The distribution of hydroxyproline in the dentine of carious human teeth

Archs oral Bid. Vol. 17, pp. 127-135, 1972. Pergamon Press. Printed in Great Britain.

THE DISTRIBUTION OF HYDROXYPROLINE IN THE DENTINE OF CARIOUS HUMAN TEETH R. S. LEVINE* Department of Oral Pathology, Dental School, St. Mary’s Row, Birmingham B4 6NN Summary-Loss of hydroxyproline from dentine matrix was used as a means of correlating the pattern of attack in the collagenous matrix due to caries, with the pattern of demineralization as shown by microradiography. Samples of dentine, 150 pm in diameter, were removed from undecalcified sections of ca.rious teeth, along lines passing through the lesion and through adjacent sound dentine. The hydroxyproline content of the samples was estimated using a method working in the range O-80 ng and the results expressed as pg/mm3 of dentine. This basis of expression gave results unaffected by density variations or contamination of the carious dentine by foreign, non-collagenous material. Results indicated an increased collagen content in sclerotic dentine beneath the lesion and at the demineralization front, compared with corresponding sound dentine, falling towards the surface of the lesion, where a concentration of intact collagen lower than that found in corresponding normal dentine was indicated. The results suggest that the organic matrices of peritubular dentine and intratubular deposits in sclerosed tubules are collagenous.

CARIOUSdestruction of dentine involves an attack on both the mineral phase and the organic matrix. It is generally agreed that partial demineralization usually precedes bacterial invasion, which in turn precedes complete breakdown of the collagenous matrix (FRANK, 1967). The chemical changes in the collagenous matrix of carious dentine compared with normal dentine have been investigated by ARMSTRONG (1961) who found, amongst other changes in amino acid composition, a reduction of 22 per cent in the hydroxyproline content on a weight basis of artificially demineralized organic residue of carious dentine, when compared with a similar weight of demineralized sound dentine. Unfortunately, comparison of the composition of a pathological tissue with that of sound tissue on such a weight basis can give results which are of limited use and difficult to interpret. Comparison of amino acid concentration per unit weight of dry, undecalcified dentine would be misleading, as unit weight of carious dentine would represent a larger volume of tissue than unit weight of sound dentine and could consequently contain more organic matrix. Expression of concentration per unit weight of artificially decalcified organic residue could lead to an apparent reduction in some or all amino acid concentrations in carious dentine as compared with sound dentine, due to contamination of the tissue by proteinaceous or non-proteinaceous organic material during the carious process. However, if the chemical composition is expressed on a basis of weight per unit volume of tissue, then a true and meaningful comparison of tissues of differing density can be made. * Present address: Department of Oral Medicine, University of Manchester, Manchester Ml5 6FH. 127

128

R. S.

LEVINE

Apart from semi-quantitative results obtained by histochemical techniques and ultrastructural observation, little is known of the distribution of changes in the organic matrix of dentine subject to carious attack. The object of the present investigation was to correlate the pattern and degree of attack upon the collagenous matrix as indicated by hydroxyproline loss, with the pattern of attack upon the mineral phase as displayed by microradiography. Hydroxyproline was chosen because it is almost unique to collagen (HARKNESS,1961) and a reduction in concentration of hydroxyproline per unit volume of carious dentine, would provide an index of attack upon the collagenous matrix, free from errors due to density variations or non-collagenous contamination. METHODS Extracted human teeth with established dentinal lesions were stored unfixed at 4°C and 100 per cent humidity, then cut longitudinally through the lesion to provide plano-parallel sections between 80-130 pm thick (Bovrs, 1968). The thickness of the section was then determined and the radiodensity was recorded by microradiography, using the method of CXXPER (1968a).With the aid of a micro-punchdevice (COOPER,1968b), disc-shaped samples, 150 pm in diameter, were removed from each section along a line from pulpal dentine outward through the lesion to the surface (SL), as determined from the microradiographic appearance. A second series of samples was punched through adjacent sound dentine from pulpal dentine to the amelo-dentinal junction (ADJ). The volume of each sample was determined from the mean cross-sectional area of the hole left in the section. From the lack of microradiographic evidence for density disturbances around punch holes, it was concluded that the volume of the hole was equal to the original volume of tissue removed. This relationship was also found to be true for samples removed from soft carious dentine. The analytical method used has been previously described (LEVINE,1971). Following demineralization, the organic residue of each sample was hydrolysed and the hydroxyproline content, using a method working in the range O-80 ng, was estimated in duplicate. The results were expressed as rg hydroxyproline per mm3 of tissue. RESULTS

