JOURNAL OF ENDODONTICS J VOL 4, NO 1, JANUARY 1978
Reactions of the Living Organism to Dead and Fixed Dead Tissue
P. C. M a k k e s , DDS, PhD; S. K. T h o d e n v a n V e l z e n , DDS, PhD; a n d P. R. W e s s e l i n k , DDS, A m s t e r d a m
T w o series of e x p e r i m e n t s w e r e c o n d u c t e d o n the r e a c t i o n s of t h e living o r g a n i s m w h e n in c o n t a c t w i t h d e a d a n d fixed d e a d tissue. T h e i n f l a m m a t o r y r e a c t i o n to necrotic tissue w a s less s e v e r e t h a n the r e a c t i o n to fixed necrotic tissue. T h e results led to the t e n t a t i v e q o n c l u s i o n that c h r o n i c p e r i a p i c a l i n f l a m m a t i o n m o s t likely is c a u s e d b y living m i c r o o r g a n i s m s .
The reactions of the living organism to contact with dead and fixed dead tissue were examined to provide an answer to what maintains the chronic periapical inflammation that accompanies a necrotic pulp, and if it is possible to prevent or to cure the inflammation by fixing the necrotic pulpal tissue with formaldehyde or glutaraldehyde. In the current literature, ~ chronic apical periodontitis is blamed on an "agent" or "agents" that have no specific definition. The supposed agent seems to develop in the root canal as a result of pulp necrosis and of the activity of microorganisms that eventually occur. '-'a Based mainly on theoretical speculations, it is supposed that contaminated dead pulpal tissue, partially broken down by autolysis and the metabolism of the microorganisms, which are subsequently killed by a disinfectant, induces an inflamma-
tion in the apical periodontal tissues; and that sterile dead pulpal tissue, per se, also induces an inflammation in the apical periodontal tissues. As a result of these suppositions, the elimination of all dead pulpal tissue is the obvious and, without a doubt, the most rational treatment of apical periodontitis. In a n u m b e r of experiments of a divergent nature, however, it has been shown that it will never be possible to clean the root canal system completely.' It is therefore suggested by several authors to fix the remaining tissue remnants with formaldehyde or glutaraldehyde, and to consider that fixation of necrotic, sterile, or contaminated pulpal tissue may eliminate the inflammation-inducing agent. 4,~ In theory, this elimination could take place via three mechanisms, as follows: by binding the fixative to the agent, which eliminates the
inflammatory potential of the agent (detoxification); by binding the fixative to the pulpal tissue, which eliminates the production of the agent; or by binding the fixative to the contents of the root canal, including the agent, which produces coherent complexes that can no longer leave the root canal. In the first part of our investigation into the reactions of the living organism to contact with dead and fixed dead tissue, we used the following tissue model, which is described elsewhere in detail.' Standardized pieces of connective tissue were cultured by the subcutaneous implantation of pieces of polyvinylalcohol sponge in rabbits. These tissue fragments were, after removal, subjected to autolysis under sterile conditions, after which some were fixed in a solution of formaldehyde or in a solution of glutaraldehyde. The tissue fragments were then reim17
JOURNAL OF ENDODONTICS I VOL 4, NO 1, JANUARY 1978
planted in the same animal from which they originated and the reaction of the organism to the implants was studied (Fig 1). Because infected pulpal tissue, from which the contaminating microorganisms were killed, may be considered foreign to the organism, in one of the experiments the autologous tissue fragments (originating from the same animal) were replaced by heterologous tissue fragments (originating from an animal from another species). The experiments 6-1~performed with this tissue model produced the following results: -Implantation of sterile, autologous, autolytic connective tissue caused a transient inflammatory reaction of mild intensity; the inserted tissue was broken down and was replaced by vital tissue. -Implantation of sterile, heterologous, autolytic muscle tissue caused an inflammatory reaction of an immunological nature; the foreign tissue was broken down and replaced by vital tissue, which resulted in a transient reaction to the implant. --Implantation of formaldehydefixed, autologous, autolytic connective tissue caused an inflammatory reaction; it was shown that the reaction had an immunological nature and was especially effected through the T-cell system and in a lesser sense through the B-cell system; the fixed tissue was probably broken down completely and replaced by vital tissue. --Implantation of glutaraldehydefixed, autologous, autolytic connective tissue caused a chronic inflammatory reaction of mild intensity and probably, in the long term, an immunological reaction; the fixed tissue was broken down very slowly (at least a year) and was replaced by vital tissue. --Implantation of glutaraldehydefixed, heterologous, autolytic muscle tissue caused an inflammatory reaction that, aside from a delayed start, 18
