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Collagenolytic activity from human deciduous pulps
JOURNAL OF ENDODONTICS
VOL 7, NO 9, SEPTEMBER 1981
Collagenolytic activity from human deciduous pulps Stanley A. Alexander, DMO
Human pulp tissue from resorbing deciduous teeth showed collagenolytic activity when the tissue was incubated on collagen gels. This activity was directly proportional to the size of the explant and its period of incubation. No activity was observed in pulp tissue obtained from completely formed premolar teeth.
Recent reports have indicated specific enzymatic activities from the periodontal a n d pulpal tissues that have been related to physiological root resorption.'" Collagenolytic activity has not been reported in pulpal tissue d u r i n g resorption, despite the fact that collagen is one of the organic c o m p o n e n t s of the d e n t i n a n d dental pulp.' Because the p u l p has been reported to participate in the resorption process, ~": it is our hypothesis that collagenolytic activity may be present as well. T h e current study investigated the source of such activity from h u m a n pulp tissue.
MATERIALS A N D METHODS Thirty children were included in this study, 14 girls and 16 boys, ranging in age from 8 years, 2 months to 12 years, 2 months. A total of 40 deciduous resorbing canines, first molars, and second molars were examined; ten fully formed first premolars were used as controls. All teeth were.judged to be cariesfree, and all procedures in this study were performed under sterile conditions. The teeth were removed as an adjunct to orthodontic therapy, using a standard surgical technique. Root resorption in the deciduous teeth was judged clinically and radiographically to have involved between 50 418
and 75% of the root in all cases. This figure was based on measurement of these teeth with a Bolev millimeter gauge and a comparison of the length derived with average lengths of deciduous root described by Kramer and Ireland? A high-speed handpiece with a diamond disk and water coolant was used to remove 5 mm of the remaining apical third of the root on all specimens. Pulp tissue was removed according to the methods of Engstrom and Ohman." Specimens were washed for three hours at 37 C in mammalian Tyrode's solution containing .5 mg/ml streptomycin, 1,000 units/ml penicillin. 40/~g/ml amphotericin B, and 75 tLg/m[ gentamycin. After the tissue was washed, it was cut into ~ctions ranging in size from 1 to 4 mm.: The detection of collagenolytic activity was accomplished by incubating deciduous and permanent pulp for seven days on reconstituted collagen gels as described by Gross and I,apierer Pulp cultures v-ere incubated at 37 C in a moist atmosphere containing 905~ oxygen and 10% carbon dioxide, and were observed dail) for seven days for lytic activity. Areas of explant and lysis were measured with an ocular micrometer. Resorption products that occurred in the area of ]ysis were collected with a micropipette and analyzed biochemically for hydroxyproline according to the methods of Stegemann. '~ Control chambers were incubated in the same manner, but they were incubated without tissue explants. Immediately after incubation, pulp tis-
sue was fixed in 10~,[: buffered formalin and routinely processed for light microscopy. A second group of pulps from deciduous and permanent specimens was fixed immediately after removal from the tooth and processed for histology in the aforementioned manner.
RESULTS T h e pulpal tissues from both the resorbing deciduous and premolar teeth appeared similar microscopicall,,' (Fig 1). This finding corroborated that of Fox a n d Iteeley.': No osteoclasts were observed. Fibroblasts and connective tissues appeared normal. Pulp tissue that was tixed i m m e d i a t e l y was identical to tissue that was cultured and then fixed in formalin, except for a slight cellular outgrowth in the latter. The degree of collagenolytic activity was measured over seven days with 4-ram-' explants (Table 1). All deciduous pulp tissue showed lvsis of collagen gels. Control premolar pulps showed no activity (Fig 2). "]'he extent of substrate lysis increased proportionately when cornpared with the size of the tissue cxplant a n d period of incubatioa (Table 2). An a p p r o x i m a t e fourfold size increase in mean explant are~
JOURNAL OF ENDODONTICS ' VOL 7, NO 9, SEPTEMBER 1981
Table 1 * Collagenolytic activity of dental pulps" in resorbing and nonresorbing teeth. Incubation period (days) Tooth
l
Deciduous canine (30) Deciduous first molar (5) Deciduous second molar (5) First premolar (10) *.+++red ,~f p u l p ~- +
rxplants
l . ~ s t s car
+ 9 9 +
Re]
-
-
.
