BIOCHEMICAL STUDY OF COLLAGEN TURNOVER INCISOR PERIODONTAL LIGAMENT
IN RAT
W. A. ORLOWSKI Department of Pathology, School of Dentistry. Fairleigh Dickinson University. 110 Fuller Place. Hackensack. NJ 07601, U.S.A. Summary--Collagen turnover in an erupting maxillary incisor of the rats was studied using biochemical techniques. The collagen was labelled with [3H]-proline and the loss of activity from hydroxyproline and proline was measured. The turnover of collagen was rapid, with a half-life of 9.5 days and a turnover time of 13.4 days. The non-collagenous components turned over more rapidly with a half-life of 6.5 days. However. after I2 days, there was little or no difference in the specific activities of hydroxyproline and proline. It would appear that after 12 days, both collagen and non-collagenous components had the same turnover rate. It appears from the gradual loss of label, that the entire ligament from the alveolar crest to the apex was labelled and was undergoing remodeling.
MATERIALS
INTRODUCTION
Autoradiographic studies suggest that collagen in the periodontal ligament of rodents has a high turnover (Stallard, 1963; Crumley, 1964; Carneiro and deMoreas, 1965). A high degree of enzyme activity was also observed by Fullmer (1966) using histochemical techniques. In an autoradiographic study of the marmoset. Skougaard. Levy and Simpson (1970) found that of all the tissues tested metabolic activity was highest in the periodontal ligament. They found there was a 50 per cent reduction in the amount of radioactivity in the first 21 days after administration of label. Rippin (1976). using autoradiographic grain counts. found collagen half-lives in the ligament of 2.5 days at the tooth apices and 6.4 days at the alveolar crest in rat molars. Sodek (1976) found that the incorporation of tritiated proline into the collagen of periodontal ligament of rat molars was 5 times higher than into gingiva which in turn was considerably higher than into skin. He also found a very rapid and efficient maturation of collagen in the periodontium. Few studies have been made on the erupting incisor. Beertsen (1975) found that the cells and fibres attached to the incisor surface migrated with the tooth as it erupted, suggesting that the remodelling was largely dependent on tooth eruption. Most of the autoradiographic studies have been performed using tritiated proline to label the collagen of the periodontium. Orlowski (1976) showed that most of the tritium activity in the oral tissues was not present in collagen but was in non-collagenous components and (Orlowski. 1977) found a rapid turnover of collagen in the pulps of rat incisors; namely a half-life of 7.5 days measured over 18 days, suggesting that the turnover of collagen is more rapid than would be expected on the basis of tooth eruption. My aim was to use biochemical techniques to determine collagen turnover in the periodontal ligament of rat incisors compared with published autoradiographic findings.
AND
METHODS
30 SpragueeDawley rats, each weighing about 2OOg, were equally divided into 5 groups. Each rat received intraperitoneally 0.7 &i of [“HI-5-proline (Amersham-Searle. North Arlington. III.) per gram of body wt. The groups of rats were killed by decapitation 2. 4. 8. 12 or 18 days and the heads collected and lyophilized. The periodontal ligament from maxillary incisors was manually dissected and stored in a dessicator at -20 C until analysed. 0.5- I mg samples from each rat were analysed separately and the means and the standard deviations determined for each group. Proline and hydroxyproline were separated by ion-exchange chromatography as described by Stern et al. (1963) and adapted by Orlowski (1976). Fractions of the eluant containing proline and hydroxyproline were collected and the concentration and radioactivity determined. The radioactivity was measured in a liquid scintillation spectrometer (Beckman SL-IOOC) with an efficiency of 40 per cent. The amount of activity in collagen was calculated from the molar ratio of proline: hvdroxyproline in type I collagen of rat skin (Piez. Eigner and Lewis. 1963). which is 1.33. As there was no loss of activity from [3H]-5-proline during hydroxylation. wc would expect that both proline and hydroxyprolinc would be labelled to the same extent. Therefore. to obtain the total activity in collagen, the activity in hydroxyproline (d.p.m./mg) was multiplied by 2.33. The half-life (T:) was calculated from the slopes of a semi-log plot of the loss of activity from the imino acids over the l&da) period. Multiplying TI b> 1.41 yields the turnover time which is equal to the average life of a prolinc or hydroxyproline molecule RESLILTS
