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Reconstituted antigen-poor collagen preparations as potential pulp-capping agents
JOURNAL OF ENDODONTICS [ VOL 6, NO 7, JULY 1980
Reconstituted antigen-poor collagen preparations as potential pulp-capping agents H. M . D i c k , DDS, MSc, a n d D . J. C a r m i c h a e l , PhD
W e t collagen s p o n g e and w e t collagen fabric are b e t t e r t o l e r a t e d as pulp c a p p i n g materials t h a n d r y collagen s p o n g e or d r y collagen fabric. D e n t i n bridge f o r m a t i o n s e e m s to o c c u r o n l y w h e n an area of surface necrosis s u b s e q u e n t l y u n d e r g o e s d y s t r o p h i c calcification.
It is generally accepted that the accidentally exposed vital dental pulp can be treated with a high degree of success using a calcium hydroxide capping agent. Although the precise mechanism by which calcium hydroxide produces this effect is not understood, it has been determined that the calcium ion from the calcium hydroxide does not contribute directly to the mineralization of the dentin in the repair process? It is probable that its effectiveness may be related, in part, to the high pH of the material, which induces a localized tissue necrosis. ~-'3After dystrophic mineralization of this necrotic tissue, reparative-type dentin is deposited on the surface of the wounded pulp and this leads eventually to dentin repair of exposed pulp. The details and possible complications of this process have been reviewed extensively.4-g A material that could induce reparative dentin formation without tissue necrosis would be an improvement over the calcium hydroxide method. As collagen fibers are able to orient hydroxyapatite crystals and therefore influence mineralization, it is reasonable to evaluate collagen experimentally as a possible pulp capping agent.
The antigenicity of native collagen, however, is a limiting factor in its use as a heterograft material. In a previously reported study, ~ lyophilized, acid-soluble skin collagen, which had been modified enzymatically to reduce its antigenicity, was applied to experimentally exposed vital canine pulps of dogs to investigate its potential for inducing the formation of a mineral bridge. Although this antigenically modified collagen could induce crystallization under in vitro conditions, it did not undergo mineralization during the eight-week experimental period. Furthermore, pulp tissue in contact with the modified collagen had an inflammatory response that was not unlike that seen in certain antigen-induced inflammatory reactions. It seemed logical, therefore, to examine pulp reaction to a three-dimensional framework of immobilized, reconstituted collagen material rendered minimally antigenic. Theoretically, the porosity of the collagen should permit infiltration of mesenchymal pulp cells and, in juxtaposition to dentin, mineralization. MATERIALS AND METHODS Two types of collagen samples were obtained. The first type was a
highly porous collagen sponge prepared by lyophilizing a mixture of bovine skin collagen, water, and glutaraldehyde that had been formed as a foam in a high-speed blender. 1'' The second type was a relatively nonporous collagen fabric formed by extrusion as described by Chvapil and others. 11 Both materials were only minimally antigenic and were ~sorbable as subcutaneous implants in eight to 12 weeks. During the experiment, these materials were at times wetted by soaking them in a phosphate buffer containing 2.96 mM calcium chloride in a solution that permitted nucleation of collagen in vitro by calcium phosphate salt? -~ Five young adult mongrel dogs weighing from 10 to 12 kg were anesthetized by injecting sodium p e n t o b a r b i t a l intraperitoneally. Their canine teeth were isolated with a rubber dam and cleansed with tincture of nitromersol (Metaphen) and alcohol. Class V type preparations were cut in the labial surface above the level of the gingiva with a water-cooled, air-driven dental handpiece. A slow-speed handpiece was then used to remove part of the floor of the cavity, exposing approximately 1 mm of the pulp. After control of bleeding with 10% H202. 641
the pulp wounds were covered with a collagen sample as described in the Table. The cavity was then sealed with zinc oxide-eugenol cement and amalgam. Dogs 1 and 2 were killed four weeks after treatment, and dogs 3, 4, and 5 were killed eight weeks after treatment. The treated teeth were removed from the jaws, sectioned horizontally approximately 3 m m above and 3 m m below the cavity preparation, and fixed for 72 hours in 10% neutral buffered Formalin. After demineralization in a formic acid-sodium citrate solution, the specimens were embedded in paraffin. Serial sections 6 to 7p, in thickness were cut and stained with hematoxylin and eosin.
