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Formation of crystals on the surface of calcium hydroxide-containing materials in vitro
JOURNALOF ENDODONTICS Copyright © 1999 by The American Association of Endodontists
Printed in U.S.A. VOL. 25, No. 8, AUGUST1999
Formation of Crystals on the Surface of Calcium Hydroxide-Containing Materials In Vitro Dimitrios Tziafas, DDS, PhD, and Nikolaos Economides, DDS, MSc, PhD
The purpose of the present study was to examine the surface of calcium hydroxide-containing materials when treated in different in vitro conditions. Five calcium hydroxide-containing materials (Dycal, Nu-Cap, Life, Sealapex, and Apexit) and two control calcium hydroxide-free materials (Roth 811 and AH26) were tested. The materials were placed onto Teflon discs or root dentin samples; maintained in distilled water or phosphate-buffered saline, or culture medium supplemented or not supplemented with fetal calf serum; incubated at 37°C in humidified atmosphere containing or not containing 5% CO2; and examined by scanning electron microscope. The results demonstrated precipitation of simple crystal units or organized crystalline structures in the calcium hydroxidecontaining specimens treated in all experimental conditions, except those maintained in distilled water without 5% CO 2. X-ray elemental microanalysis of the different crystalline structures showed one or two peaks corresponding to calcium or calcium and phosphorus. These data indicate that the crystals formed by reactions of calcium ions released from the calcium hydroxide-containing materials with the environmental ions might modify the material surface, especially in the presence of substrate adhesion molecules, such as fibronectin. This modification might play an important role in the regulation of cell adhesion and the initiation of new matrix synthesis.
generally accepted, the exact mechanism of its action is not yet fully understood (7). The biological effect of calcium hydroxide-containing materials, either as capping agents or as root canal sealers, has been correlated with the release of calcium and hydroxyl ions from the calcium hydroxide molecule. The mitogenic activity of calcium ions has been previously demonstrated (8, 9). Calcium ions also have a favorable role in cell migration, differentiation, and mineralization (8, 10, 11). On the other hand, the hydroxyl ions maintain a local state of tissue alkalinity, which creates a favorable environment for cell division and matrix formation (1 l). The surface of calcium hydroxide-containing material is important to the adjacent tissue response. Extracellular matrix molecules absorbed onto the surface can provide positional signals able to modulate cell activity (12). Preliminary experiments have shown that calcium ions are released from the calcium hydroxide-containing materials and form anorganic precipitations when exposed to pulp tissue in vivo (13) or to cell culture in vitro (14, 15). However, the effect of these precipitants onto the surface structure of these materials is still unknown. The purpose of the present study was to evaluate the surface of calcium hydroxide-containing materials when treated in different in vitro conditions without cells.
M A T E R I A L S AND M E T H O D S Five calcium hydroxide-containing materials, three pulp capping liners (Dycal, Caulk Lab, Milford, DE, Life, Kerr/Sybron, Torino, Italy; and Nu-Cap, Coe Co., Chicago, IL), two root canal sealers (Sealapex, Kerr/Sybron, Torino, Italy, and Apexit, Vivadent/Ivoclaar, Schaan, Liechtenstein), and two control calcium hydroxide-free materials, root canal sealers (Roth 811, Roth International, Chicago, IL; and AH26, DeTrey/Dentsply, Konstanz, Germany) were tested. The materials were mixed according to the manufacturer's instructions and placed either onto Teflon discs of 10 mm in diameter, or onto root dentin slices of 7 mm in diameter that were previously treated with 5% EDTA solution for 1 h. Three specimens of each material were prepared and incubated for 10 days at 37°C in a humidified atmosphere containing or not containing 5% CO> and then placed in 5 ml of the following solutions: 1. Distilled water 2. Phosphate-buffered saline (PBS; pH 7.2) supplemented or not supplemented with fetal calf serum (FCS; Gibco, Glasgow, UK)
Calcium hydroxide has been established for the last 3 decades as the most popular material for restricting bacterial contamination and providing the appropriate chemical stimulation for tissue healing in various endodontic situations where formation of calcific barriers are necessary. It is the usual dental material for direct pulp capping, pulpotomy, apical closure, root fractures, or resorption therapeutic procedures (1-3). It has also been introduced as an intracanal medication and root canal sealer in endodontic therapy (4-6). Although the beneficial properties of calcium hydroxide are 539
540
Tziafas and Economides
FIG 1. No crystal formation on AH26 surface (original magnification x500).