~tee weeks'ingrowthof connective tissue in
subcutaneous
polyvinyb-alcoho] /
I
i ~I
sterile transfer of PVA-spongewith ingrowntissueto 10rnl physiological satlne
48 hours' ~
]
~
~
autolysis
tissue specimen, after
r ~
bacterla|oglca! exarnlnoHon, transferred to 25 ml formaldehydesolution (
I 1 days' f[xatlon
/
i
r ~
transfer of fixed tissue specimen
to 25 m| physloIog~ca|saline )
2 x 12hours' washing
fixed tissue specimen ~mplantedagainst
r ~
femur or tibia
,(
Fig 1--Diagram showing subsequent steps of experiment in which formalde,~yde-fixed tissue was implanted. Procedurefor gtutaratdehyde-fixed tissue was identical In procedurefor ut~xed tissue, implantation of specimen was done directly after 48-hour period of autolysis. differed little or none from the reaction to the implantation of unfixed tissue; the fixed tissue was probably also broken down and replaced very slowly, which resulted in a longlasting inflammation. These results gave reason to question the value of the suppositions, expressed in the literature, that the
cause of chronic periapical inflammation is situated in the sterile necrotic pulp and that fixation of the necrotic tissue will prevent or cure this inflammation. However, the spatial relationship of the experimental tissue model with the host tissues gives several severe limitations as compared with the dental root
JOURNAL OF ENDODONTICS I VOL 4, NO 1, JANUARY 1978
canal and its surrounding tissues. Because the clinical relevance of the experimental results was limited by this factor, we decided to repeat the experiments using a model that would allow a better comparison with the dental root canal. This second model consisted of a polyethylene tube with a length of 15 ram, closed at both ends with inlay wax and with four small holes in the sidewalls of the tube, each with a diameter of 0.6 m m (Fig 2). ~1 The tubes were filled with isologous (originating from a genetical identical animal) and homologous (originating from an animal from the same species), unfixed or fixed, muscle tissue and were subsequently implanted in the subcutaneous connective tissue of rats. The homologous muscle tissue in this model served as a substitute for the compilation of foreign proteins that can be found in contaminated pulpal tissue. The several experiments '~1:~ (P. C. Makkes, S. K. Thoden van Velzen, and A. Van den Hooff, unpublished data, 1977) that we performed with this model produced the following results: - B o t h isologous and homologous, sterile, autolytic muscle tissue caused
a slight, transient, inflammatory reaction around the tube model; the autolytic tissue was not broken down or replaced because ingrowth of the host tissue through the holes of the tube only takes place over a very short distance (Fig 3). --Isologous and homologous muscle tissue, fixed with formaldehyde, caused a chronic inflammatory reaction; ingrowth of host tissue did not occur within the limited time of the experiment (Fig 4). --Isologous and homologous mus-
cle tissue, fixed with glutaraldehyde, caused severe and chronic inflammatory reactions characterized by great numbers of eosinophilic granulocytes and by necrosis of the host tissues; ingrowth of host tissue also did not occur within the experimental time (Fig 5). Provided that any scientific experiment with an experimental model includes unavoidable restrictions, it seems warranted to connect the following conclusions to the results of our studies: the chronic periapical
Fig 3--Ingrowth of connective tissue in po~ethylene tube containing homologous sterile necrotic tissue after 21 days of implantation: t, original site of tube; m, muscle tissue of host animal (H&E, orig mag
x 4o).
Fig 2--Schematic drawing of polyethylene tube used as model for dental root canal.
19
JOURNAL OF ENDODONTICS I VOL 4, NO 1, JANUARY 1978
Fig 4--Part of tissues surrounding polyethylene tube containing formaldehyde-fixed homologous tissue after 21 days of implantation: t, original site of tube; m, muscle tissue of host animal; n, necrotic musclefragments of host animal (H&E, orig mag x lo).