RESULTS After four weeks, the pulp next to the area capped with the wet collagen sponge showed mild to moderate edema and congestion but a notable lack of polymorphonuclear cell infiltration. A few giant cells and moderate numbers of macrophages were present at the interface of the wet collagen sponge and the pulp. Increased secondary dentin formation was apparent peripheral to the area of injury (Fig 1). At eight weeks, the pulps treated with the wet collagen sponge showed changes only at the site of injury.
Fig 1--Four weeks after pulp capping with collagen sponge (WCS) soaked in phosphate buffer, pulp tissue shows minimal inflammatory changes. Reparative dentin (SD) is forming at margin of lesion (H&E, orig mag x lO).
Three of the four pulps showed a zone of necrosis at the interface of the wet collagen sponge and the pulp, and two showed reparative dentin formation associated with the zone of necrosis (Fig 2). The fourth pulp showed a well-defined zone of inflammation at the interface with no evidence of necrosis or calcification. At four weeks, pulps treated with the dry collagen sponge showed moderate to noticeable edema and congestion with infiltration of polymorphonuclear leukocytes. Some extravasation of RBCs had occurred and hemosiderin-laden macrophages were seen throughout the pulp tissue. Dentin formation was arrested and some areas of the odontoblast layer had undergone fibrosis (Fig 3). One of the four pulps was totally replaced by an eosinophilic plasma coagulum
Table 9 Collagen samples applied to pulp wounds in dogs.
Animal no..
1, 2, 3, 4 5
642
Fig 2-Eight weeks after pulp capping with collagen sponge soaked in phosphate buffer (WCS), zone of reparative dentin (RD) is forming deep to necrotic zone (NZ) (H&E, orig mag X 40).
Maxillary and mandibular right canines Wet collagen sponge Wet collagen fabric
Maxillary and mandibular left canines Dry collagen sponge Dry collagen fabric
Fig 3-Four weeks after treatment with d~y collagen sponge, mixed inflammatory cell infiltration is seen throughout pulp tissue (P). Odontoblast layer has undergone generalized fibrosis (Y) (H&E, orig mag • 25). with a few interspersed inflammatory cells. Eight weeks after capping of the exposed pulp with the dry collagen sponge, the histologic picture of the pulp was almost normal, except for the exposure site, where fibrosis was apparent along the pulp wall, and a sharply demarcated zone of polymorphonuclear leukocytes, giant cells, and macrophages was seen at the interface of the dry collagen sponge and the pulp. In one tooth there was reparative dentin formation at the margin of the injury but
JOURNAL OF E N D O D O N T I C S [ VOL 6, N O 7, JULY 1980
pulp treated successfully with calcium hydroxide showed a prominent zone of necrosis with dystrophic calcification on the pulp surface four weeks after injury. By six weeks, a well-defined reparative dentin bridge was forming (Fig 4). DISCUSSION AND CONCLUSIONS
Fig 4--Six weeks after treatment with calcium hydroxide, well-defined bridge of reparative dentin (RD) is forming in association with poorly defined zone of tissue necrosis (NZ) (H&E, orig mag • 40).