Journal of Endodontics
FIG 2. A calcium hydroxide-containing material placed on Teflon discs in distilled water and cultivated for 10 days without 5% CO2. Cubic or parallelepipedic crystals have been deposited onto the surface of Sealapex (original magnification x 1000).
3. Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented or not supplemented with FCS and penicillin (100 IU/ml). The specimens were dehydrated in the room environment and grated alcohols, coated with evaporating carbon, and examined under a scanning electron microscope (JEOL JSM-840 A, Tokyo, Japan). X-ray microprobe analysis associated with the scanning electron microscopic examination was performed lbr chemical analysis of the specimens. RESULTS
Control Materials The surface of the calcium hydroxide-free endodontic materials (AH26 and Roth 811) did not exhibit any change in the surface morphology and chemical composition throughout all experimental conditions. No crystal formation or any other type of anorganic deposition was observed in any of the specimens (Fig. 1).
Calcium Hydroxide-Containing Materials Specimens with calcium hydroxide-containing materials placed in the control condition (distilled water without CO 2) revealed the typical surface structure of freshly mixed materials (Fig. 2). Anorganic depositions were not found in this condition. In contrast, all specimens with calcium hydroxide-containing materials in all experimental conditions demonstrated precipitation of simple crystal units or formation of crystalline structures on their surfaces. The morphology, organization, and chemical composition of the crystals depended on both: the material and experimental condition. Differences between specimens dehydrated in room atmosphere or alcohols were not noticed. In all specimens of calcium hydroxide-containing materials treated in distilled water in the presence of CO 2, similar large crystal units of parallelepipedic or rhombic form, ranging from 20 to 30/xm in diameter and intergr0wn of crystalline structures, were observed (Fig. 3). When the materials had been placed onto dentin slices, the crystals were mainly found around them. The X-ray microanalysis of the crystals showed only one peak corresponding to calcium. Specimens treated with PBS or DMEM, in the presence or
FIG 3. Calcium hydroxide-containing material on Teflon disc with 5% CO2. Cubic crystals deposited in Apexit (original magnification ×80).
absence of CO 2, exhibited formation of crystals differing in shape and site. The structures were ranged from single rod-shaped or parallelepipedic crystals to more complicated oblong forms with 5 to 40/xm in length. The most characteristic variety in the form of the deposited crystals was found in Sealapex specimens (Fig. 4). In contrast, Dycal exhibited numerous oblong crystalline structures 1 to 3 /xm in width and 20 to 40/zm in length (Fig. 5). The X-ray microanalysis of the microcrystals revealed two peaks corresponding to calcium and phosphorous. However, the peak intensity of phosphorous was higher in Dycal than in Sealapex. In general, the most complicated forms of crystalline structures were seen when fetal calf serum had been added in the culture medium (Fig. 6). DISCUSSION These experiments clearly showed that cultivation of the five calcium hydroxide-containing materials tested herein under different environmental conditions induced formation of crystalline structures onto the material surfaces. The same culture conditions had no effect on the surface morphology of the two calcium hydroxide-free endodontic materials used in this study as controls: Roth 811 and AH26. The size, shape, and composition of the
Vol. 25, No. 8, August 1999
FIG 4. Calcium hydroxide-containing materials placed on Teflon discs with no PBS supplement with FCS and cultivated for 10 days with 5% CO2. Synthetic crystalline structures have been growth onto the surface of Sealapex (original magnification ×950).
