and collagen denaturation, a reduction in the strength ofthe dentine may occur (Causton & Johnson, 1979). It seems clear that the integrity of collagen in the surface layer of dentine is compromised by 7M phosphoric acid treatment. The amount of collagen solubilized by trypsin, when pretreated with phosphoric acid, decreased with increasing duration of exposure to tannie acid, which is a well-known tanning agent. Furthermore, phosphoric acid-pretreated dentine collagen was shown to regain its normal resistance to tryptie digestion after exposure to tannie acid for 2h. The mechanism whereby tannic acid protects dentine collagen from the action of trypsin is the subject of some speeulation at present. The acid may act as an astringent, changing the tertiary conformation of collagen and rendering it resistant to trypsin. It is also possible that this acid is able to inactivate trypsin directly, or to create unfavourable conditions for the action of trypsin by ehelating the catalytic cations (White, 1956). In addition, since tannic acid is unlikely to form stable intermolecular crosslinks in collagen under the physiological conditions employed in the study, it may form reversible, readily dissociated cross-links, and thus insolubilize dentine collagen, so preventing digestion by trypsin. Although the mechanism whereby tannic acid protects dentine collagen from the action of trypsin is unclear, it would appear to improve the resistance of dentine collagen etched with phosphoric acid to enzymatic digestion. Further studies using mineralized dentine collagen are in progress to investigate the potential benefits of tannic acid as a dentine conditioner in composite resin therapy. Acknowledgements
The authors are grateful to Dr S. Kashket for providing laboratory facilities. This study was supported in part by PHS grant RR-()54(S3 from the National Institute of Health. References C'AKMKHAHt. D..J.. DoDD, CM. & VKI.S. A. (1977) The solubilization of bone and dentin collagens by pepsin. Effect of cross-linkages and tion-collagcti components. Biochimua et Bioplnsica Acta, 491. 177. CAUSTON. B.E. & JOHNSON. N . W . (1979) Changes in the dentine of huirian teeth following extraction and their implication for in-vitro studies of adhesion to tootli substanee. Archives of Oral Biology, 24. 229. ERtKSEN. H.M. & LHIDAL. T.I, (1979) Monkey pulpal respotise to composite resin restorations in cavities treated with various cleansing agents. Scandinavian Journal of Dental Research. 87. 3(19. GLiMCHtK. M..I. (1981) On the form and function of bone: from molecules to organs. Wolffs law revisited. In: The Chemistry and Biology of Mineralized Connective Tissues (Ed. A. Vcis), p. 617. Elsevier & North Holland. New York. KuBOKt, Y.. TsuzAKi. M.. Liu. C.F. & MECHANIC G.L. (1981) Location of the intcrmolceular crosslinks in bovine dentin collagen, solubilization with trypsin and isolation of cross-link peptides containing dihydroxylysinonorleucinc and pyridinoline. Biochemical and Biophysical Research Communications, 102. 119. NHUMAN. R.E. & TYTHLL. A.A. (1930) Action of protcolytic enzymes on collagen. Proceedings ofthe Society for Experimental Biology and Medicine, 13, 409. OKAMOTO. Y. (1985) Influence of composite resin on organic matrix in dentine. Quintessence, 4. 1097. PiEz, K.A. (1967) Soluble collagen and the components resulting from its denaturation. In: Treatise on Collagen I (Ed. G.N. Ramachandran). p. 207. Academic Press. New York. STANLEY, H.R.. GOING. R.E. & CHAUNCEY, H . H . (1975) Human pulp response to acid pretreatment
512
y. Okamoto et al.
of dentin and to composite restoration. Journal of the American Dental Association. 91, 817. M.L. (1976) Crosslinking. In: Biochemistry of Collagen (Eds G.N. Ramaehandran & A.H. Reddi), p. 137. Plenum Press, New York. VoLPiN, D. & VEIS, A. (1973) Cyanogen bromide peptides from insoluble skin and dentin bovine eollagens. Biochemistry, 12, 1452. WHITE, T . (1956) The scope of vegetable tannin ehemistry. In: Symposium on the Chemistry of Vegetable Tannins (Ed. Society of Leather Trades' Chemists), p. 7. Croydon, London. WoESSNER, J.F. (1961) The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Archives of Biochemistry and Biophysics., 93, 440. TANZER,