there was no evidence of a dentin bridge. In dog 5, the two pulps treated with the dry collagen sponge were necrotic. The two pulps treated with the wet collagen fabric showed diffuse infiltration with chronic and acute inflammatory cells and peripheral areas of fibrosis, irregular dentin formation, and dentin resorption. At the interface between the pulp and the wet collagen fabric, several fragments of the fabric appeared to have undergone calcification and were covered with a reparative-type dentin. The results of our previous study 9 were used for purposes of comparison. The experimental method of exposing the pulp in canines of dogs was similar in all respects to the present experimental method except that the exposed pulp was treated with calcium hydroxide (Dycal). The
The results of this study indicate that, as pulp-capping materials, wet collagen sponge and wet collagen fabric are tolerated better than dry collagen sponge and dry collagen fabric, and that the collagen sponge product is tolerated better than the collagen fabric product. The pulps treated with wet collagen sponge showed a low-grade inflammatory response with evidence of only localized necrosis in all of the teeth tested. At eight weeks, three of four pulps showed a zone of necrosis at the interface of the wet collagen sponge and pulp, and two also showed early calcified bridge formation. The degenerative pulp reactions that are often seen in pulp treated with calcium hydroxide ~'9 were not seen in pulps treated with collagen sponge, which suggests that collagen sponge is relatively less irritating to the pulp tissue. However, if we compare the results of the current study with the results with calcium hydroxide in the earlier study, 9 we must make the following conclusion: calcium hydroxide is a more effective promoter of dentin bridge repair in experimentally exposed dog canine pulps than are the collagen materials that we tested in this study. Comparison of the reaction of injured pulp to the collagen gels used in the earlier experimenff with the reaction to the collagen sponge and the collagen fabric used in the cur-
rent experiment indicates that the pulp tissue is less tolerant to the collagen gel. We have not determined that the collagen gel is more of an irritant because of its higher antigenicity, but we suspect that it may be so. Unfortunately, there is also no evidence from the results of this study that the suggested cytophilic nature of the three-dimensional collagen sponge in any way enhances healing of the wounded pulp surface. In all of the collagen materials tested so far, it appears that formation of the clentin bridge occurs only when an area of surface necrosis is formed which subsequently undergoes dystrophic calcification. The rationale for using altered native collagen to enhance reparative dentin formation in wounded pulp tissue may be pursued from two perspectives. First, it is known that collagen fibers can catalyze calcium phosphate crystallization from physiologic concentrations of calcium and phosphate ions in vitro. Second, a three-dimensional collagenous framework can be formed which is "liked" by cells and, therefore, m a y be used as a framework in which the healing process may be initiated--a role not dissimilar to the fibrin clot seen in normal tissue healing (M. Chvapil, personal communication, June 1974). These two principles have been proved in bone reconstruction experiments and in tissue culture systems, but they do not appear to apply when the collagen materials are used as pulp-capping agents. This study was supported by grant MT3540 from the Canadian MRG. Dr. Dick is professorand chairman, division of oral pathology, Faculty of Dentistry, and Dr. Garmichael is director of graduate studies 643
JOURNAL OF ENDODONTICS I VOL 6, NO 7, JULY 1980
and research, Faculty of Dentistry, University of Alberta. Requests for reprints should be directed to Dr. Dick, 5085 Dentistry-Pharmacy Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2N8.
References 1. Sciaky, 1., and Pisanti, S. Localization of calcium placed over amputated pulps in dog's teeth. J Dent Res 39:1128-1132, 1960. 2. Clarke, N.G. The morphology of the reparative dentine bridge. Oral Surg 29:746752, 1970. 3. Nyborg, H. Healing processes in the pulp on capping. A morphologic study. Experiments on surgical lesions of the pulp in dog and man. Acta Odontol Scand (suppl 16), 13:9-130, 1955.
644
4. Ripp, N. Pulp capping. A review. NY State Dent J 42 (5):285-290, 1976. 5. Tronstad, L. Reactions of the exposed pulp to Dycal treatment. Oral Surg 38:945953, 1974. 6. McWalter, G.M.; EI-Kafrawy, A.H.; and Mitchell, D.F. Long-term study of pulp capping in monkeys with three agents. JADA 93(1):105-110, 1976. 7. Joos, R.W. Calcium hydroxide as a pulpcapping agent. Northwest Dent 53:362-365, 1974. 8. Schroder, U. Effect of an extra-pulpal blood clot on healing following experimental pulpotomy and capping with calcium hydroxide. Odontol Revy 24:257-268, 1973. 9. Carmichael, D.J.; Dick, H.M.; and
Dodd, C.M. Histologic effects of antigenically altered collagen as a heterograft for mammalian pulp exposures. Arch Oral Biol 19(12):1121-1126, 1974. 10. Chvapil, M., and Holusa, R. Experimental experiences with the collagen sponge as hemostaticum and tampon. J Biomed Mater Res 2:245-264, 1968. 11. Chvapil, M.; Owen, J.A.; and Clark, D.S. Effect of collagen crosslinking on the rate of resorption of implanted collagen tubing in rabbits. J Biomed Mater Res 11(2):297-314, 1977. 12. Davis, N.R. and Walker, T.E. The r01e of carboxyl groups in collagen calcification. Biochem Biophys Res Commun 48:1656-1662, 1972.