Fig 5-Part of tissues surrounding polyethylene tube containing glutaraldehydefixed isologous tissue after 21 days of implantation: t, original site of tube; c, conglomeration containing granulocytes andfat cells (H&E, orig mag • 10).
20
inflammation is not caused by the necrotic pulpal tissue, per se; in the case of a chronic periapical inflammation, the necrotic pulpal tissue is contaminated with living microorganisms; and fixation with formaldehyde or glutaraldehyde of contaminated pulpal tissue remnants kills the microorganisms but makes the disinfected contents of the pulp cavity noxious, which perhaps leads to chronic inflammatory processes in the periapical tissues. SUMMARY
Two series of experiments that involved the reactions of the living organism to contact with dead and fixed dead tissue led us to the following conclusion: chronic periapical inflammation is not caused by necrotic pulpal tissue, per se, but most probably by products of the metabolism of living microorganisms, which are contaminating the necrotic pulpal tissue; and fixation of contaminated pulpal tissue remnants will perhaps lead to chronic inflammatory reactions in the periodontal tissues.
JOURNAL OF ENDODONTICS t VOL 4, NO 1, JANUARY 1978
This paper was presented at the annual meeting of the American Association of Endodontists on April 15, 1977, in Houston, Tex. The authors thank Mr. L. Kenter and his colleagues, Mr. A. J. Lammens, Dr. A. D. P. Heyboer, Mr. A. J. Dons, Mr. J. P. L. Rijss, and also Dr. S. R. Fox, and Ms. V. Bressers. Dr. Makkes is assistant professor, Dr. Thoden van Velzen is professor and department head, and Dr. Wesselink is instructor in the department of cariology and endodontology of the University of Amsterdam, Requests for reprints should be directed to Dr. Makkes, Tandheelkunde, Louwesweg 1, Amsterdam, The Netherlands.
References 1. Thoden van Velzen, S.K. The influence of dead and fixed-dead tissue in the living organism. I. Problem statement and selection of a method of im,estigation. Neth Dent J 82:6, 1975.
2. Ogilvie, A.L. Periapical pathosis. In Ingle, J.I. (ed.). Endodontics, ed 2. Philadelphia, Lea & Febiger, 1976, pp 394-397. 3. Seltzer, S. Endodontology. Biologic considerations in endodontic procedures. New York, McGraw-Hill, 1971, pp 197-209. 4. Buckley, J.P. The chemistry of pulp decomposition, with a rational treatment for this condition and its sequelae9 Am Dent J 3:764, 1904. 5. 's-Gravenmade, EJ. Some biochemical considerations of fixation in endodontics. J Endod 1:233 July 1975. 6. Thoden van Velzen, S.K., and Van den Hooff, A. The influence of dead and fixeddead tissue in the living organism. II. The tissue reaction to implantation of autologous dead tissue. Neth Dent J 82:23, 1975. 7. Thoden van Velzen S.K., and Van den Hooff, A. The influence of dead and fixeddead tissue in the living organism. III. The tissue reaction to implantation of autologous dead tissue fixed with formaldehyde or glutaraldehyde. Neth Dent J 82:6, 1975.
8. Thoden van Velzen, S.K., and Feltkamp-Vroom, T,M. Immunologic consequences of formaldhyde fixation of autologous tissue implants. J Endod 3:179 May 1977. 9. Thoden van Velzen, S.K., and Van den Hooff, A. Long-term results of the implantation of glutaraldehyde-fixed tissue. Oral Surg, to be published. 10. Wesselink, P.R.; Thoden van Velzen, S.K.; Van den Hooff, A. Tissue reaction to implantation of unfixed and glutaraldehydefixed heterologous tissue. J Endod 3:229 June 1977. 11. Makkes, P. C., and others. Polyethylene tubes as a model for the root canal. Oral Surg 44:293 Aug 1977. 12. Makkes, P. C.; Thoden van Velzen, S.K.; and Van den Hooff, A. The response of the living organism to dead and fixed dead, enclosed isologous tissue. Oral Surg, to be published. 13. Makkes, P. G.; Thoden van Velzen, S.K.; and Van den Hooff, A. The response of the living organism to dead and fixed dead, enclosed homologous tissue. Oral Surg, to be published.