+,as a p p r o , + t m a t e ] ~ +
}~lIs/*etl
] /lid
2
2 Into
3
4
5
+ +
+ + + +
+ + + + + 4-
+ + + + + + + +
+ + + +
+
+ +
+ + a-
+ + m +
+ + + +
. I mm
. -
-
.ll+ltltld the
|.,,,,ix t~f i~(.] bclx+t~+ll $ alfl(] 4 t n n l . m n m d
+ + + +
. Nt+ h s l s o l ~ t , l l a ~ t r n ~ r l
tissue
+ + ,
-
+
-
Ix,i,
I.x+ls o f g e l bet++r
o l ttt+l l ) + ' ~ l r l n l n g b t + l l e d t h t h e n x s u c 2 .~lld
+ l l l l l l art+llTIO t h e
tissue
l h c tl,++.tle
Table 2 9 Relationship between size of pulp explant area of lysis, and hydroxyproline released from collagen gel. Numlx:r of Cuhures*
Explant area range (in ram")
Mean explant area (in mine)
Mean area (,f lvsis (nun')
Hydroxyprolim' !,ug)
8 8 8
0.5 to 1.2 1.8 to 2.6 3.3 to ,1.2
0.9 _+ 0.2 2.3 _"- 0.:3 3.8 -'- 0.3
12.6 ~ 2.1 21.6 _+ 3.7 41+).2 • 6.4
4.9 _"- 0.2 7.3 ': 0.2 10.0 - 0.6
+Incubation
pcru*]
w a s *.~'vetl d . ~
fol all c u]turc~ obsvrsed
---"
( 4
"
"1
"
~s
,..,. 4
o
"~
DISCUSSION Physiological resorption is a n a t u ral process whereby the p e r m a n e n t dentition replaces the deciduous teeth. T h e removal of collagen during resorption a n d exfoliation of the deciduous tooth has been reported and has been a t t r i b u t e d to the periodontal tissue activity."' Examples of
rlal
")""
Ip'~
~
,.
,
/
9
"~-
,.~
. -r
.
.
"
9
I
." ,.I~
~
--
:,.-.
I
.
9
"
"%"
~+,
-~,
.j
~
"
..t'" ~ "
" . , 9t'
9 9" , ,
.i
'. '~
4. z
. ~;,,.
- . ,
~.
collagenolytic activity have occurred elsewherc ira a m p h i b i a n regeneration,'" tadpole m e t a m o r p h o s i s ? ' and in w o u n d healing.' ..... Human collagenase activity has been identified within the periodontal tissues.' ..... Although collagenolytic activity has been observed d u r i n g bovine and h u m a n root resorption,' :~'":':' no data are available that localize this activity to h u m a n dental pulp. O u r results indicate that shortterm c u l t u r e s of pulpal tissue from h u m a n resorbing deciduous teeth produce an enzyme capable of degrading collagen u n d e r physiological conditions. Little, if any, inflammato W infiltrate was observed within the pulp of resorbing teeth, despite reports of its presence d u r i n g resorption in adjacent tissues. "'-'~.:' This
.
+~
9 "~t'
resulted in a 3()0~. increase in gel lysis. Furthermore, the a m o u n t , of collagen solubilized as measured by release of hydroxyproline was correlated with tissue size a n d with the area of lysis, A fimrfold increase in mean explant area resulted in an increase in measurable hydroxyproline from 4.9 _+ 0.2 >g to 10.0 + 0.6 Ixg after seven days of i n c u b a t i o n .
.
k
.T "%.