The disappearance periodontal ligament 1163
of
total tritium activity from was rapid over the Is-day
W. A. Orlowski
1164
Table 1. Change
Days 2 4 8 12 18
Total activity* 7157 5385 3263 1886 1222
+ f i + f
756 717 583 198 276
in tritium
Activity in collagent 2255 2374 1740 860 556
activity
in periodontal
Activity in non-collagenous substances*
(34”,,) (46”,,) (55”“) (491:J (54”;J
4324 2775 1449 893 483
ligament
Sp. activity OH-prolinef 75.0 74.7 46.2 35.4 23.7
& & k * +
9.3 7.3 5.0 3.5 2.8
Sp. activity prolinef 143.7 99.6 63.8 35.3 22.7
f + F * +
8.2 21.4 9.7 4.7 3.3
* Expressed in d.p.m./mg of dry wt of tissue. t Expressed in d.p.m./mg of dry wt (see Materials and Methods). In parenthesis the values are expressed as a percentage of the total activity which is equal to: activity in collagen/(activity in collagen + activity in non-collagenous substances). 1 Expressed in d.p.m./pg of the imino acid. Proline values include both collagenous and non-collagenous proline.
period. During the first 12 days the loss of activity was more rapid from proline than hydroxyproline (Table 1, and Fig. I), although afterwards the radioactivity was lost at the same rate from both (Fig. 1). The proportion of activity in collagen increased from 30 per cent at day 2 to 50 per cent at day 8 to day 18 after administration of label. The half-lives determined by least-square linear regression analysis were 9.5, 6.5 and 5.2 days for hydroxyproline, total proline and non-collagenous proline, respectively. The corresponding turnover times (half-life x 1.41) were: 13.5, 9.2 and 7.3 days, respectively. DISCUSSION
The collagen turnover for periodontal ligament was slower than that of the pulp of the incisor, but considerably faster than in the gingiva. The turnover time for the dental pulp was 9.5 days (Orlowski, 1977), whereas in the gingiva there was a sharp drop in the specific activity in the first 4 days post-injection. However, thereafter no change could be detected, PERIODONTAL LIGAMENT RAT INCISOR A - PROLINE 0 - HYDROXYPROLINE Cl - NON-COLLAGENOUS
PROL .INE
1 day for collagen of the rat molar. a paradoxical finding as the incisor erupts more rapidly. His study suggests that there was no relationship or inverse relationship between tooth eruption and collagen turnover. Whether his approach has yielded more accurate results than mine is still to be determined.
Periodontal
ligament turnover
The possibility that recycled labelled proline occurs locally in the cell or tissue and thus may yield longer half-lives was suggested by Jackson and Heininger (1974) and Sodek (1976). However, the data can be otherwise interpreted and are contradicted by a wellcontrolled study of Klein (1969). Jackson and Heininger (1975) later showed that the half-lives for collagen turnover measured with “0 label, which is not re-utilized. were much shorter than when measured with [3H]-proline. suggesting that there was reutilization of [3H]-proline. However, the prevailing
opinion is that the intracellular pool of proline is in equilibrium with the extracellular and plasma pool of the imino acid. Re-utilization of proline under such conditions. although present. would be greatly limited because of the markedly-reduced activity of proline after 38 h and its high turnover in plasma. It is unclear to what extent the remodelling of collagen librcs in periodontal ligament in the incisor is related to tooth eruption. Melcher (1967) showed that the fihrcs attached to bone and teeth remained stable during tooth eruption and therefore concluded that remodelling had to take place in the intermediate plexus
The gradual
with time from both in my study suggests that the entire ligament was labelled and was being remodelled rather than being primarily synthesi/ed in the region of the end of the root and then migrating with the tooth during eruption. If most of the collagen of the ligament was synthesized with the tooth and then degraded as it migrated coronally. little change uould bc expected in the specific activity of collagen until eruption of the newly-synthesized portion of the tooth. probably l&l5 days after administration of label. Main and Adams (1965) found that the rat incisor erupts at a rate of approximatel! 0.9mm in a 24-h period. collagen
and
loss of activity
non-collagenous
substances
A~,kno\~/t,clgrr,Icnr\~~Thisinvestigation was supported by a U.S.P.H.S. Grant No. DE-03178 and No. DE-04023 from the National
Institute
of Dental
Research.
