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Extraction of collagen fractions from bovine and rabbit dental follicle, papilla and pulp
SHORT
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EXTRACTION OF COLLAGEN FRACTIONS FROM BOVINE AND RABBIT DENTAL FOLLICLE, PAPILLA AND PULP C. A. SHUITLEWORTH, J. I.. WARD and P. N. HIRSCHMANN Department of Medical Biochemistry and Turner Dental School, University of Manchester, Manchester Ml3 9PT. England Summary-Age-related changes in collagen solubility were found in bovine dental papilla and dental pulp. Increased amounts of insoluble collagen in mature dental pulp were associated with completion of root formation. In the pulp of the continually erupting rabbit incisor tooth. increase in the amount of newly synthesized collagen led to an increase in soluble
collagen.
The dental pulp and its precursor. the dental papilla, are loose connective tissues specialized for the formation and subsequent maintenance of dentine. These functions are related, in their turn. to the properties of the extracellular components of this tissue. Histologically, the extracellular matrix of pulp appears to be relatively homogeneous; fine reticulin fibres predominate and collagen fibres with 64nm periodicity are few (Han, 1968). Orlowski (1974) confirmed by chemical analyses that there are only small amounts of collagen in the bovine dental pulp (16.8 per cent); he also demonstrated that the pulp contains large amounts of acid mucopolysaceharides and glycoproteins in the proportions usually associated with fine collagenous fibres (Schwartz, 1966). We have been concerned with the characterisation and quantitation of the collagen species present in dental pulp, and have demonstrated the presence of Type I and III collagens in dental papilla (Hirschmann and Shuttleworth, 1977). The work of Nowack et al. (1976) appears to justify equating Type III collagen with what is identified histologically as reticulin. The relative proportions of collagen and reticulin in connective tissues appear to vary with development, maturation and ageing. Bovine tissues were obtained fresh from the abattoir; using mid-term foetal jaws. the dental papilla and dental follicle were separated from incisor and molar teeth in which root formation had not commenced The teeth were retained and a portion of mandibular bone was also taken from the same animals. Mature pulps were obtained from permanent molar teeth. Rabbit pulps from erupted incisor and molar teeth were kindly supplied by Dr. A. Smith, Department of Dental Sciences. University of Liverpool. The teeth and bone samples were demineralized in IO per cent EDTA. pH 7.5, and then treated in an identical manner to the pulp samples. Bone, dentine. pulp and follicle were washed with copious amounts of water, chopped into fine pieces and treated with
I mol/litre NaCI. 0.05 mol/litre tris. pH 7.5. to extract neutral salt-soluble collagen (NSSC) until the super-
natant was clear. The residue was then treated with 0.1 mol/litre acetic acid to extract acid-soluble collagen (ASC). The insoluble residues remaining aRer this extraction were digested with pepsin (enzyme: collagen, I: IO) at 4°C for 24 h and a pepsin-solubilized and a pepsin-resistant fraction obtained. The various collagen fractions were dialysed against 0.1 mol/litrc acetic acid, freeze-dried and the amount of collagen in each extract was estimated by hydroxyproline analysis using the method of Wocssner (1961). The various extracts showed differences in the extractability of collagen from each of the tissues examined. As expected. demineralized bone and dentine were insoluble in neutral-salt and acid solutions. and only small amounts of collagen could be solubilized from these tissues by pepsin treatment. The resi-
dues remaining after these three treatments accounted for 96.4 per cent of dentine collagen and 90.1 per cent of mandibular bone collagen. Table I compares the solubility of collagen from the soft connective tissues in the two animal species. The two foetal tissues, dental follicle and dental papilla. differed in their response to the various extractants. When these tissues were treated with salt and acid solutions. more collagen (45 per cent of the total) was extracted from papilla than from follicle (28 per cent of the total). On the other hand. more collagen from papilla (17.8 per cent) was resistant to pepsin-treatment than from follicle (6.2 per cent). Second, comparing dental papilla and mature dental pulp, collagen is less readily extracted from pulp. We consider that this is due to an age-related increase in cross-linking and fibre organisation. In both papilla and mature pulp. similar percentages of the insoluble collagen were solubilizcd by pepsin. indicating that the proportion of pepsin-soluble fibres increases when root formation is complete. In rabbit, there were marked dilfcrences in solubility between incisor and molar pulps. In the molar pulp, 56.3 per cent of total collagen was pcpsin-solublc and only 7.4 per cent pepsin-resistant. whcrcas, in incisors almost 90 per cent was neutral-salt and acid soluble. and less than I per cent pepsin-resistant.