Reconstituted antigen-poor collagen preparations as potential pulp-capping agents H. M . D i c k , DDS, MSc, a n d D . J. C a r m i c h a e l , PhD
W e t collagen s p o n g e and w e t collagen fabric are b e t t e r t o l e r a t e d as pulp c a p p i n g materials t h a n d r y collagen s p o n g e or d r y collagen fabric. D e n t i n bridge f o r m a t i o n s e e m s to o c c u r o n l y w h e n an area of surface necrosis s u b s e q u e n t l y u n d e r g o e s d y s t r o p h i c calcification.
It is generally accepted that the accidentally exposed vital dental pulp can be treated with a high degree of success using a calcium hydroxide capping agent. Although the precise mechanism by which calcium hydroxide produces this effect is not understood, it has been determined that the calcium ion from the calcium hydroxide does not contribute directly to the mineralization of the dentin in the repair process? It is probable that its effectiveness may be related, in part, to the high pH of the material, which induces a localized tissue necrosis. ~-'3After dystrophic mineralization of this necrotic tissue, reparative-type dentin is deposited on the surface of the wounded pulp and this leads eventually to dentin repair of exposed pulp. The details and possible complications of this process have been reviewed extensively.4-g A material that could induce reparative dentin formation without tissue necrosis would be an improvement over the calcium hydroxide method. As collagen fibers are able to orient hydroxyapatite crystals and therefore influence mineralization, it is reasonable to evaluate collagen experimentally as a possible pulp capping agent.
The antigenicity of native collagen, however, is a limiting factor in its use as a heterograft material. In a previously reported study, ~ lyophilized, acid-soluble skin collagen, which had been modified enzymatically to reduce its antigenicity, was applied to experimentally exposed vital canine pulps of dogs to investigate its potential for inducing the formation of a mineral bridge. Although this antigenically modified collagen could induce crystallization under in vitro conditions, it did not undergo mineralization during the eight-week experimental period. Furthermore, pulp tissue in contact with the modified collagen had an inflammatory response that was not unlike that seen in certain antigen-induced inflammatory reactions. It seemed logical, therefore, to examine pulp reaction to a three-dimensional framework of immobilized, reconstituted collagen material rendered minimally antigenic. Theoretically, the porosity of the collagen should permit infiltration of mesenchymal pulp cells and, in juxtaposition to dentin, mineralization. MATERIALS AND METHODS Two types of collagen samples were obtained. The first type was a
highly porous collagen sponge prepared by lyophilizing a mixture of bovine skin collagen, water, and glutaraldehyde that had been formed as a foam in a high-speed blender. 1'' The second type was a relatively nonporous collagen fabric formed by extrusion as described by Chvapil and others. 11 Both materials were only minimally antigenic and were ~sorbable as subcutaneous implants in eight to 12 weeks. During the experiment, these materials were at times wetted by soaking them in a phosphate buffer containing 2.96 mM calcium chloride in a solution that permitted nucleation of collagen in vitro by calcium phosphate salt? -~ Five young adult mongrel dogs weighing from 10 to 12 kg were anesthetized by injecting sodium p e n t o b a r b i t a l intraperitoneally. Their canine teeth were isolated with a rubber dam and cleansed with tincture of nitromersol (Metaphen) and alcohol. Class V type preparations were cut in the labial surface above the level of the gingiva with a water-cooled, air-driven dental handpiece. A slow-speed handpiece was then used to remove part of the floor of the cavity, exposing approximately 1 mm of the pulp. After control of bleeding with 10% H202. 641
the pulp wounds were covered with a collagen sample as described in the Table. The cavity was then sealed with zinc oxide-eugenol cement and amalgam. Dogs 1 and 2 were killed four weeks after treatment, and dogs 3, 4, and 5 were killed eight weeks after treatment. The treated teeth were removed from the jaws, sectioned horizontally approximately 3 m m above and 3 m m below the cavity preparation, and fixed for 72 hours in 10% neutral buffered Formalin. After demineralization in a formic acid-sodium citrate solution, the specimens were embedded in paraffin. Serial sections 6 to 7p, in thickness were cut and stained with hematoxylin and eosin.
