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In-vivo incorporation of [3H]-proline into rabbit dental pulp collagen
An hr orcrl &of. Vol. 24, pp. 613 lo 615 Papamon PressLtd 1979Printed m Great Britam
IN-VII/O
INCORPORATION OF [3H]-PROLINE RABBIT DENTAL PULP COLLAGEN
INTO
C. A. SHUTTLEWORTH, J. L. WARD and P. N. HIRSCHMANN* Department of Medical Biochemistry, Medical School, Oxford Road, Manchester M 13 9PT, and Turner Dental School, University of Manchester, England
Summary-Collagen metabolism of rabbit dental pulp was studied by following the incorporation of [3H]-proline into pulp collagen. The specific activity of hydroxyproline was studied in various collagen fractions in rabbit incisor and molar teeth of different ages. The specific activity of the collagen fractions was consistently higher in incisor than in molar pulps at all ages, except for the pepsin-resistant fraction. In the molar at most ages, the pepsin-resistant fraction had a specific activity equal to, or greater than, that in the fractions solubilized by either acid or pepsin. The results demonstrate the high metabolic activity of dental pulp, and rapid incorporation of new collagen into the insoluble fibre network.
INTRODUCTION Dental pulp differs from other connective
tissues in the composition of its extracellular matrix, and thus Its function. Histologically, the extracellular matrix of pulp appears to be relatively homogeneous; fine reciculin fibres predominate and collagen fibres are few I Han, 1968). We have previously reported on both the extractability (Shuttleworth, Ward and Hirschmann, 1978a) and the nature (Shuttleworth, Ward and Hirschmann, 1978b) of the collagen moieties in dental pulp. Linde (1973) and Orlowski (1977) demonstrated the high metabolic activity of the matrix components of the pulp. We have followed the in vivo incorporation of [3H)-proline into the various collagen fractions of rabbit tooth pulp and examined the changes in incorporation during post-natal growth and development.
Na citrate buffer and radioactivity in the isolated fractions analysed as described by Shuttleworth and Forrest (1975). Hydroxyproline was estimated colorimetrically by the method of Woessner (1961).
MATERIALS AND METHODS
Rabbits, ranging in age from 2 to 14 months (except for a preliminary experiment with a 3-yr-old rabbit) were given an intraperitoneal injection of 5OO@i of [‘HI-proline (Radiochemicals, Amersham, Bucks.). The animals were killed 24 h later, molar and incisor teeth were extracted and the dental pulps removed. Three animals were used for each age group; the pulps from each rabbit were analysed separately. Pulps were extracted as previously described (Shuttleworth et al., 1978a) to give neutral salt-soluble collagen (NSSC), acid-soluble collagen (ASC), pepsinsoluble (PS) and pepsin-insoluble fractions (PI). Isolated fractions were dissolved in 0.1 mol/litre acetic acid, dialysed against the same solution and freezedried. Amino acid analysis
Collagen samples were hydrolysed in 6M-HCl at 105°C for 24 h, and radioactive proline and hydroxyproline separated on the Jeol 6AH amino acid analyser using SO-cm column, at 53°C and 6O”C, 0.21 M .* Present address: The Dental Hospital at Leeds, Clareodon
Way, Leeds LS2 9LU, England. 613
RESULTS The preliminary
experiment
with a mature rabbit
indicated that radioactive proline was incorporated into the pulp collagens in vivo (Fig. 1). The specific
activity of the neutral salt-soluble collagen from both incisor and molar pulps was higher than that of the acid-soluble fraction, a situation known to occur in other tissues (Jackson and Bentley, 1960; Klein, 1969; Heikkinen, 1968). After digestion with pepsin, the pepsin-resistant fraction had a higher specific activity than the pepsin-soluble one. The specific activity of the collagen fractions was higher in incisor than molar teeth, except for the pepsin-resistant fraction which was higher in molars. This finding is consistent with a rapid incorporation of newly-synthesized collagen into the pepsin-soluble fibre network; and supports our previous suggestion (Shuttleworth et al., 1978a) that the collagen of molar pulps becomes cross-linked faster than that of incisors. The composition of the collagen fractions varied with age (Table 1). The highest specific activity was