21
Reactions of the Living Organism to Dead and Fixed Dead Tissue
P. C. M a k k e s , DDS, PhD; S. K. T h o d e n v a n V e l z e n , DDS, PhD; a n d P. R. W e s s e l i n k , DDS, A m s t e r d a m
T w o series of e x p e r i m e n t s w e r e c o n d u c t e d o n the r e a c t i o n s of t h e living o r g a n i s m w h e n in c o n t a c t w i t h d e a d a n d fixed d e a d tissue. T h e i n f l a m m a t o r y r e a c t i o n to necrotic tissue w a s less s e v e r e t h a n the r e a c t i o n to fixed necrotic tissue. T h e results led to the t e n t a t i v e q o n c l u s i o n that c h r o n i c p e r i a p i c a l i n f l a m m a t i o n m o s t likely is c a u s e d b y living m i c r o o r g a n i s m s .
The reactions of the living organism to contact with dead and fixed dead tissue were examined to provide an answer to what maintains the chronic periapical inflammation that accompanies a necrotic pulp, and if it is possible to prevent or to cure the inflammation by fixing the necrotic pulpal tissue with formaldehyde or glutaraldehyde. In the current literature, ~ chronic apical periodontitis is blamed on an "agent" or "agents" that have no specific definition. The supposed agent seems to develop in the root canal as a result of pulp necrosis and of the activity of microorganisms that eventually occur. '-'a Based mainly on theoretical speculations, it is supposed that contaminated dead pulpal tissue, partially broken down by autolysis and the metabolism of the microorganisms, which are subsequently killed by a disinfectant, induces an inflamma-
tion in the apical periodontal tissues; and that sterile dead pulpal tissue, per se, also induces an inflammation in the apical periodontal tissues. As a result of these suppositions, the elimination of all dead pulpal tissue is the obvious and, without a doubt, the most rational treatment of apical periodontitis. In a n u m b e r of experiments of a divergent nature, however, it has been shown that it will never be possible to clean the root canal system completely.' It is therefore suggested by several authors to fix the remaining tissue remnants with formaldehyde or glutaraldehyde, and to consider that fixation of necrotic, sterile, or contaminated pulpal tissue may eliminate the inflammation-inducing agent. 4,~ In theory, this elimination could take place via three mechanisms, as follows: by binding the fixative to the agent, which eliminates the
inflammatory potential of the agent (detoxification); by binding the fixative to the pulpal tissue, which eliminates the production of the agent; or by binding the fixative to the contents of the root canal, including the agent, which produces coherent complexes that can no longer leave the root canal. In the first part of our investigation into the reactions of the living organism to contact with dead and fixed dead tissue, we used the following tissue model, which is described elsewhere in detail.' Standardized pieces of connective tissue were cultured by the subcutaneous implantation of pieces of polyvinylalcohol sponge in rabbits. These tissue fragments were, after removal, subjected to autolysis under sterile conditions, after which some were fixed in a solution of formaldehyde or in a solution of glutaraldehyde. The tissue fragments were then reim17
JOURNAL OF ENDODONTICS I VOL 4, NO 1, JANUARY 1978
planted in the same animal from which they originated and the reaction of the organism to the implants was studied (Fig 1). Because infected pulpal tissue, from which the contaminating microorganisms were killed, may be considered foreign to the organism, in one of the experiments the autologous tissue fragments (originating from the same animal) were replaced by heterologous tissue fragments (originating from an animal from another species). The experiments 6-1~performed with this tissue model produced the following results: -Implantation of sterile, autologous, autolytic connective tissue caused a transient inflammatory reaction of mild intensity; the inserted tissue was broken down and was replaced by vital tissue. -Implantation of sterile, heterologous, autolytic muscle tissue caused an inflammatory reaction of an immunological nature; the foreign tissue was broken down and replaced by vital tissue, which resulted in a transient reaction to the implant. --Implantation of formaldehydefixed, autologous, autolytic connective tissue caused an inflammatory reaction; it was shown that the reaction had an immunological nature and was especially effected through the T-cell system and in a lesser sense through the B-cell system; the fixed tissue was probably broken down completely and replaced by vital tissue. --Implantation of glutaraldehydefixed, autologous, autolytic connective tissue caused a chronic inflammatory reaction of mild intensity and probably, in the long term, an immunological reaction; the fixed tissue was broken down very slowly (at least a year) and was replaced by vital tissue. --Implantation of glutaraldehydefixed, heterologous, autolytic muscle tissue caused an inflammatory reaction that, aside from a delayed start, 18