RESULTS After four weeks, the pulp next to the area capped with the wet collagen sponge showed mild to moderate edema and congestion but a notable lack of polymorphonuclear cell infiltration. A few giant cells and moderate numbers of macrophages were present at the interface of the wet collagen sponge and the pulp. Increased secondary dentin formation was apparent peripheral to the area of injury (Fig 1). At eight weeks, the pulps treated with the wet collagen sponge showed changes only at the site of injury.
Fig 1--Four weeks after pulp capping with collagen sponge (WCS) soaked in phosphate buffer, pulp tissue shows minimal inflammatory changes. Reparative dentin (SD) is forming at margin of lesion (H&E, orig mag x lO).
Three of the four pulps showed a zone of necrosis at the interface of the wet collagen sponge and the pulp, and two showed reparative dentin formation associated with the zone of necrosis (Fig 2). The fourth pulp showed a well-defined zone of inflammation at the interface with no evidence of necrosis or calcification. At four weeks, pulps treated with the dry collagen sponge showed moderate to noticeable edema and congestion with infiltration of polymorphonuclear leukocytes. Some extravasation of RBCs had occurred and hemosiderin-laden macrophages were seen throughout the pulp tissue. Dentin formation was arrested and some areas of the odontoblast layer had undergone fibrosis (Fig 3). One of the four pulps was totally replaced by an eosinophilic plasma coagulum
Table 9 Collagen samples applied to pulp wounds in dogs.
Animal no..
1, 2, 3, 4 5
642
Fig 2-Eight weeks after pulp capping with collagen sponge soaked in phosphate buffer (WCS), zone of reparative dentin (RD) is forming deep to necrotic zone (NZ) (H&E, orig mag X 40).
Maxillary and mandibular right canines Wet collagen sponge Wet collagen fabric
Maxillary and mandibular left canines Dry collagen sponge Dry collagen fabric
Fig 3-Four weeks after treatment with d~y collagen sponge, mixed inflammatory cell infiltration is seen throughout pulp tissue (P). Odontoblast layer has undergone generalized fibrosis (Y) (H&E, orig mag • 25). with a few interspersed inflammatory cells. Eight weeks after capping of the exposed pulp with the dry collagen sponge, the histologic picture of the pulp was almost normal, except for the exposure site, where fibrosis was apparent along the pulp wall, and a sharply demarcated zone of polymorphonuclear leukocytes, giant cells, and macrophages was seen at the interface of the dry collagen sponge and the pulp. In one tooth there was reparative dentin formation at the margin of the injury but
JOURNAL OF E N D O D O N T I C S [ VOL 6, N O 7, JULY 1980
pulp treated successfully with calcium hydroxide showed a prominent zone of necrosis with dystrophic calcification on the pulp surface four weeks after injury. By six weeks, a well-defined reparative dentin bridge was forming (Fig 4). DISCUSSION AND CONCLUSIONS
Fig 4--Six weeks after treatment with calcium hydroxide, well-defined bridge of reparative dentin (RD) is forming in association with poorly defined zone of tissue necrosis (NZ) (H&E, orig mag • 40).