associated with the neutral salt-soluble fraction in both molar and incisor teeth. Except for the 1Cmonth rabbits, the fraction resistant to pepsin had a higher specific activity than that solubilized by pepsin at all stages in both molar and incisor pulp. In the incisor, the specific activity of the acid-soluble extract was higher than that of the pepsin-soluble fraction at all ages studied, and significantly higher than the pepsin-insoluble fraction at 2 and 14 months. There was no such relationship in the molar pulps, where, apart from the 2-month-old rabbits, the specific activity of the pepsin-insoluble fraction was equal to, or greater than, that of the pepsin-soluble and acid-soluble fractions. These findings indicate that collagen once synthesized is rapidly incorporated into the fibre
C. A. Shuttleworth, J. L. Ward and P. N. Hirschmann
614
Collogen
IB
Molar
III
Incisor
fraction
Fig. 1. Histogram showing the specific activity of neutral salt-soluble collagen (N), acid-soluble collagen (A), pepsin-soluble (S) and pepsin-insoluble (I), isolated from molar and incisor teeth of 3-yr-old rabbit after injection of 500 &i [3H]-proline. network, and that this process is independent of the animal’s stage of development. In the three soluble collagen fractions, the specific activity was higher in incisor than molar pulps at all the ages studied; in contrast, the specific activity of the pepsin-resistant fraction varied with age. Thus it was higher in incisors at 2 and 4 months; at subsequent stages, on the other hand, it was higher in the molars (Table 1). DISCUSSION
We showed previously that the highest proportion of the total hydroxyproline is in the pepsin-soluble fraction (Shuttleworth et al., 1978a) therefore, in view of the low specific activity found in this fraction in the present experiment, the bulk of this material is probably derived from mature collagen fibre breakdown Table 1. Effect of age on the incorporation Age (months)
NSSC*
ASCt PSI PIfi
Molar Incisor Molar Incisor Molar Incisor Molar Incisor
Collagen types: * Neutral salt-soluble. t Acid-soluble. $ Pepsin-soluble. 5 Pepsin-insoluble.
rather than from collagen in the process of being incorporated into the fibre network. In our previous study, we also showed that the solubility of collagen in the pulps of bovine molars decreases as development proceeds. In our present study, the differences noted between molar and incisor teeth in the rabbit which reflect the different functional demands on the two types of teeth. There are differences in the turnover of collagen in the pulps of rat incisor (Orlowski, 1977) and molar teeth (Orlowski and Doyle, 1976), but Uitto and Antila (1971) found no significant differences in the rate of collagen biosynthesis between rabbit molar and incisor pulps. Our present results show that higher total specific activity of collagen from incisor teeth than that from molars. Rat molars erupt at a far slower rate than the incisors (Ness, 1964; Schour and Massler, 1949), but of [3H]-proline into rabbit dental pulp
4 2 6 Specific activity x lo-* dpm/pmole hydroxyproline (mean * SD) 48.5 + 128.5 f 27.1 1 34.2 f 6.7 f 9.7 1 10.8 + 17.1 +
8.3 30.4 5.2 6.3 1.8 3.4 1.2 6.4
18.1 + 6.2 55.5 f 18.6 7.1 f 2.4 11.3 + 2.6 5.1 f 2.4 6.1 + 2.8 12.0 k 4.2 16.9 & 5.6
49.0 f 126.6 + 12.5 f 18.0 ) 3.7 + 4.5 + 15.9 + 14.8 f
9.6 25.4 3.5 3.0 1.6 2.3 4.9 5.1
14
78.1 + 150.7 + 5.1 + 26.9 + 6.0 + 10.5 + 7.4 + 5.6 +
23.2 28.4 1.2 9.7 4.0 2.8 2.6 1.5
Dental
pulp collagen synthesis
information on rabbit molars is lacking. The difference in total specific activity in rabbit pulps could reflect a difference in the eruption rate. The eruption rate of rat molars decreases markedly once these teeth are functional : subsequent eruption, compensating for attrition, is by. means of deposition of dental cementurn (Ness, 1964). The fibre content of dental pulp increases with age, ah hough the number of fibroblasts decreases and the blood vessel walls become much thicker (Seltzer and Bender, 1975; Quigley, 1973; Bernick and Nedelman, 1975). There is, however, disagreement as to the precise location and nature of the fibrosis. Bhussry (1968) fo.md an increase in the fibrous component of both human and rat dental pulps during growth and development. There were increases in the concentration of