~tee weeks'ingrowthof connective tissue in
subcutaneous
polyvinyb-alcoho] /
I
i ~I
sterile transfer of PVA-spongewith ingrowntissueto 10rnl physiological satlne
48 hours' ~
]
~
~
autolysis
tissue specimen, after
r ~
bacterla|oglca! exarnlnoHon, transferred to 25 ml formaldehydesolution (
I 1 days' f[xatlon
/
i
r ~
transfer of fixed tissue specimen
to 25 m| physloIog~ca|saline )
2 x 12hours' washing
fixed tissue specimen ~mplantedagainst
r ~
femur or tibia
,(
Fig 1--Diagram showing subsequent steps of experiment in which formalde,~yde-fixed tissue was implanted. Procedurefor gtutaratdehyde-fixed tissue was identical In procedurefor ut~xed tissue, implantation of specimen was done directly after 48-hour period of autolysis. differed little or none from the reaction to the implantation of unfixed tissue; the fixed tissue was probably also broken down and replaced very slowly, which resulted in a longlasting inflammation. These results gave reason to question the value of the suppositions, expressed in the literature, that the
cause of chronic periapical inflammation is situated in the sterile necrotic pulp and that fixation of the necrotic tissue will prevent or cure this inflammation. However, the spatial relationship of the experimental tissue model with the host tissues gives several severe limitations as compared with the dental root
JOURNAL OF ENDODONTICS I VOL 4, NO 1, JANUARY 1978
canal and its surrounding tissues. Because the clinical relevance of the experimental results was limited by this factor, we decided to repeat the experiments using a model that would allow a better comparison with the dental root canal. This second model consisted of a polyethylene tube with a length of 15 ram, closed at both ends with inlay wax and with four small holes in the sidewalls of the tube, each with a diameter of 0.6 m m (Fig 2). ~1 The tubes were filled with isologous (originating from a genetical identical animal) and homologous (originating from an animal from the same species), unfixed or fixed, muscle tissue and were subsequently implanted in the subcutaneous connective tissue of rats. The homologous muscle tissue in this model served as a substitute for the compilation of foreign proteins that can be found in contaminated pulpal tissue. The several experiments '~1:~ (P. C. Makkes, S. K. Thoden van Velzen, and A. Van den Hooff, unpublished data, 1977) that we performed with this model produced the following results: - B o t h isologous and homologous, sterile, autolytic muscle tissue caused
a slight, transient, inflammatory reaction around the tube model; the autolytic tissue was not broken down or replaced because ingrowth of the host tissue through the holes of the tube only takes place over a very short distance (Fig 3). --Isologous and homologous muscle tissue, fixed with formaldehyde, caused a chronic inflammatory reaction; ingrowth of host tissue did not occur within the limited time of the experiment (Fig 4). --Isologous and homologous mus-
cle tissue, fixed with glutaraldehyde, caused severe and chronic inflammatory reactions characterized by great numbers of eosinophilic granulocytes and by necrosis of the host tissues; ingrowth of host tissue also did not occur within the experimental time (Fig 5). Provided that any scientific experiment with an experimental model includes unavoidable restrictions, it seems warranted to connect the following conclusions to the results of our studies: the chronic periapical
Fig 3--Ingrowth of connective tissue in po~ethylene tube containing homologous sterile necrotic tissue after 21 days of implantation: t, original site of tube; m, muscle tissue of host animal (H&E, orig mag
x 4o).
Fig 2--Schematic drawing of polyethylene tube used as model for dental root canal.
19
JOURNAL OF ENDODONTICS I VOL 4, NO 1, JANUARY 1978
Fig 4--Part of tissues surrounding polyethylene tube containing formaldehyde-fixed homologous tissue after 21 days of implantation: t, original site of tube; m, muscle tissue of host animal; n, necrotic musclefragments of host animal (H&E, orig mag x lo).