II65 REFERENCES
Beertsen W. 1975. Migration of fibroblasts in the periodontal ligament of the mouse incisor as revealed by autoradiography. Archs oral Biol. 20. 659-666. Beertsen W. and Everts V. 1977. Collagen remodeling in the periodontal ligament of the mouse incisor. ./. I/~,,II. RH.. 56, Special Issue A. Abstract 215. Carneiro J. and deMoreas F. F. 1965. Radioautographic visualization of collagen metabolism rn periodontal tissues of the mouse. .ilrc,hs ortrl Biol. IO, 833 X45. Crumley P. J. 1964. Collagen formation in normal and stressed periodontium. Periodontics 2, 53 61. Fullmer H. M. 1969. Histochemlcal studies on the pertodontium. J. drnt. Rcs. 41. 225 234. Gould B. S. and Woessner J. G. Jr. 1957. The influcncc of ascorbic acid on the proline. hydroxyproline. glycine and collagen content of regenerating guinea pig -skin. .I. hiol. Chm. 226, 289~ 300. c‘linkcl
Chr.
Acta 51, 163~ 161.
Jackson S. H. and Heininger J. A. 1975. Proline recycling during collagen metabolism as determined by concurrent O”and H ‘-labeling. Biochirm hioph~~s. -lctcl 381. 359 -367. Klein L. 1969. Reversible transformation of fibrous collagen to a soluble state ifi viw. B-w. mm1 .Ac,od. Su. I:.S.A. 62. 920 927. Main J. H. P. and Adams D. A. 1965. Measurement of the rate of eruption of the rat incisor. .4wh orcrl. Brol. IO, 999m 1008. Melcher A. H. 1967. Remodeling of the periodontal ligament during eruption of the rat incisor. .4rch ortrl Biol. 12, 1649 1651. Melcher A. and Correia M. A. 1971. Remodeling of pcriodontal hgament m erupting molars of mature rats. J. puiodmt. Rca. 6. 1 IX 125. Orlowski W. A. 1976. The incorporation of Hz-proline into the periodontium of a rat. J. pwodottt. Rr.s. I I. 96 100. Orlowski W. A. 1977. The turnover of collagen in the dental pulp of rat incisors. J. dcrlt Rcs. 56. 437 440. Orlowski W. A. 197X. Metabollhm of gin+al collagen 111 a rat. .I. (/~,II. Rc\. 57. 329 333. Piez K.. Eigner E. and Lewis M. 1963. The chromatographic ceparation and amino acid composltlon of the subunit\ of \cveral collagcns. Biochc~/tri.xtr~~ ( 11Irr/~.)2. 5X 66. Rippin J. W. 1976. Collagen turnover in the periodontal ligament under normal and altered functional forces. I. Young rat molars. J. prriodont. Rex II. 101 107. Sicher H. 1942. Tooth eruption: axial movement of teeth with limited growth. J. dent. Rcs. 21, 395 401. Skougaard M. R., Levy B. M. and Simpson J. 1970. (‘01. lagen metabolism in skin and periodontal membranes of the marmoset. SU&. J. dertt. Rcs. 78, 256 262. Sodek J. 1976. A new approach to assessing collagen turnover by using a microassay: a highly efticlent and rapid turnover of collagen in rat periodontal tissues. Birxhcwr. J. 160. 243 746. Sodek J. 1977. A comparison of the rates of synthesis and turnover of collagen and non-collagen proteins m adult rat periodontal tissues and skin using a microassaq. Arch ovul Biol. 22. 655-665. Stallard R. 1963. The utiliration of H’-proline bq the connective tissue elements of the perlodontium. Pr,riodo,lrlcs 1. 1X5- 188. Stern B. D.. Mechanic G. L. Glimcher J. J. and Goldhabcr P. 1962. The resorption of bone collagen in tissue culture. Bioc,hc,~~. hioph~~. RPS. C~WHUH. 13. I37 143.