.a'~
"~'~
F~g I- Pulp m.,'uefiom mot of resorbmg deHduous camne s&nvmg normal morphology ( I I & E , orzg mag • 160).
finding is in agreement with similar studies of connective tissue resorption. : ' ' ' ' ~ l'ibroblast activity has been proposed as being responsible for the collagenolytic activity seen in other tissues in the absence of inflammation"::' a n d is most probably the source of the activity in pulp as well. T h e stimulus for collagenolysis is still u n k n o w n , a l t h o u g h the pressures associated with orthodontic tooth movement have been observed to 419
IOURNAL OF ENDODONTICS ' VOL 7, NO 9, SEPTEMBER 1981
t,'zg 2-Pulp tzssue frora root of resorbing dectduous canine ( R ) and from root of premolar (N) incubated on collagen substrate for seven days. Note area of lysis (arrow) surrounding deciduous pulp, indicating collagenolyttc actw-
it)',
s t i m u l a t e p e r i o d o n t a l l i g a m e n t res o r p t i o n of collagen?'-' It is p r o p o s e d that tooth movement associated with eruption may stimulate collagenolytic a c t i v i t y in t h e p u l p a l tissues. W e conclude, however, that one compon e n t o f root r e s o r p t i o n rests w i t h i n t h e d e c i d u o u s p u l p a n d is r e s p o n s i b l e in p a r t for t h e e x f o l i a t i o n o f t h e d e c i d u o u s t e e t h . F u r t h e r m o r e , it h a s been shown that collagenolytic activity d e r i v e d f r o m t h e p e r i o d o n t a l ligam e n t is r e s p o n s i b l e for t h e d e c r e a s e d amounts of dentin hydroxyproline p r o d u c t i o n seen in t h e r o o t s o f d e c i d uous teeth? The human deciduous d e n t a l p u l p a n d its a s s o c i a t e d collagenolytic activity may be responsible for t h e s e r e p o r t e d d e c r e a s e s as well. l)r. Alexander is assistant professor of children's dentistry,, State University of New York at Stony Brook, Stony Brook, NY 11794. Requests for reprints should be directed to the author.
References 1. Alexander, S.A., and Swerdloff, M. Collagenloytic activity of periodontal ligament during human deciduous root resorption. Pediatr Dent 1:226-229, 1979. 2. Alexander, S.A., and Swerdloff, M. Identification and localization of a mucopolysaccharidase in human deciduous teeth. J Dent Res 59:594-601, 1980. 3. Alexander, S.A., and Swerdloff, M. Collagenolytic activity of periodontal ligament and hydroxproline content during human
420
deciduous root resorption. J Periodont Res 15:434-443, 1980. 4. Alexander, S.A.; Swerdloff, M.; and Caputo, l,. Dental pulp mucopolysaccharidam: identification and role in tooth resorption. J l)cnt Res 59:14114-1408. 1980. 5. Sicher, H., and Bhaskar, S.N. Orban's oral histology and emb~'olog':', ed 7. St. Louis, C. V. Mosby (do., 1972, pp,97-159. 6. Weatherell, J.A., and Hargreaves, J.A. Effect of resorption on the: fluoride content of human deciduous teeth Arch Oral Biul 11:749-756, 1966. 7. Snskolne. W.A, and Bimstein, E. A histomorphological study of the shedding process of human deciduous teeth at various chronological stages. Arch Oral Biol 22:331-335, 1977. 8. Kramer, W.A., and Ireland, R.I,. Measurements of the primats' teeth. J Dent Child 26:252-261. 1959. 9 Engstrom, H., and Ohman, A, Studies on the innervation of human teeth. J Dent Res 39:799-809, 1960. 10. Gross, J., and lapiere, C.M. Collagenolyric activity in amphibian tissues: a tissue culture assay. Proe Nat Acad Sci 48:10141022, 1962. l l. Stegemann, H. Mikrobestimmung yon hydroxyprolin mot chloramin-T and p-l)imethylaminubenzaldehyd. Z Physiol Chem 311:41-45, 1958. 12. Fox, A.G., and Heely, J.D. I listolngical study of pulps of human primal' teeth. Arch Oral Biol 25:103-110, 1980. 13. Grillo, |I.C., and others. Collagenolytic activity in regenerating forelimbs of the aduh newt (Triturus viridescens). Develop Biol 17:571-583, 1968. 14. Eisen, AZ., and (;ross, J. 'Fhc role' of epithelium and mesenehyme in the production of a collagenolytic enzyme and a hyaluronidase in the anuran tadpole. Develop Biol