A total of 9 carious permanent and deciduous teeth were investigated, one section from each tooth being analysed. All sections showed a similar pattern of results, a typical example is illustrated below. A section of a grossly carious upper permanent molar is shown in Fig. 1, a microradiograph of the section is shown in Fig. 2 and the section after sampling is shown in Fig. 3. Series I of eight samples (l-8) was punched through the lesion and numbered from the pulp to the surface of the lesion (S.L.). Series II of seven samples (9-15) was punched through adjacent sound dentine and numbered from pulp to ADJ. Results of the analysis of the samples are shown in Table 1 and are displayed as spatial distribution graphs, together with microradiographic density tracings across the area sampled, in Figs. 4a and b. It was demonstrated previously (LEVINE, 1971) that a hydroxyproline gradient exists from the plaque to the ADJ. Consequentlyin this investigationit was necessary to compare hydroxyproline concentration in the lesion (Series I) with that at the corresponding point on the gradient in sound dentine of the same tooth (Series II). Such a comparison indicated that the hydroxyproline level increased from the sclerotic dentine beneath the lesion to a point a little beyond the demineralization front, reaching a level higher than that found in the corresponding sound dentine. The

THE DISTRIBUTION OF HYDROXYPROLINE IN CARIOUSDENTINE

129

TABLE 1. HYDROXYPROLINECONCENTRATIONOF SAMPLESREMOVEDFROM SECTION SHOWN IN FIG. 1 &g/mm3). Series I Sample Hydroxyproline

(rg/mm3)

1

2

3

4

5

6

7

8

29.0

34.8

44.5

53.4

59.8

51.1

55.8

45.3

Series II

Sample

9

Hydroxyproline

&g/mm3)

37.5

10

11

12

13

14

15

43.6

42.0

45.7

45.7

50.7

51.7

hydroxyproline concentration then fell to a level below that of the corresponding sound dentine at the surface of the lesion. In order to analyse the results statistically, 5 zones were recognised in each series. Series I-curious

Zone Zone Zone Zone

A B C D

Zone E

dentine

Pulpal dentine beneath the lesion. The sclerotic or hypermineralized area beneath the lesion. The demineralization front, as determined from the radiodensity tracing. The centre of the lesion, the point mid-way between the demineralization front and the surface of the lesion. The surface of the lesion.

Series II- -sound dentine

Zone A-E

in series II corresponded

topographically

with those in series I.

The mean hydroxyproline concentration found in each zone is displayed in Fig. 5. The differences between corresponding zones in the lesion (I) and sound dentine (II) for each section were compared using paired t-tests. The results of this statistical analysis indicated a significant increase in concentration of Zones B and C in the lesion of 21 per cent and 30 per cent respectively when compared with sound dentine. DISCUSSION

The present findings of a mean reduction in hydroxyproline concentration of only 4 per cent (Zone E) in carious dentine on a volume basis, may be compared to the reduction of 22 per cent observed by ARMSTRONG (1961) on a weight basis. The effect of the different modes of expression must be examined. Whereas Armstrong expressed his results as g amino acid per 100 g organic residue after complete, artificial decalcification, the present results refer to the concentration per unit volume of existing tissue. Possible explanations for the reduction may be considered with respect to the different basis of expression. (a) Contamination of curious dentine by a non-proteinaceous organic substance. This A.O.B. 17/1--r

130

R. S. LEVINE M

I\\_/__ 4

SERIES I

0 PULP

1'0

2'0 H.M.

2’6 S.L.