argyrophilic reticulin fibres and in the thickness of ble,od vessel walls. The increase in collagen fibres was less well marked. Cahen (1970) noted the presence of two collagen fibre types in human dental pulp, one of 75.0 nm dia. and the other, unstriated, of 15.0 nm. The number of the larger tibres increased in older pulps. Hernick and Nedelman (1975) localized the fibrosis to the blood vessel walls and nerve fibre sheaths of human pulp. Our finding, that in 3-yr-old rabbits there is rapid incorporation of newly synthesized collagen into the insoluble fibre network, supports this morphological evidence of an increase in fibre content in the older tissues. Orlowski and Doyle (1976) found that rat dental pulps, both incisor and molar, had a higher specific activity than other oral tissues such as periodontal ligament. We have not studied other oral tissues but histochemical studies (Goggins and Fullmer, 1966, 1967), glycosaminolglycan turnover (Linde, 1973) and our present findings on the incorporation of [ ‘HI-proline into collagen all demonstrate the high metabolic activity of dental pulp. We showed the presence of type I and III collagen in dental pulp (Shuttleworth et al., 1978b). However, wt: could not examine the relative rates of synthesis of these two collagen species using the techniques employed here. REFERENCES
S. and Nedelman C. 1975. The effect of aging on The human dental pulp. J. Endodont. 1, 88-94. Bhussry B. R. 1968. Modification of the dental pulp organ during development and aging. In: Biology of the Dental Pulp Organ (Edited by Finn S. B.) pp. 145-168. UniverBernick
sity of Alabama
Press, Birmingham.
615
Cahen P. M. 1970. Electronmicroscopic study of human dental pulp. J. dent. Res. 46, 688. Abstract. Goggins J. F. and Fullmer H. N. 1966. Dehydrogenase histochemistry of the rat molar pulp. Archs oral Biol. 11, 136551370. Goggins J. R. and Fullmer H. M. 1967. Enzyme histochemistry of the rat molar pulp. Archs orat Biol. 12, 639644. Han S. S. 1968. The fine structure of cells and intercellular substances. In: Biology of the Dental Pulp Organ (Edited by Finn S. B.) pp. 1033144. University of Alabama Press, Birmingham. Heikkinen E. 1968. Transformation of rat skin collagen. With special reference to the aging process. Acta physiol. stand. Suppl. 317, l-69. Jackson D. S. and Bentley J. P. 1960. Significance of soluble collagens. J. biophys. biochem. Cytol. 7, 3743. Klein L. 1969. Reversible transformation of fibrous collagen to a soluble state in viuo. Proc. Narn Acad. Sci. 62, 920-921. Linde A. 1973. Glycosaminoglycan turnover and synthesis in the rat incisor pulp. Scund. J. dent. Res. 181, 1455154. Ness A. L. 1964. Movement and forces in tooth eruption. Adc. oral Biol. 1, 33-75. Orlowski W. A. 1977. The turnover of collaeen in dental pulp of rat incisors. 3. dent. Res. 56, 43744vO. Orlowski W. A. and Doyle J. L. 1976. Collagen metabolism in the pulps of rat teeth. Archs oral Biol. 21, 391-392. Quigley M. B. 1973. The Biology of the Human Dental Pulp (Edited Siskin M.) p. 114. C. V. Mosby, St. Louis. Schour I. and Massler M. 1949. The Rut in Lahoratorq Incestigarion 2nd edn (Edited by Farris E. J. and Griffiths J. Q.) p. 118. Lippincott, London. Seltzer S. and Bender I. B. 1975. The Dental Pulp: Biological Considerations in Dental Procedures. 2nd edn. Lippincott, Philadelphia. Shuttleworth C. A. and Forrest L. 1975. Changes in guinea-pig dermal collagen during development. Eur. J. Biochem. 55, 391-395. Shuttleworth C. A., Ward J. L. and Hirschmann P. N. 1978a. Extraction of collagen fractions from bovine and rabbit dental follicle, papilla and pulp. Archs oral Biol. 23, 235-236. Shuttleworth C. A., Ward J. L. and Hirschmann P. N. 1978b. The presence of type 111 collagen in the developing tooth. Biochim. biophys. Acta 535, 348-355. Sodek J. 1977. A comparison of the rates of synthesis and turnover of collagen and non-collagen proteins in adult rat periodontal tissues and skin using a microassay. Archs oral Biol. 22, 655-665. Uitto V. J. and Antila R. 1971. Characterization of collagen biosynthesis in rabbit dental pulp in vitro. Actu odont. stand. 29, 609-617. Woessner J. G. 1961. The determination of hydroxyproline in tissues and protein samples containing small proportions of the amino acid. Archs Biochem. Biophys. 93, 440-447.