12:408-418, 1965. 15. 1)onoff, R.B.; McLennan. J.E.: and (]rillo, H.C. Preparation and propcrtlcs of collagenases from epithelium and mcsenchyme of healing mammalian v,ounds. Biochim Biophys Acta 227:639-653, 1971 16. Donnff, R.B., and Grillo. II.C. The effects of skin grafting on healing open wounds in rabbits. ,J Surg Res 19:163-167, 1975. 17. Donoff, RB. Wound healing: bir ical events and potential roh' of collagenase. J Oral Surg 28:356-362, 1970. 18. Golub, I,.M., and others. ('olla~en,lytic activity of crevicular fluid from pericoronal gingival flaps. J Dent Res 55:177-181, 1976. 19. Golub, LM., and others. Some characteristics of collagenase activity in gingival crevicular fluid and its relationship to gingival diseases in humans. J Dent Res 55:1040-1057, 1976. 20. Chrismer, P. Collagenase in the human periodontal ligament. J Perindontnl 51:.t55461. 1980. 21. Morita, tl., and nthcrs. The collagcnolyric activity during root resorption of bovine deciduous teeth. Arch Oral Biol 15:503-508, 19711. 22. Ozaki, T., amt others. Collagenulytic activity during tooth movement in the rabbit. Arch Oral Biol 16:1123-1126, 1971. 23. Bernick, S.; Rabinowitch. BZ.: and Rutherford, R.L. Microscopic studies of the teeth of a six-year-old boy. Tooth absorpcionAnat Res 105:249-258, 1949. 24. Obersztyn, A. Experimental investiga" lion of factors causing resorption of deciduous teeth..1 Dent Res 42:660-674, 1963. 25. Depnrter, D.A., and TenCate. A.R. Collagen resorption by periodontal ligament fibroblasts at the hard tissue-ligament inter" faces of the: mouse perindontmm. J Periodon~ tol 51:429-432, 1980.
VOL 7, NO 9, SEPTEMBER 1981
Collagenolytic activity from human deciduous pulps Stanley A. Alexander, DMO
Human pulp tissue from resorbing deciduous teeth showed collagenolytic activity when the tissue was incubated on collagen gels. This activity was directly proportional to the size of the explant and its period of incubation. No activity was observed in pulp tissue obtained from completely formed premolar teeth.
Recent reports have indicated specific enzymatic activities from the periodontal a n d pulpal tissues that have been related to physiological root resorption.'" Collagenolytic activity has not been reported in pulpal tissue d u r i n g resorption, despite the fact that collagen is one of the organic c o m p o n e n t s of the d e n t i n a n d dental pulp.' Because the p u l p has been reported to participate in the resorption process, ~": it is our hypothesis that collagenolytic activity may be present as well. T h e current study investigated the source of such activity from h u m a n pulp tissue.
MATERIALS A N D METHODS Thirty children were included in this study, 14 girls and 16 boys, ranging in age from 8 years, 2 months to 12 years, 2 months. A total of 40 deciduous resorbing canines, first molars, and second molars were examined; ten fully formed first premolars were used as controls. All teeth were.judged to be cariesfree, and all procedures in this study were performed under sterile conditions. The teeth were removed as an adjunct to orthodontic therapy, using a standard surgical technique. Root resorption in the deciduous teeth was judged clinically and radiographically to have involved between 50 418
and 75% of the root in all cases. This figure was based on measurement of these teeth with a Bolev millimeter gauge and a comparison of the length derived with average lengths of deciduous root described by Kramer and Ireland? A high-speed handpiece with a diamond disk and water coolant was used to remove 5 mm of the remaining apical third of the root on all specimens. Pulp tissue was removed according to the methods of Engstrom and Ohman." Specimens were washed for three hours at 37 C in mammalian Tyrode's solution containing .5 mg/ml streptomycin, 1,000 units/ml penicillin. 40/~g/ml amphotericin B, and 75 tLg/m[ gentamycin. After the tissue was washed, it was cut into ~ctions ranging in size from 1 to 4 mm.: The detection of collagenolytic activity was accomplished by incubating deciduous and permanent pulp for seven days on reconstituted collagen gels as described by Gross and I,apierer Pulp cultures v-ere incubated at 37 C in a moist atmosphere containing 905~ oxygen and 10% carbon dioxide, and were observed dail) for seven days for lytic activity. Areas of explant and lysis were measured with an ocular micrometer. Resorption products that occurred in the area of ]ysis were collected with a micropipette and analyzed biochemically for hydroxyproline according to the methods of Stegemann. '~ Control chambers were incubated in the same manner, but they were incubated without tissue explants. Immediately after incubation, pulp tis-
sue was fixed in 10~,[: buffered formalin and routinely processed for light microscopy. A second group of pulps from deciduous and permanent specimens was fixed immediately after removal from the tooth and processed for histology in the aforementioned manner.