FIG. 4a.

would reduce all amino acids present in the organic residue by an equal proportion when expressed on a weight basis. However, since Armstrong’s results account for almost all the organic residue as amino acids, such contamination must be small. The results of the present investigation are unaffected by such contamination. (b) Contamination of curious dentine by non-collagenous proteins, e.g. of bacterial or salivary origin. Since such proteins are highly unlikely to contain hydroxyproline, the proportion of this amino acid per unit weight of organic residue would be lower. A contamination of organic matrix by one quarter of its own weight of foreign protein could reduce the hydroxyproline concentration by 20 per cent in itself. Again the results of the present investigation are unaffected. (c) Destruction or mod$cation of hydroxyproline units in situ. This would result in a lower concentration using either basis of expression. (d) Liberation from the organic matrix of soluble peptide fractions containing relatively high concentrations of hydroxyproline. Since soluble elements of the samples were removed prior to analysis in both investigations, the effect would be a reduction in hydroxyproline concentration using either basis of expression. (e) So&ion of a fraction of the collagenous matrix having a normal amino acid

THE DISTRIBUTION

OF HYDROXYPROLINE

I

I

0 PULP

IN CARIOIJS

DENTINE

131

SERIESII

I 1

1

1'0

2’9.0

I

3:o 3-l A,D.J.

KM.

FIG. 4b

FIGS. 4a and b. Distribution of hydroxyproline and corresponding microradiographic density in the section shown in Figs. 1-3. Samples of series I (Fig. 4a) were taken through the lesion from the pulpal dentine to the surface (S.L.). Samples of series II (Fig. 4b) were taken through sound dentine from the pulpal region to the amelodentinal junction (A.D.J.). The arrow indicates increasing radiodensity.

composition, e.g. as in gelation of collagen. This would result in no apparent change in hydroxyproline content per unit weight of organic residue, but would give a corresponding reduction in concentration per unit volume of tissue. Since the hydroxyproline concentration of mammalian collagen is relatively constant (EASTOE, 1967), it would appear from the above analysis that the reduction in hydroxyproline concentration found in the present investigation must represent an attack upon some or all of the collagenous matrix. Further, the increase in hydroxyproline concentration in Zones B and C can only be due to an increased collagen deposition in the tissue. Because of this evidence for increased deposition of collagen in sclerosed dentine, any comparison of sound and carious dentine must take into account the possibility that the collagen concentration of the carious dentine was raised, relative to corresponding sound dentine, before it became carious. Adding the mean increase in hydroxyproline concentration found in Zone C to that found in sound dentine in Zone E, it would appear possible that a true reduction in the order of 25 per cent could have occurred in the tissue at the surface of the lesions, as illustrated in Fig. 6.

132

R. S.LEVINE ZONES IN DENTINE

CAVITY

60504030-

P
OJ PULP

A.D,J./S,L,

0 l

-

CARIOUS DENTINE, SERIES CORRESPONDING

I

SOUND DENT!@

SERIES

II

FIG.5.Mean hydroxyproline distribution in carious dentine and corresponding sound dentine related to topographical zones. S-significant difference. NS-difference not significant. O--Carious dentine, series I. @-Corresponding sound dentine, series II.

The nature of the increased collagen content of Zones B and C may now be disdiscussed in the light of established concepts of dentine physiology and ultrastructural observation. BRADFORD (1960) suggested that dentinal sclerosis as seen beneath carious lesions and areas of attrition and as an age change in the roots of teeth, is a result of total or partial occlusion on the dentinal tubules by centripetal deposition of peritubular dentine. Although this concept has generally been supported by ultrastructural observation (FRANK, 1967), it sometimes appears that the organic matrix within some occluded tubules is much more dense than the fine fibrillar network seen in normal peritubulardentine (NALBANDIAN,GONZALES and SOGNNAES,~~~O;JOHNSON,TAYLOR and BERMAN, 1969).

THE DISTRIBUTION

OF HYDROXYPROLINE

IN CARIOUS

133

DENTINE

80 -

“$ ‘, z.

60 -

PULP

58’1 55.5 04X

25x

s. L.