Fig 5-Part of tissues surrounding polyethylene tube containing glutaraldehydefixed isologous tissue after 21 days of implantation: t, original site of tube; c, conglomeration containing granulocytes andfat cells (H&E, orig mag • 10).
20
inflammation is not caused by the necrotic pulpal tissue, per se; in the case of a chronic periapical inflammation, the necrotic pulpal tissue is contaminated with living microorganisms; and fixation with formaldehyde or glutaraldehyde of contaminated pulpal tissue remnants kills the microorganisms but makes the disinfected contents of the pulp cavity noxious, which perhaps leads to chronic inflammatory processes in the periapical tissues. SUMMARY
Two series of experiments that involved the reactions of the living organism to contact with dead and fixed dead tissue led us to the following conclusion: chronic periapical inflammation is not caused by necrotic pulpal tissue, per se, but most probably by products of the metabolism of living microorganisms, which are contaminating the necrotic pulpal tissue; and fixation of contaminated pulpal tissue remnants will perhaps lead to chronic inflammatory reactions in the periodontal tissues.
JOURNAL OF ENDODONTICS t VOL 4, NO 1, JANUARY 1978
This paper was presented at the annual meeting of the American Association of Endodontists on April 15, 1977, in Houston, Tex. The authors thank Mr. L. Kenter and his colleagues, Mr. A. J. Lammens, Dr. A. D. P. Heyboer, Mr. A. J. Dons, Mr. J. P. L. Rijss, and also Dr. S. R. Fox, and Ms. V. Bressers. Dr. Makkes is assistant professor, Dr. Thoden van Velzen is professor and department head, and Dr. Wesselink is instructor in the department of cariology and endodontology of the University of Amsterdam, Requests for reprints should be directed to Dr. Makkes, Tandheelkunde, Louwesweg 1, Amsterdam, The Netherlands.
References 1. Thoden van Velzen, S.K. The influence of dead and fixed-dead tissue in the living organism. I. Problem statement and selection of a method of im,estigation. Neth Dent J 82:6, 1975.
2. Ogilvie, A.L. Periapical pathosis. In Ingle, J.I. (ed.). Endodontics, ed 2. Philadelphia, Lea & Febiger, 1976, pp 394-397. 3. Seltzer, S. Endodontology. Biologic considerations in endodontic procedures. New York, McGraw-Hill, 1971, pp 197-209. 4. Buckley, J.P. The chemistry of pulp decomposition, with a rational treatment for this condition and its sequelae9 Am Dent J 3:764, 1904. 5. 's-Gravenmade, EJ. Some biochemical considerations of fixation in endodontics. J Endod 1:233 July 1975. 6. Thoden van Velzen, S.K., and Van den Hooff, A. The influence of dead and fixeddead tissue in the living organism. II. The tissue reaction to implantation of autologous dead tissue. Neth Dent J 82:23, 1975. 7. Thoden van Velzen S.K., and Van den Hooff, A. The influence of dead and fixeddead tissue in the living organism. III. The tissue reaction to implantation of autologous dead tissue fixed with formaldehyde or glutaraldehyde. Neth Dent J 82:6, 1975.
8. Thoden van Velzen, S.K., and Feltkamp-Vroom, T,M. Immunologic consequences of formaldhyde fixation of autologous tissue implants. J Endod 3:179 May 1977. 9. Thoden van Velzen, S.K., and Van den Hooff, A. Long-term results of the implantation of glutaraldehyde-fixed tissue. Oral Surg, to be published. 10. Wesselink, P.R.; Thoden van Velzen, S.K.; Van den Hooff, A. Tissue reaction to implantation of unfixed and glutaraldehydefixed heterologous tissue. J Endod 3:229 June 1977. 11. Makkes, P. C., and others. Polyethylene tubes as a model for the root canal. Oral Surg 44:293 Aug 1977. 12. Makkes, P. C.; Thoden van Velzen, S.K.; and Van den Hooff, A. The response of the living organism to dead and fixed dead, enclosed isologous tissue. Oral Surg, to be published. 13. Makkes, P. G.; Thoden van Velzen, S.K.; and Van den Hooff, A. The response of the living organism to dead and fixed dead, enclosed homologous tissue. Oral Surg, to be published.
21