IN-VII/O
INCORPORATION OF [3H]-PROLINE RABBIT DENTAL PULP COLLAGEN
INTO
C. A. SHUTTLEWORTH, J. L. WARD and P. N. HIRSCHMANN* Department of Medical Biochemistry, Medical School, Oxford Road, Manchester M 13 9PT, and Turner Dental School, University of Manchester, England
Summary-Collagen metabolism of rabbit dental pulp was studied by following the incorporation of [3H]-proline into pulp collagen. The specific activity of hydroxyproline was studied in various collagen fractions in rabbit incisor and molar teeth of different ages. The specific activity of the collagen fractions was consistently higher in incisor than in molar pulps at all ages, except for the pepsin-resistant fraction. In the molar at most ages, the pepsin-resistant fraction had a specific activity equal to, or greater than, that in the fractions solubilized by either acid or pepsin. The results demonstrate the high metabolic activity of dental pulp, and rapid incorporation of new collagen into the insoluble fibre network.
INTRODUCTION Dental pulp differs from other connective
tissues in the composition of its extracellular matrix, and thus Its function. Histologically, the extracellular matrix of pulp appears to be relatively homogeneous; fine reciculin fibres predominate and collagen fibres are few I Han, 1968). We have previously reported on both the extractability (Shuttleworth, Ward and Hirschmann, 1978a) and the nature (Shuttleworth, Ward and Hirschmann, 1978b) of the collagen moieties in dental pulp. Linde (1973) and Orlowski (1977) demonstrated the high metabolic activity of the matrix components of the pulp. We have followed the in vivo incorporation of [3H)-proline into the various collagen fractions of rabbit tooth pulp and examined the changes in incorporation during post-natal growth and development.
Na citrate buffer and radioactivity in the isolated fractions analysed as described by Shuttleworth and Forrest (1975). Hydroxyproline was estimated colorimetrically by the method of Woessner (1961).
MATERIALS AND METHODS
Rabbits, ranging in age from 2 to 14 months (except for a preliminary experiment with a 3-yr-old rabbit) were given an intraperitoneal injection of 5OO@i of [‘HI-proline (Radiochemicals, Amersham, Bucks.). The animals were killed 24 h later, molar and incisor teeth were extracted and the dental pulps removed. Three animals were used for each age group; the pulps from each rabbit were analysed separately. Pulps were extracted as previously described (Shuttleworth et al., 1978a) to give neutral salt-soluble collagen (NSSC), acid-soluble collagen (ASC), pepsinsoluble (PS) and pepsin-insoluble fractions (PI). Isolated fractions were dissolved in 0.1 mol/litre acetic acid, dialysed against the same solution and freezedried. Amino acid analysis
Collagen samples were hydrolysed in 6M-HCl at 105°C for 24 h, and radioactive proline and hydroxyproline separated on the Jeol 6AH amino acid analyser using SO-cm column, at 53°C and 6O”C, 0.21 M .* Present address: The Dental Hospital at Leeds, Clareodon
Way, Leeds LS2 9LU, England. 613
RESULTS The preliminary
experiment
with a mature rabbit
indicated that radioactive proline was incorporated into the pulp collagens in vivo (Fig. 1). The specific
activity of the neutral salt-soluble collagen from both incisor and molar pulps was higher than that of the acid-soluble fraction, a situation known to occur in other tissues (Jackson and Bentley, 1960; Klein, 1969; Heikkinen, 1968). After digestion with pepsin, the pepsin-resistant fraction had a higher specific activity than the pepsin-soluble one. The specific activity of the collagen fractions was higher in incisor than molar teeth, except for the pepsin-resistant fraction which was higher in molars. This finding is consistent with a rapid incorporation of newly-synthesized collagen into the pepsin-soluble fibre network; and supports our previous suggestion (Shuttleworth et al., 1978a) that the collagen of molar pulps becomes cross-linked faster than that of incisors. The composition of the collagen fractions varied with age (Table 1). The highest specific activity was associated with the neutral salt-soluble fraction in both molar and incisor teeth. Except for the 1Cmonth rabbits, the fraction resistant to pepsin had a higher specific activity than that solubilized by pepsin at all stages in both molar and incisor pulp. In the incisor, the specific activity of the acid-soluble extract was higher than that of the pepsin-soluble fraction at all ages studied, and significantly higher than the pepsin-insoluble fraction at 2 and 14 months. There was no such relationship in the molar pulps, where, apart from the 2-month-old rabbits, the specific activity of the pepsin-insoluble fraction was equal to, or greater than, that of the pepsin-soluble and acid-soluble fractions. These findings indicate that collagen once synthesized is rapidly incorporated into the fibre
C. A. Shuttleworth, J. L. Ward and P. N. Hirschmann
614
Collogen
IB
Molar
III
Incisor
fraction