FIG. 6. A comparison of the mean hydroxyproline distribution in the carious lesion (broken line), with that in sound dentine (continuous line). The hatched area represents the reduction in concentration that could occur if the carious tissue had become sclerosed prior to attack. The cross-hatched area represents the reduction found in comparison with corresponding normal dentine.

The chemical nature of the fibrillar matrix of peritubular dentine has been the subject of much controversy. JOHANSENand PARKS (1962) demonstrated fine fibres showing collagen-like crossbanding in the demineralized peritubular zone of sound dentine, while FRANK (1966) elegantly displayed collagen fibres in the peri-odontoblastic space within dentinal tubules. However, TAKUMA(1967) and JOHNSON,TAYLOR and BERMAN(1969) found the peritubular matrix to be composed of a fine network of structureless fibrils, with no evidence for a collagenous nature. Furthermore, BLAKE (1958) claimed that peritubular dentine is an example of calcification in a non-collagenous matrix while HABERMANet al. (1967) came to a similar conclusion for intratubular calcification. In carious dentine, a remarkable persistence of the collagenous intertubular matrix is seen (JOHANSENand PARKS, 1961) while, from ultrastructural observation, TAKUMA et al., (1967) report an increase in the density of organic material within peritubular dentine of partially demineralized carious dentine. A granular organic residue, seen in the lumen of such tubules by TAKUMAet al., was also noted by SYMONS(1970), who suggested that it represents a calcifiable matrix which in some tubules may have calcified before being decalcified by the carious process. It would thus appear that, as part of the sclerotic reaction to caries, an increased amount of organic material is laid down within the tissue and especially in the peritubular and intratubular areas. This process may continue even after the first signs of demineralization (cf. Fig. 4a). In the light of present findings, it may be concluded that the material is collagenous in nature and it is suggested that it arises owing to the increased deposition of normal or modified peritubular dentine which consequently supports the view that this material has a fibrous matrix and is composed largely or totally of collagen. Acknowledgement-The author is grateful to Dr. S. L. ROWLEYfor his advice in the preparation of this communication.

134

R. S. LEVINE R&urn&--La perte d’hydroxyleproline de la matrice dentinaire est utilis& comme un moyen de comparer le mode d’attaque de la matrice collagenique par la carie, avec le type de demineralisation, demontre par microradiographie. Des echantillons de dentine, de 150 pm de diametre, sont prelevts sur des coupes non decalcifiees de dents cariees le long de lignes passant a travers la lesion et la dentine saine adjacente. Le contenu en hydroxyleproline des echantillons est determinee a l’aide dune methode sensible de O-80 ng et les resultats sont exprimes en pg/mm3 de dentine. Ces resuhats ne sont ainsi pas modifies par des variations de densite ou par une contamination de dentine cariee par du materiel &ranger, non collagbnique. Une augmentation du contenue en collagene est notee dam la dentine sclerotique, situ&e sous la lesion et au niveau du front de demineralisation, si on la compare avec la dentine saine. Vers la surface de la lesion, une concentration moins &levee de collagtne est notee par rapport a la dentine normale correspondante. Ces resultats indiquent que les matrices organiques de la dentine pericanaliculaire et intracanaliculaire, dans les canalicules scleroses, sont de nature collagenique.

Zusammenfassung-Der Hydroxyprolinverlust aus der Dentinmatrix wurde als MaBstab bentitzt, urn den Verlauf des kariosen Angriffes auf die Kollagenmatrix mit der mikroradiographisch nachweisbaren Demineralisation zu vergleichen. Dentinproben im Durchmesser von 150 pm wurden unentkalkten Schnitten karioser Zahne entlang einer Linie, die durch die kariiise L&ion und durch das benachbarte gesunde Dentin verlauft, entnommen. Der Hydroxyprolingehalt der Proben wurde mit Hilfe einer Methode im Bereich zwischen O-80 ng bestimmt und die Ergebnisse als pg/mm3 Dentin ausgedriickt. Diese Ergebnisse wurden nicht durch Variationen der Dichte oder durch Kontamination des kariosen Dentins mit fremdem, nichtkollagenem Material beeintrlchtigt. Die Ergebnisse lassen einen erhijhten Kollagengehalt im sklerotischen Dentin unter der L&ion und an der Demineralisationsfront im Vergleich zum entsprechenden gesunden Dentin erkennen. Die Werte fallen zur Oberflache der Llsion hin ab, wo die Konzentration intakten Kollagens niedriger als im entsprechenden normalen Dentin gefunden wurde. Die Ergebnisse legen nahe, da8 die organische Matrix von Ablagerungen peritubularen und intratubularen Dentins in den sklerosierten Tubuli kollagener Natur sind.