IN RAT
W. A. ORLOWSKI Department of Pathology, School of Dentistry. Fairleigh Dickinson University. 110 Fuller Place. Hackensack. NJ 07601, U.S.A. Summary--Collagen turnover in an erupting maxillary incisor of the rats was studied using biochemical techniques. The collagen was labelled with [3H]-proline and the loss of activity from hydroxyproline and proline was measured. The turnover of collagen was rapid, with a half-life of 9.5 days and a turnover time of 13.4 days. The non-collagenous components turned over more rapidly with a half-life of 6.5 days. However. after I2 days, there was little or no difference in the specific activities of hydroxyproline and proline. It would appear that after 12 days, both collagen and non-collagenous components had the same turnover rate. It appears from the gradual loss of label, that the entire ligament from the alveolar crest to the apex was labelled and was undergoing remodeling.
MATERIALS
INTRODUCTION
Autoradiographic studies suggest that collagen in the periodontal ligament of rodents has a high turnover (Stallard, 1963; Crumley, 1964; Carneiro and deMoreas, 1965). A high degree of enzyme activity was also observed by Fullmer (1966) using histochemical techniques. In an autoradiographic study of the marmoset. Skougaard. Levy and Simpson (1970) found that of all the tissues tested metabolic activity was highest in the periodontal ligament. They found there was a 50 per cent reduction in the amount of radioactivity in the first 21 days after administration of label. Rippin (1976). using autoradiographic grain counts. found collagen half-lives in the ligament of 2.5 days at the tooth apices and 6.4 days at the alveolar crest in rat molars. Sodek (1976) found that the incorporation of tritiated proline into the collagen of periodontal ligament of rat molars was 5 times higher than into gingiva which in turn was considerably higher than into skin. He also found a very rapid and efficient maturation of collagen in the periodontium. Few studies have been made on the erupting incisor. Beertsen (1975) found that the cells and fibres attached to the incisor surface migrated with the tooth as it erupted, suggesting that the remodelling was largely dependent on tooth eruption. Most of the autoradiographic studies have been performed using tritiated proline to label the collagen of the periodontium. Orlowski (1976) showed that most of the tritium activity in the oral tissues was not present in collagen but was in non-collagenous components and (Orlowski. 1977) found a rapid turnover of collagen in the pulps of rat incisors; namely a half-life of 7.5 days measured over 18 days, suggesting that the turnover of collagen is more rapid than would be expected on the basis of tooth eruption. My aim was to use biochemical techniques to determine collagen turnover in the periodontal ligament of rat incisors compared with published autoradiographic findings.
AND
METHODS
30 SpragueeDawley rats, each weighing about 2OOg, were equally divided into 5 groups. Each rat received intraperitoneally 0.7 &i of [“HI-5-proline (Amersham-Searle. North Arlington. III.) per gram of body wt. The groups of rats were killed by decapitation 2. 4. 8. 12 or 18 days and the heads collected and lyophilized. The periodontal ligament from maxillary incisors was manually dissected and stored in a dessicator at -20 C until analysed. 0.5- I mg samples from each rat were analysed separately and the means and the standard deviations determined for each group. Proline and hydroxyproline were separated by ion-exchange chromatography as described by Stern et al. (1963) and adapted by Orlowski (1976). Fractions of the eluant containing proline and hydroxyproline were collected and the concentration and radioactivity determined. The radioactivity was measured in a liquid scintillation spectrometer (Beckman SL-IOOC) with an efficiency of 40 per cent. The amount of activity in collagen was calculated from the molar ratio of proline: hvdroxyproline in type I collagen of rat skin (Piez. Eigner and Lewis. 1963). which is 1.33. As there was no loss of activity from [3H]-5-proline during hydroxylation. wc would expect that both proline and hydroxyprolinc would be labelled to the same extent. Therefore. to obtain the total activity in collagen, the activity in hydroxyproline (d.p.m./mg) was multiplied by 2.33. The half-life (T:) was calculated from the slopes of a semi-log plot of the loss of activity from the imino acids over the l&da) period. Multiplying TI b> 1.41 yields the turnover time which is equal to the average life of a prolinc or hydroxyproline molecule RESLILTS