RESULTS T h e pulpal tissues from both the resorbing deciduous and premolar teeth appeared similar microscopicall,,' (Fig 1). This finding corroborated that of Fox a n d Iteeley.': No osteoclasts were observed. Fibroblasts and connective tissues appeared normal. Pulp tissue that was tixed i m m e d i a t e l y was identical to tissue that was cultured and then fixed in formalin, except for a slight cellular outgrowth in the latter. The degree of collagenolytic activity was measured over seven days with 4-ram-' explants (Table 1). All deciduous pulp tissue showed lvsis of collagen gels. Control premolar pulps showed no activity (Fig 2). "]'he extent of substrate lysis increased proportionately when cornpared with the size of the tissue cxplant a n d period of incubatioa (Table 2). An a p p r o x i m a t e fourfold size increase in mean explant are~
JOURNAL OF ENDODONTICS ' VOL 7, NO 9, SEPTEMBER 1981
Table 1 * Collagenolytic activity of dental pulps" in resorbing and nonresorbing teeth. Incubation period (days) Tooth
l
Deciduous canine (30) Deciduous first molar (5) Deciduous second molar (5) First premolar (10) *.+++red ,~f p u l p ~- +
rxplants
l . ~ s t s car
+ 9 9 +
Re]
-
-
.
+,as a p p r o , + t m a t e ] ~ +
}~lIs/*etl
] /lid
2
2 Into
3
4
5
+ +
+ + + +
+ + + + + 4-
+ + + + + + + +
+ + + +
+
+ +
+ + a-
+ + m +
+ + + +
. I mm
. -
-
.ll+ltltld the
|.,,,,ix t~f i~(.] bclx+t~+ll $ alfl(] 4 t n n l . m n m d
+ + + +
. Nt+ h s l s o l ~ t , l l a ~ t r n ~ r l
tissue
+ + ,
-
+
-
Ix,i,
I.x+ls o f g e l bet++r
o l ttt+l l ) + ' ~ l r l n l n g b t + l l e d t h t h e n x s u c 2 .~lld
+ l l l l l l art+llTIO t h e
tissue
l h c tl,++.tle
Table 2 9 Relationship between size of pulp explant area of lysis, and hydroxyproline released from collagen gel. Numlx:r of Cuhures*
Explant area range (in ram")
Mean explant area (in mine)
Mean area (,f lvsis (nun')
Hydroxyprolim' !,ug)
8 8 8
0.5 to 1.2 1.8 to 2.6 3.3 to ,1.2
0.9 _+ 0.2 2.3 _"- 0.:3 3.8 -'- 0.3
12.6 ~ 2.1 21.6 _+ 3.7 41+).2 • 6.4
4.9 _"- 0.2 7.3 ': 0.2 10.0 - 0.6
+Incubation
pcru*]
w a s *.~'vetl d . ~
fol all c u]turc~ obsvrsed
---"
( 4
"
"1
"
~s
,..,. 4
o
"~
DISCUSSION Physiological resorption is a n a t u ral process whereby the p e r m a n e n t dentition replaces the deciduous teeth. T h e removal of collagen during resorption a n d exfoliation of the deciduous tooth has been reported and has been a t t r i b u t e d to the periodontal tissue activity."' Examples of
rlal
")""
Ip'~
~
,.