REFERENCES ARMSTRONG,W. G. 1961. A quantitative comparison of amino acid composition of sound dentine, carious dentine and the collagenase resistant fraction of carious dentine. Archs oral Biol. 5, 115124. BLAKE,G. C. 1958. The peritubular translucent zone in human dentine. Br. dent. J. 104, 57-64. Bovrs, S. C. 1968. The preparation of plano-parallel sections of calcified tissues. Br. dent. J. 125, 502-505. BRADFORD,E. W. 1960. The dentine, a barrier to caries. Br. dent. J. 109, 387-398. COOPER,W. E. G. 1968a. A microchemical, microradiographic and histological investigation of amelogenesis in the pig. Archs oral Biol. 13, 2748. COOPER,W. E. G. 1968b. An apparatus for the removal of 25 pm diameter samples from tooth sections. Archs oral Biol. 13, 835-837. EASTOE,J. E. 1967. In Structural and Chemical Organisation of the Teeth, (edited by MILES,A. E. W.). Vol. II, p. 285. Academic Press, London. FRANK, R. M. 1966. Etude au microscope Clectronique de l’odontoblaste et du canalicule dentaire humain. Archs oral Biol. 11, 179-199. FRANK, R. M. 1967. Ultrastructure of the teeth from the point of view of mineralisation, demineralisation and remineralisation. Int. dent. J. 17, 661-683. HABERMAN,S., BOUSCHOR,C., MATTHEWS,L. and SAUNDERS,E. 1967. Fine structure of soft carious dentine. Oral Surg. 24, 216-223. HARKNESS,R. D. 1961. Biological functions of collagen. Biol. Rev. 36, 399463.

THE DISTRIBUTIONOF HYDROXYPROLINEIN CARIOUSDENTINE

135

JOHANSEN, E. and PARKS, H. F. 1961. Electron-microscopic observations on soft carious human dentine. J. dent. Res. 40, 235-248. JOHANSEN, E. and PARKS, H. F. 1962. Electron-microscopic observations on sound human dentine.

Archs oral Biol. 7, 185-l 92. JOHNSON, N. W., TAYLOR, B. R. and BERMAN, D. S. 1969. Response of deciduous dentine to caries studied by correlated light and electron-microscopy. Caries Rex 3, 348-368. LEVINE, R. S. 1971. The distribution of hydroxyproline in sound human coronal dentine. Archs oral

Biol. 16, 473-478. NALBANDIAN,J., GONZALES, F. and SOGNNAES, R. F. (1960). Sclerotic age changes in root dentine of human teeth as observed by optical, electron and x-ray microscopy. J. dent. Res. 39, 598-607. SYMONS,N. B. B. 1970. Electron-microscopic study of human carious dentine. Archs oral Biol. 15, 239-251. TAKUMA, S. 1967. Ultrastructure of dentinogenesis. In Structural and Chemical Organisation of the Teeth, (edited by MILES, A. E. W.). Vol. I, p. 359-366. Academic Press, London. TAKUMA, S., SUNOHMRA, H., SEKIGUCHI, K. and EGAWA, I. 1967. Electron microscopy of carious lesions in human teeth. Bull. Tokyo dent. Coil. 8, 143-165.

PLATE

I OVERLEAF

THE DISTRIBUTION

OF HYDROXYPROLINE

IN CARIOUS

DENTINE

FIG. I. Section of grossly carious molar in reflected light. FIG. 2. Microradiograph FIG. 3. Section

shown

of section shown in Fig. I.

in Fig. 1 after sampling. Additional holes are due to the loss of samples. Transmitted light.

PLATE

1

A.O.B. f.p. 136