The disappearance periodontal ligament 1163
of
total tritium activity from was rapid over the Is-day
W. A. Orlowski
1164
Table 1. Change
Days 2 4 8 12 18
Total activity* 7157 5385 3263 1886 1222
+ f i + f
756 717 583 198 276
in tritium
Activity in collagent 2255 2374 1740 860 556
activity
in periodontal
Activity in non-collagenous substances*
(34”,,) (46”,,) (55”“) (491:J (54”;J
4324 2775 1449 893 483
ligament
Sp. activity OH-prolinef 75.0 74.7 46.2 35.4 23.7
& & k * +
9.3 7.3 5.0 3.5 2.8
Sp. activity prolinef 143.7 99.6 63.8 35.3 22.7
f + F * +
8.2 21.4 9.7 4.7 3.3
* Expressed in d.p.m./mg of dry wt of tissue. t Expressed in d.p.m./mg of dry wt (see Materials and Methods). In parenthesis the values are expressed as a percentage of the total activity which is equal to: activity in collagen/(activity in collagen + activity in non-collagenous substances). 1 Expressed in d.p.m./pg of the imino acid. Proline values include both collagenous and non-collagenous proline.
period. During the first 12 days the loss of activity was more rapid from proline than hydroxyproline (Table 1, and Fig. I), although afterwards the radioactivity was lost at the same rate from both (Fig. 1). The proportion of activity in collagen increased from 30 per cent at day 2 to 50 per cent at day 8 to day 18 after administration of label. The half-lives determined by least-square linear regression analysis were 9.5, 6.5 and 5.2 days for hydroxyproline, total proline and non-collagenous proline, respectively. The corresponding turnover times (half-life x 1.41) were: 13.5, 9.2 and 7.3 days, respectively. DISCUSSION
The collagen turnover for periodontal ligament was slower than that of the pulp of the incisor, but considerably faster than in the gingiva. The turnover time for the dental pulp was 9.5 days (Orlowski, 1977), whereas in the gingiva there was a sharp drop in the specific activity in the first 4 days post-injection. However, thereafter no change could be detected, PERIODONTAL LIGAMENT RAT INCISOR A - PROLINE 0 - HYDROXYPROLINE Cl - NON-COLLAGENOUS
PROL .INE
1 day for collagen of the rat molar. a paradoxical finding as the incisor erupts more rapidly. His study suggests that there was no relationship or inverse relationship between tooth eruption and collagen turnover. Whether his approach has yielded more accurate results than mine is still to be determined.
Periodontal
ligament turnover
The possibility that recycled labelled proline occurs locally in the cell or tissue and thus may yield longer half-lives was suggested by Jackson and Heininger (1974) and Sodek (1976). However, the data can be otherwise interpreted and are contradicted by a wellcontrolled study of Klein (1969). Jackson and Heininger (1975) later showed that the half-lives for collagen turnover measured with “0 label, which is not re-utilized. were much shorter than when measured with [3H]-proline. suggesting that there was reutilization of [3H]-proline. However, the prevailing
opinion is that the intracellular pool of proline is in equilibrium with the extracellular and plasma pool of the imino acid. Re-utilization of proline under such conditions. although present. would be greatly limited because of the markedly-reduced activity of proline after 38 h and its high turnover in plasma. It is unclear to what extent the remodelling of collagen librcs in periodontal ligament in the incisor is related to tooth eruption. Melcher (1967) showed that the fihrcs attached to bone and teeth remained stable during tooth eruption and therefore concluded that remodelling had to take place in the intermediate plexus
The gradual
with time from both in my study suggests that the entire ligament was labelled and was being remodelled rather than being primarily synthesi/ed in the region of the end of the root and then migrating with the tooth during eruption. If most of the collagen of the ligament was synthesized with the tooth and then degraded as it migrated coronally. little change uould bc expected in the specific activity of collagen until eruption of the newly-synthesized portion of the tooth. probably l&l5 days after administration of label. Main and Adams (1965) found that the rat incisor erupts at a rate of approximatel! 0.9mm in a 24-h period. collagen
and
loss of activity
non-collagenous
substances
A~,kno\~/t,clgrr,Icnr\~~Thisinvestigation was supported by a U.S.P.H.S. Grant No. DE-03178 and No. DE-04023 from the National
Institute
of Dental
Research.