,
/
9
"~-
,.~
. -r
.
.
"
9
I
." ,.I~
~
--
:,.-.
I
.
9
"
"%"
~+,
-~,
.j
~
"
..t'" ~ "
" . , 9t'
9 9" , ,
.i
'. '~
4. z
. ~;,,.
- . ,
~.
collagenolytic activity have occurred elsewherc ira a m p h i b i a n regeneration,'" tadpole m e t a m o r p h o s i s ? ' and in w o u n d healing.' ..... Human collagenase activity has been identified within the periodontal tissues.' ..... Although collagenolytic activity has been observed d u r i n g bovine and h u m a n root resorption,' :~'":':' no data are available that localize this activity to h u m a n dental pulp. O u r results indicate that shortterm c u l t u r e s of pulpal tissue from h u m a n resorbing deciduous teeth produce an enzyme capable of degrading collagen u n d e r physiological conditions. Little, if any, inflammato W infiltrate was observed within the pulp of resorbing teeth, despite reports of its presence d u r i n g resorption in adjacent tissues. "'-'~.:' This
.
+~
9 "~t'
resulted in a 3()0~. increase in gel lysis. Furthermore, the a m o u n t , of collagen solubilized as measured by release of hydroxyproline was correlated with tissue size a n d with the area of lysis, A fimrfold increase in mean explant area resulted in an increase in measurable hydroxyproline from 4.9 _+ 0.2 >g to 10.0 + 0.6 Ixg after seven days of i n c u b a t i o n .
.
k
.T "%.
.a'~
"~'~
F~g I- Pulp m.,'uefiom mot of resorbmg deHduous camne s&nvmg normal morphology ( I I & E , orzg mag • 160).
finding is in agreement with similar studies of connective tissue resorption. : ' ' ' ' ~ l'ibroblast activity has been proposed as being responsible for the collagenolytic activity seen in other tissues in the absence of inflammation"::' a n d is most probably the source of the activity in pulp as well. T h e stimulus for collagenolysis is still u n k n o w n , a l t h o u g h the pressures associated with orthodontic tooth movement have been observed to 419
IOURNAL OF ENDODONTICS ' VOL 7, NO 9, SEPTEMBER 1981
t,'zg 2-Pulp tzssue frora root of resorbing dectduous canine ( R ) and from root of premolar (N) incubated on collagen substrate for seven days. Note area of lysis (arrow) surrounding deciduous pulp, indicating collagenolyttc actw-
it)',
s t i m u l a t e p e r i o d o n t a l l i g a m e n t res o r p t i o n of collagen?'-' It is p r o p o s e d that tooth movement associated with eruption may stimulate collagenolytic a c t i v i t y in t h e p u l p a l tissues. W e conclude, however, that one compon e n t o f root r e s o r p t i o n rests w i t h i n t h e d e c i d u o u s p u l p a n d is r e s p o n s i b l e in p a r t for t h e e x f o l i a t i o n o f t h e d e c i d u o u s t e e t h . F u r t h e r m o r e , it h a s been shown that collagenolytic activity d e r i v e d f r o m t h e p e r i o d o n t a l ligam e n t is r e s p o n s i b l e for t h e d e c r e a s e d amounts of dentin hydroxyproline p r o d u c t i o n seen in t h e r o o t s o f d e c i d uous teeth? The human deciduous d e n t a l p u l p a n d its a s s o c i a t e d collagenolytic activity may be responsible for t h e s e r e p o r t e d d e c r e a s e s as well. l)r. Alexander is assistant professor of children's dentistry,, State University of New York at Stony Brook, Stony Brook, NY 11794. Requests for reprints should be directed to the author.