235
C. A. Shuttleworth.
236
Table
I. Solubility
J. L. Ward
and P. N. Hirschmann
of collagen. expressed as a percentage of the total collagen. in follicle. papilla and pulp Bovine
Rabbit Incisor
Molar
Molar Pulp
Pulp
78.9
Follicle
Papilla
Pulp
5.1
23.4
8.6
27.6
23.2 65.3 6.2
22.1 36.6 17.8
I.1 55.1 35.2
8.8 56.3
Neutral-salt soluble collagen Acid-soluble collagen Pepsin-soluble Pepsin residue
Uitto and Antila (1971) found more collagen in rabbit molar pulps than in those from incisors: however they found no significant differences in the rate of collagen biosynthesis between the two tissues. Their findings and ours suggest that. in molars, pulp collagen becomes cross-linked faster than in incisors. Orlowski and Doyle (1976) injected labelled proline into rats and found that the specific activity of hydroxyproline was higher in the continuously growing inesor than in the molars, which are of limited growth. supporting our interpretation of the increased proportion of NSSC in the incisor, as due to a higher rate of collagen synthesis associated with the continuous eruption of this tooth. Our findings show that the solubility of collagen in the molar pulp ducrcases as dcvclopmcnt proceeds. This appears not to occur in the rabbit incisor. The low solubility of collagen in bovine molar pulp is probably due to grcatcr incorporation of soluble collagen into the insoluble network. In the continually erupting rabbit incisor tooth. the greater proportion of collagen extracted with neutral-salt and acid solutions is presumably related to more newly synthcsizcd collagen. The ditfercnces in collagen solubility arc presumably in turn related to the different functional demands on thcsc two teeth.
7.4
IO.0 10.2 0.8
REFERENCES
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. 103-144. University of Alabama Press. Birmingham. Hirschmann P. N. and Shuttleworth C. A. 1977. Collagen heterogeneity in the developing tooth. /. dmr. Rcs.. In press. IADR. Brit. Div. Abs 44. Nowack H.. Gay S.. Wick G.. Becker V. and Timpl R. 1976. Preparation and use in immunology of antibodies specific for Type 1 and III collagen and procollagen. J. immun. Methods 12. 117-124. Orlowski W. A. 1974. Analysis of collagen, acid mucopolysaccharides and glycoproteins in bovine and porcine pulp. Archs oral Biol. 19. 255-258. Orlowski W. A. and Doyle J. L. 1976. Collagen metabolism in the pulps of rat teeth. Archs orul tliol. 21. 391-392. Schwartz W. 1966. Morphology and differentiation of conncctivr tissue fibrcs. In: Connecrire Tissue Symposium (Edited by Tunbridgc R. E.) pp. 144-156. L)lackwcll. Oxford. Uitto V. and Antila R. 1971. Charactcrisation biosynthesis in rabbtt dental pulp in cirro. scund. 29. 609-6 I 7.
of collagen Acru odonr
Wocssncr J. G. 1961. The dctcrmination of hydroxyprolinc in tissurs and protein. Samples containing small proportions of the amino acid. Archs Biochem. Biophrs. . 93. 440-447.
COMMUNICATIONS
EXTRACTION OF COLLAGEN FRACTIONS FROM BOVINE AND RABBIT DENTAL FOLLICLE, PAPILLA AND PULP C. A. SHUITLEWORTH, J. I.. WARD and P. N. HIRSCHMANN Department of Medical Biochemistry and Turner Dental School, University of Manchester, Manchester Ml3 9PT. England Summary-Age-related changes in collagen solubility were found in bovine dental papilla and dental pulp. Increased amounts of insoluble collagen in mature dental pulp were associated with completion of root formation. In the pulp of the continually erupting rabbit incisor tooth. increase in the amount of newly synthesized collagen led to an increase in soluble
collagen.
The dental pulp and its precursor. the dental papilla, are loose connective tissues specialized for the formation and subsequent maintenance of dentine. These functions are related, in their turn. to the properties of the extracellular components of this tissue. Histologically, the extracellular matrix of pulp appears to be relatively homogeneous; fine reticulin fibres predominate and collagen fibres with 64nm periodicity are few (Han, 1968). Orlowski (1974) confirmed by chemical analyses that there are only small amounts of collagen in the bovine dental pulp (16.8 per cent); he also demonstrated that the pulp contains large amounts of acid mucopolysaceharides and glycoproteins in the proportions usually associated with fine collagenous fibres (Schwartz, 1966). We have been concerned with the characterisation and quantitation of the collagen species present in dental pulp, and have demonstrated the presence of Type I and III collagens in dental papilla (Hirschmann and Shuttleworth, 1977). The work of Nowack et al. (1976) appears to justify equating Type III collagen with what is identified histologically as reticulin. The relative proportions of collagen and reticulin in connective tissues appear to vary with development, maturation and ageing. Bovine tissues were obtained fresh from the abattoir; using mid-term foetal jaws. the dental papilla and dental follicle were separated from incisor and molar teeth in which root formation had not commenced The teeth were retained and a portion of mandibular bone was also taken from the same animals. Mature pulps were obtained from permanent molar teeth. Rabbit pulps from erupted incisor and molar teeth were kindly supplied by Dr. A. Smith, Department of Dental Sciences. University of Liverpool. The teeth and bone samples were demineralized in IO per cent EDTA. pH 7.5, and then treated in an identical manner to the pulp samples. Bone, dentine. pulp and follicle were washed with copious amounts of water, chopped into fine pieces and treated with