II65 REFERENCES
Beertsen W. 1975. Migration of fibroblasts in the periodontal ligament of the mouse incisor as revealed by autoradiography. Archs oral Biol. 20. 659-666. Beertsen W. and Everts V. 1977. Collagen remodeling in the periodontal ligament of the mouse incisor. ./. I/~,,II. RH.. 56, Special Issue A. Abstract 215. Carneiro J. and deMoreas F. F. 1965. Radioautographic visualization of collagen metabolism rn periodontal tissues of the mouse. .ilrc,hs ortrl Biol. IO, 833 X45. Crumley P. J. 1964. Collagen formation in normal and stressed periodontium. Periodontics 2, 53 61. Fullmer H. M. 1969. Histochemlcal studies on the pertodontium. J. drnt. Rcs. 41. 225 234. Gould B. S. and Woessner J. G. Jr. 1957. The influcncc of ascorbic acid on the proline. hydroxyproline. glycine and collagen content of regenerating guinea pig -skin. .I. hiol. Chm. 226, 289~ 300. c‘linkcl
Chr.
Acta 51, 163~ 161.
Jackson S. H. and Heininger J. A. 1975. Proline recycling during collagen metabolism as determined by concurrent O”and H ‘-labeling. Biochirm hioph~~s. -lctcl 381. 359 -367. Klein L. 1969. Reversible transformation of fibrous collagen to a soluble state ifi viw. B-w. mm1 .Ac,od. Su. I:.S.A. 62. 920 927. Main J. H. P. and Adams D. A. 1965. Measurement of the rate of eruption of the rat incisor. .4wh orcrl. Brol. IO, 999m 1008. Melcher A. H. 1967. Remodeling of the periodontal ligament during eruption of the rat incisor. .4rch ortrl Biol. 12, 1649 1651. Melcher A. and Correia M. A. 1971. Remodeling of pcriodontal hgament m erupting molars of mature rats. J. puiodmt. Rca. 6. 1 IX 125. Orlowski W. A. 1976. The incorporation of Hz-proline into the periodontium of a rat. J. pwodottt. Rr.s. I I. 96 100. Orlowski W. A. 1977. The turnover of collagen in the dental pulp of rat incisors. J. dcrlt Rcs. 56. 437 440. Orlowski W. A. 197X. Metabollhm of gin+al collagen 111 a rat. .I. (/~,II. Rc\. 57. 329 333. Piez K.. Eigner E. and Lewis M. 1963. The chromatographic ceparation and amino acid composltlon of the subunit\ of \cveral collagcns. Biochc~/tri.xtr~~ ( 11Irr/~.)2. 5X 66. Rippin J. W. 1976. Collagen turnover in the periodontal ligament under normal and altered functional forces. I. Young rat molars. J. prriodont. Rex II. 101 107. Sicher H. 1942. Tooth eruption: axial movement of teeth with limited growth. J. dent. Rcs. 21, 395 401. Skougaard M. R., Levy B. M. and Simpson J. 1970. (‘01. lagen metabolism in skin and periodontal membranes of the marmoset. SU&. J. dertt. Rcs. 78, 256 262. Sodek J. 1976. A new approach to assessing collagen turnover by using a microassay: a highly efticlent and rapid turnover of collagen in rat periodontal tissues. Birxhcwr. J. 160. 243 746. Sodek J. 1977. A comparison of the rates of synthesis and turnover of collagen and non-collagen proteins m adult rat periodontal tissues and skin using a microassaq. Arch ovul Biol. 22. 655-665. Stallard R. 1963. The utiliration of H’-proline bq the connective tissue elements of the perlodontium. Pr,riodo,lrlcs 1. 1X5- 188. Stern B. D.. Mechanic G. L. Glimcher J. J. and Goldhabcr P. 1962. The resorption of bone collagen in tissue culture. Bioc,hc,~~. hioph~~. RPS. C~WHUH. 13. I37 143.