Fig. 1. Histogram showing the specific activity of neutral salt-soluble collagen (N), acid-soluble collagen (A), pepsin-soluble (S) and pepsin-insoluble (I), isolated from molar and incisor teeth of 3-yr-old rabbit after injection of 500 &i [3H]-proline. network, and that this process is independent of the animal’s stage of development. In the three soluble collagen fractions, the specific activity was higher in incisor than molar pulps at all the ages studied; in contrast, the specific activity of the pepsin-resistant fraction varied with age. Thus it was higher in incisors at 2 and 4 months; at subsequent stages, on the other hand, it was higher in the molars (Table 1). DISCUSSION
We showed previously that the highest proportion of the total hydroxyproline is in the pepsin-soluble fraction (Shuttleworth et al., 1978a) therefore, in view of the low specific activity found in this fraction in the present experiment, the bulk of this material is probably derived from mature collagen fibre breakdown Table 1. Effect of age on the incorporation Age (months)
NSSC*
ASCt PSI PIfi
Molar Incisor Molar Incisor Molar Incisor Molar Incisor
Collagen types: * Neutral salt-soluble. t Acid-soluble. $ Pepsin-soluble. 5 Pepsin-insoluble.
rather than from collagen in the process of being incorporated into the fibre network. In our previous study, we also showed that the solubility of collagen in the pulps of bovine molars decreases as development proceeds. In our present study, the differences noted between molar and incisor teeth in the rabbit which reflect the different functional demands on the two types of teeth. There are differences in the turnover of collagen in the pulps of rat incisor (Orlowski, 1977) and molar teeth (Orlowski and Doyle, 1976), but Uitto and Antila (1971) found no significant differences in the rate of collagen biosynthesis between rabbit molar and incisor pulps. Our present results show that higher total specific activity of collagen from incisor teeth than that from molars. Rat molars erupt at a far slower rate than the incisors (Ness, 1964; Schour and Massler, 1949), but of [3H]-proline into rabbit dental pulp
4 2 6 Specific activity x lo-* dpm/pmole hydroxyproline (mean * SD) 48.5 + 128.5 f 27.1 1 34.2 f 6.7 f 9.7 1 10.8 + 17.1 +
8.3 30.4 5.2 6.3 1.8 3.4 1.2 6.4
18.1 + 6.2 55.5 f 18.6 7.1 f 2.4 11.3 + 2.6 5.1 f 2.4 6.1 + 2.8 12.0 k 4.2 16.9 & 5.6
49.0 f 126.6 + 12.5 f 18.0 ) 3.7 + 4.5 + 15.9 + 14.8 f
9.6 25.4 3.5 3.0 1.6 2.3 4.9 5.1
14
78.1 + 150.7 + 5.1 + 26.9 + 6.0 + 10.5 + 7.4 + 5.6 +
23.2 28.4 1.2 9.7 4.0 2.8 2.6 1.5
Dental
pulp collagen synthesis
information on rabbit molars is lacking. The difference in total specific activity in rabbit pulps could reflect a difference in the eruption rate. The eruption rate of rat molars decreases markedly once these teeth are functional : subsequent eruption, compensating for attrition, is by. means of deposition of dental cementurn (Ness, 1964). The fibre content of dental pulp increases with age, ah hough the number of fibroblasts decreases and the blood vessel walls become much thicker (Seltzer and Bender, 1975; Quigley, 1973; Bernick and Nedelman, 1975). There is, however, disagreement as to the precise location and nature of the fibrosis. Bhussry (1968) fo.md an increase in the fibrous component of both human and rat dental pulps during growth and development. There were increases in the concentration of argyrophilic reticulin fibres and in the thickness of ble,od vessel walls. The increase in collagen fibres was less well marked. Cahen (1970) noted the presence of two collagen fibre types in human dental pulp, one of 75.0 nm dia. and the other, unstriated, of 15.0 nm. The number of the larger tibres increased in older pulps. Hernick and Nedelman (1975) localized the fibrosis to the blood vessel walls and nerve fibre sheaths of human pulp. Our finding, that in 3-yr-old rabbits there is rapid incorporation of newly synthesized collagen into the insoluble fibre network, supports this morphological evidence of an increase in fibre content in the older tissues. Orlowski and Doyle (1976) found that rat dental pulps, both incisor and molar, had a higher specific activity than other oral tissues such as periodontal ligament. We have not studied other oral tissues but histochemical studies (Goggins and Fullmer, 1966, 1967), glycosaminolglycan turnover (Linde, 1973) and our present findings on the incorporation of [ ‘HI-proline into collagen all demonstrate the high metabolic activity of dental pulp. We showed the presence of type I and III collagen in dental pulp (Shuttleworth et al., 1978b). However, wt: could not examine the relative rates of synthesis of these two collagen species using the techniques employed here. REFERENCES
S. and Nedelman C. 1975. The effect of aging on The human dental pulp. J. Endodont. 1, 88-94. Bhussry B. R. 1968. Modification of the dental pulp organ during development and aging. In: Biology of the Dental Pulp Organ (Edited by Finn S. B.) pp. 145-168. UniverBernick
sity of Alabama
Press, Birmingham.