References 1. Alexander, S.A., and Swerdloff, M. Collagenloytic activity of periodontal ligament during human deciduous root resorption. Pediatr Dent 1:226-229, 1979. 2. Alexander, S.A., and Swerdloff, M. Identification and localization of a mucopolysaccharidase in human deciduous teeth. J Dent Res 59:594-601, 1980. 3. Alexander, S.A., and Swerdloff, M. Collagenolytic activity of periodontal ligament and hydroxproline content during human
420
deciduous root resorption. J Periodont Res 15:434-443, 1980. 4. Alexander, S.A.; Swerdloff, M.; and Caputo, l,. Dental pulp mucopolysaccharidam: identification and role in tooth resorption. J l)cnt Res 59:14114-1408. 1980. 5. Sicher, H., and Bhaskar, S.N. Orban's oral histology and emb~'olog':', ed 7. St. Louis, C. V. Mosby (do., 1972, pp,97-159. 6. Weatherell, J.A., and Hargreaves, J.A. Effect of resorption on the: fluoride content of human deciduous teeth Arch Oral Biul 11:749-756, 1966. 7. Snskolne. W.A, and Bimstein, E. A histomorphological study of the shedding process of human deciduous teeth at various chronological stages. Arch Oral Biol 22:331-335, 1977. 8. Kramer, W.A., and Ireland, R.I,. Measurements of the primats' teeth. J Dent Child 26:252-261. 1959. 9 Engstrom, H., and Ohman, A, Studies on the innervation of human teeth. J Dent Res 39:799-809, 1960. 10. Gross, J., and lapiere, C.M. Collagenolyric activity in amphibian tissues: a tissue culture assay. Proe Nat Acad Sci 48:10141022, 1962. l l. Stegemann, H. Mikrobestimmung yon hydroxyprolin mot chloramin-T and p-l)imethylaminubenzaldehyd. Z Physiol Chem 311:41-45, 1958. 12. Fox, A.G., and Heely, J.D. I listolngical study of pulps of human primal' teeth. Arch Oral Biol 25:103-110, 1980. 13. Grillo, |I.C., and others. Collagenolytic activity in regenerating forelimbs of the aduh newt (Triturus viridescens). Develop Biol 17:571-583, 1968. 14. Eisen, AZ., and (;ross, J. 'Fhc role' of epithelium and mesenehyme in the production of a collagenolytic enzyme and a hyaluronidase in the anuran tadpole. Develop Biol
12:408-418, 1965. 15. 1)onoff, R.B.; McLennan. J.E.: and (]rillo, H.C. Preparation and propcrtlcs of collagenases from epithelium and mcsenchyme of healing mammalian v,ounds. Biochim Biophys Acta 227:639-653, 1971 16. Donnff, R.B., and Grillo. II.C. The effects of skin grafting on healing open wounds in rabbits. ,J Surg Res 19:163-167, 1975. 17. Donoff, RB. Wound healing: bir ical events and potential roh' of collagenase. J Oral Surg 28:356-362, 1970. 18. Golub, I,.M., and others. ('olla~en,lytic activity of crevicular fluid from pericoronal gingival flaps. J Dent Res 55:177-181, 1976. 19. Golub, LM., and others. Some characteristics of collagenase activity in gingival crevicular fluid and its relationship to gingival diseases in humans. J Dent Res 55:1040-1057, 1976. 20. Chrismer, P. Collagenase in the human periodontal ligament. J Perindontnl 51:.t55461. 1980. 21. Morita, tl., and nthcrs. The collagcnolyric activity during root resorption of bovine deciduous teeth. Arch Oral Biol 15:503-508, 19711. 22. Ozaki, T., amt others. Collagenulytic activity during tooth movement in the rabbit. Arch Oral Biol 16:1123-1126, 1971. 23. Bernick, S.; Rabinowitch. BZ.: and Rutherford, R.L. Microscopic studies of the teeth of a six-year-old boy. Tooth absorpcionAnat Res 105:249-258, 1949. 24. Obersztyn, A. Experimental investiga" lion of factors causing resorption of deciduous teeth..1 Dent Res 42:660-674, 1963. 25. Depnrter, D.A., and TenCate. A.R. Collagen resorption by periodontal ligament fibroblasts at the hard tissue-ligament inter" faces of the: mouse perindontmm. J Periodon~ tol 51:429-432, 1980.