I mol/litre NaCI. 0.05 mol/litre tris. pH 7.5. to extract neutral salt-soluble collagen (NSSC) until the super-
natant was clear. The residue was then treated with 0.1 mol/litre acetic acid to extract acid-soluble collagen (ASC). The insoluble residues remaining aRer this extraction were digested with pepsin (enzyme: collagen, I: IO) at 4°C for 24 h and a pepsin-solubilized and a pepsin-resistant fraction obtained. The various collagen fractions were dialysed against 0.1 mol/litrc acetic acid, freeze-dried and the amount of collagen in each extract was estimated by hydroxyproline analysis using the method of Wocssner (1961). The various extracts showed differences in the extractability of collagen from each of the tissues examined. As expected. demineralized bone and dentine were insoluble in neutral-salt and acid solutions. and only small amounts of collagen could be solubilized from these tissues by pepsin treatment. The resi-
dues remaining after these three treatments accounted for 96.4 per cent of dentine collagen and 90.1 per cent of mandibular bone collagen. Table I compares the solubility of collagen from the soft connective tissues in the two animal species. The two foetal tissues, dental follicle and dental papilla. differed in their response to the various extractants. When these tissues were treated with salt and acid solutions. more collagen (45 per cent of the total) was extracted from papilla than from follicle (28 per cent of the total). On the other hand. more collagen from papilla (17.8 per cent) was resistant to pepsin-treatment than from follicle (6.2 per cent). Second, comparing dental papilla and mature dental pulp, collagen is less readily extracted from pulp. We consider that this is due to an age-related increase in cross-linking and fibre organisation. In both papilla and mature pulp. similar percentages of the insoluble collagen were solubilizcd by pepsin. indicating that the proportion of pepsin-soluble fibres increases when root formation is complete. In rabbit, there were marked dilfcrences in solubility between incisor and molar pulps. In the molar pulp, 56.3 per cent of total collagen was pcpsin-solublc and only 7.4 per cent pepsin-resistant. whcrcas, in incisors almost 90 per cent was neutral-salt and acid soluble. and less than I per cent pepsin-resistant.
235
C. A. Shuttleworth.
236
Table
I. Solubility
J. L. Ward
and P. N. Hirschmann
of collagen. expressed as a percentage of the total collagen. in follicle. papilla and pulp Bovine
Rabbit Incisor
Molar
Molar Pulp
Pulp
78.9
Follicle
Papilla
Pulp
5.1
23.4
8.6
27.6
23.2 65.3 6.2
22.1 36.6 17.8
I.1 55.1 35.2
8.8 56.3
Neutral-salt soluble collagen Acid-soluble collagen Pepsin-soluble Pepsin residue
Uitto and Antila (1971) found more collagen in rabbit molar pulps than in those from incisors: however they found no significant differences in the rate of collagen biosynthesis between the two tissues. Their findings and ours suggest that. in molars, pulp collagen becomes cross-linked faster than in incisors. Orlowski and Doyle (1976) injected labelled proline into rats and found that the specific activity of hydroxyproline was higher in the continuously growing inesor than in the molars, which are of limited growth. supporting our interpretation of the increased proportion of NSSC in the incisor, as due to a higher rate of collagen synthesis associated with the continuous eruption of this tooth. Our findings show that the solubility of collagen in the molar pulp ducrcases as dcvclopmcnt proceeds. This appears not to occur in the rabbit incisor. The low solubility of collagen in bovine molar pulp is probably due to grcatcr incorporation of soluble collagen into the insoluble network. In the continually erupting rabbit incisor tooth. the greater proportion of collagen extracted with neutral-salt and acid solutions is presumably related to more newly synthcsizcd collagen. The ditfercnces in collagen solubility arc presumably in turn related to the different functional demands on thcsc two teeth.
7.4
IO.0 10.2 0.8
REFERENCES
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. 103-144. University of Alabama Press. Birmingham. Hirschmann P. N. and Shuttleworth C. A. 1977. Collagen heterogeneity in the developing tooth. /. dmr. Rcs.. In press. IADR. Brit. Div. Abs 44. Nowack H.. Gay S.. Wick G.. Becker V. and Timpl R. 1976. Preparation and use in immunology of antibodies specific for Type 1 and III collagen and procollagen. J. immun. Methods 12. 117-124. Orlowski W. A. 1974. Analysis of collagen, acid mucopolysaccharides and glycoproteins in bovine and porcine pulp. Archs oral Biol. 19. 255-258. Orlowski W. A. and Doyle J. L. 1976. Collagen metabolism in the pulps of rat teeth. Archs orul tliol. 21. 391-392. Schwartz W. 1966. Morphology and differentiation of conncctivr tissue fibrcs. In: Connecrire Tissue Symposium (Edited by Tunbridgc R. E.) pp. 144-156. L)lackwcll. Oxford. Uitto V. and Antila R. 1971. Charactcrisation biosynthesis in rabbtt dental pulp in cirro. scund. 29. 609-6 I 7.
of collagen Acru odonr
Wocssncr J. G. 1961. The dctcrmination of hydroxyprolinc in tissurs and protein. Samples containing small proportions of the amino acid. Archs Biochem. Biophrs. . 93. 440-447.