615
Cahen P. M. 1970. Electronmicroscopic study of human dental pulp. J. dent. Res. 46, 688. Abstract. Goggins J. F. and Fullmer H. N. 1966. Dehydrogenase histochemistry of the rat molar pulp. Archs oral Biol. 11, 136551370. Goggins J. R. and Fullmer H. M. 1967. Enzyme histochemistry of the rat molar pulp. Archs orat Biol. 12, 639644. Han S. S. 1968. The fine structure of cells and intercellular substances. In: Biology of the Dental Pulp Organ (Edited by Finn S. B.) pp. 1033144. University of Alabama Press, Birmingham. Heikkinen E. 1968. Transformation of rat skin collagen. With special reference to the aging process. Acta physiol. stand. Suppl. 317, l-69. Jackson D. S. and Bentley J. P. 1960. Significance of soluble collagens. J. biophys. biochem. Cytol. 7, 3743. Klein L. 1969. Reversible transformation of fibrous collagen to a soluble state in viuo. Proc. Narn Acad. Sci. 62, 920-921. Linde A. 1973. Glycosaminoglycan turnover and synthesis in the rat incisor pulp. Scund. J. dent. Res. 181, 1455154. Ness A. L. 1964. Movement and forces in tooth eruption. Adc. oral Biol. 1, 33-75. Orlowski W. A. 1977. The turnover of collaeen in dental pulp of rat incisors. 3. dent. Res. 56, 43744vO. Orlowski W. A. and Doyle J. L. 1976. Collagen metabolism in the pulps of rat teeth. Archs oral Biol. 21, 391-392. Quigley M. B. 1973. The Biology of the Human Dental Pulp (Edited Siskin M.) p. 114. C. V. Mosby, St. Louis. Schour I. and Massler M. 1949. The Rut in Lahoratorq Incestigarion 2nd edn (Edited by Farris E. J. and Griffiths J. Q.) p. 118. Lippincott, London. Seltzer S. and Bender I. B. 1975. The Dental Pulp: Biological Considerations in Dental Procedures. 2nd edn. Lippincott, Philadelphia. Shuttleworth C. A. and Forrest L. 1975. Changes in guinea-pig dermal collagen during development. Eur. J. Biochem. 55, 391-395. Shuttleworth C. A., Ward J. L. and Hirschmann P. N. 1978a. Extraction of collagen fractions from bovine and rabbit dental follicle, papilla and pulp. Archs oral Biol. 23, 235-236. Shuttleworth C. A., Ward J. L. and Hirschmann P. N. 1978b. The presence of type 111 collagen in the developing tooth. Biochim. biophys. Acta 535, 348-355. Sodek J. 1977. A comparison of the rates of synthesis and turnover of collagen and non-collagen proteins in adult rat periodontal tissues and skin using a microassay. Archs oral Biol. 22, 655-665. Uitto V. J. and Antila R. 1971. Characterization of collagen biosynthesis in rabbit dental pulp in vitro. Actu odont. stand. 29, 609-617. Woessner J. G. 1961. The determination of hydroxyproline in tissues and protein samples containing small proportions of the amino acid. Archs Biochem. Biophys. 93, 440-447.