there was no evidence of a dentin bridge. In dog 5, the two pulps treated with the dry collagen sponge were necrotic. The two pulps treated with the wet collagen fabric showed diffuse infiltration with chronic and acute inflammatory cells and peripheral areas of fibrosis, irregular dentin formation, and dentin resorption. At the interface between the pulp and the wet collagen fabric, several fragments of the fabric appeared to have undergone calcification and were covered with a reparative-type dentin. The results of our previous study 9 were used for purposes of comparison. The experimental method of exposing the pulp in canines of dogs was similar in all respects to the present experimental method except that the exposed pulp was treated with calcium hydroxide (Dycal). The
The results of this study indicate that, as pulp-capping materials, wet collagen sponge and wet collagen fabric are tolerated better than dry collagen sponge and dry collagen fabric, and that the collagen sponge product is tolerated better than the collagen fabric product. The pulps treated with wet collagen sponge showed a low-grade inflammatory response with evidence of only localized necrosis in all of the teeth tested. At eight weeks, three of four pulps showed a zone of necrosis at the interface of the wet collagen sponge and pulp, and two also showed early calcified bridge formation. The degenerative pulp reactions that are often seen in pulp treated with calcium hydroxide ~'9 were not seen in pulps treated with collagen sponge, which suggests that collagen sponge is relatively less irritating to the pulp tissue. However, if we compare the results of the current study with the results with calcium hydroxide in the earlier study, 9 we must make the following conclusion: calcium hydroxide is a more effective promoter of dentin bridge repair in experimentally exposed dog canine pulps than are the collagen materials that we tested in this study. Comparison of the reaction of injured pulp to the collagen gels used in the earlier experimenff with the reaction to the collagen sponge and the collagen fabric used in the cur-
rent experiment indicates that the pulp tissue is less tolerant to the collagen gel. We have not determined that the collagen gel is more of an irritant because of its higher antigenicity, but we suspect that it may be so. Unfortunately, there is also no evidence from the results of this study that the suggested cytophilic nature of the three-dimensional collagen sponge in any way enhances healing of the wounded pulp surface. In all of the collagen materials tested so far, it appears that formation of the clentin bridge occurs only when an area of surface necrosis is formed which subsequently undergoes dystrophic calcification. The rationale for using altered native collagen to enhance reparative dentin formation in wounded pulp tissue may be pursued from two perspectives. First, it is known that collagen fibers can catalyze calcium phosphate crystallization from physiologic concentrations of calcium and phosphate ions in vitro. Second, a three-dimensional collagenous framework can be formed which is "liked" by cells and, therefore, m a y be used as a framework in which the healing process may be initiated--a role not dissimilar to the fibrin clot seen in normal tissue healing (M. Chvapil, personal communication, June 1974). These two principles have been proved in bone reconstruction experiments and in tissue culture systems, but they do not appear to apply when the collagen materials are used as pulp-capping agents. This study was supported by grant MT3540 from the Canadian MRG. Dr. Dick is professorand chairman, division of oral pathology, Faculty of Dentistry, and Dr. Garmichael is director of graduate studies 643
JOURNAL OF ENDODONTICS I VOL 6, NO 7, JULY 1980
and research, Faculty of Dentistry, University of Alberta. Requests for reprints should be directed to Dr. Dick, 5085 Dentistry-Pharmacy Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2N8.
References 1. Sciaky, 1., and Pisanti, S. Localization of calcium placed over amputated pulps in dog's teeth. J Dent Res 39:1128-1132, 1960. 2. Clarke, N.G. The morphology of the reparative dentine bridge. Oral Surg 29:746752, 1970. 3. Nyborg, H. Healing processes in the pulp on capping. A morphologic study. Experiments on surgical lesions of the pulp in dog and man. Acta Odontol Scand (suppl 16), 13:9-130, 1955.
644
4. Ripp, N. Pulp capping. A review. NY State Dent J 42 (5):285-290, 1976. 5. Tronstad, L. Reactions of the exposed pulp to Dycal treatment. Oral Surg 38:945953, 1974. 6. McWalter, G.M.; EI-Kafrawy, A.H.; and Mitchell, D.F. Long-term study of pulp capping in monkeys with three agents. JADA 93(1):105-110, 1976. 7. Joos, R.W. Calcium hydroxide as a pulpcapping agent. Northwest Dent 53:362-365, 1974. 8. Schroder, U. Effect of an extra-pulpal blood clot on healing following experimental pulpotomy and capping with calcium hydroxide. Odontol Revy 24:257-268, 1973. 9. Carmichael, D.J.; Dick, H.M.; and
Dodd, C.M. Histologic effects of antigenically altered collagen as a heterograft for mammalian pulp exposures. Arch Oral Biol 19(12):1121-1126, 1974. 10. Chvapil, M., and Holusa, R. Experimental experiences with the collagen sponge as hemostaticum and tampon. J Biomed Mater Res 2:245-264, 1968. 11. Chvapil, M.; Owen, J.A.; and Clark, D.S. Effect of collagen crosslinking on the rate of resorption of implanted collagen tubing in rabbits. J Biomed Mater Res 11(2):297-314, 1977. 12. Davis, N.R. and Walker, T.E. The r01e of carboxyl groups in collagen calcification. Biochem Biophys Res Commun 48:1656-1662, 1972.