FIG 5. Calcium hydroxide-containing materials placed on Teflon discs in DMEM supplemented with FCS and cultivated for 10 days with 5% CO2. Simple crystals or synthetic forms of crystalline structures have been deposited onto the surface of Sealapex (original magnification × 1500).
simple crystal units or the complex crystalline structures depended mainly on the environmental condition rather than the material itself, whereas the number of precipitants was related to the material used. In general, Sealapex showed a variety in the form of deposited crystals, whereas on the Dycal surface, we found the most organized forms of crystalline structures. The more simple form of crystals was found after treatment of calcium hydroxide-containing materials in distilled water in the presence of CO 2. Similar treatment in the absence of CO 2 did not show any change on the material's surface. This fact indicates that the previous precipitations had been formed by reaction of the calcium ions released from the setting materials with the CO 2. The cubic shape of the crystals, as well as the presence of only one peak corresponding to calcium in their microanalysis, confirmed the calcium carbonate nature of these crystals. Groups of calcium phosphate crystals were found on calcium hydroxide-containing materials treated in PBS or DMEM solutions. Microanalysis of their surface morphology also showed phosphorus-free crystals when CO 2 had been added in the culture chamber. The most complicated forms of crystalline structures
Crystal Formation onto Calcium Hydroxide Materials
541
FIG 6. Calcium hydroxide-containing materials placed on Teflon discs in DMEM supplemented with FCS and cultivated for 10 days with 5% CO2. Simple crystals or synthetic forms on the surface of Apexit (original magnification ×1000).
were found in the presence of FCS, a very common supplement in the culture media. It seems reasonable to suggest that the adhesive effect of the substrate adhesive molecule fibronectin contained in the FCS might result in formation of the complex crystalline structures. Strong interaction between crystals and fibronectin has been previously demonstrated (15, 16). Formation of calcium carbonate or calcium phosphate crystals on the surface of the calcium hydroxide-containing materials has been previously noticed in studies either in vitro (14, 15) or in vivo (13, 16). However, this is the first systematic analysis of surface morphology of calcium hydroxide-containing materials in various in vitro conditions without cells. The mechanism by which calcium hydroxide-containing materials stimulate tissue repair by hard tissue formation during the vital pulp therapy or in other endodontic situations remains an open question. Cell adhesion onto a mechanical support leading to polarized secretion of extracellular matrix has been recognized as a critical step in hard tissue formation during pulp or periodontal tissue wound healing (12, 17, 18). In vitro (14) or in vivo (16) observations have shown that fibronectin-mediated pulp cell attachment onto microcrystals can initiate differentiation into hard tissue-forming cells. Accumulation of endogenous growth factors onto the fibronectin-rich crystal surface can change the materialtissue interface into a dynamic biologically active substrate able to stimulate specific expression in the interacting cells (17). Therefore, it seems possible to hypothesize a role for crystals formed after application of calcium hydroxide-containing materials onto a wound surface in the regulation of cell adhesion and initiation of polarized matrix deposition. Synergistic effects of calcium and hydroxyl ions on cell proliferation, migration, or matrix synthesis of course cannot be excluded. In conclusion, present experiments indicate that various forms of crystals can be formed by the reaction of calcium ions released from the calcium hydroxide-containing materials with the free ions in the surrounding tissue fluids. Organization and chemical composition of the material-tissue interface did not remain constant, but depended basically on the environmental conditions. The possible role of crystals and their molecular interactions with extracellular matrix molecules formed after placement of the calcium hydroxide-containing materials at endodontic wound healing situations remains to be further investigated.
542
Tziafas and Econornides
Dr. Tziafas is assistant professor and Dr. Economides is a research fellow, Department of Endodontology, Dental School, Aristotle University Thessaloniki, Thessaloniki, Greece. Address requests for reprints to Dr. Nikolaos Economides, 8 Agiou Georgiou Square (Rotonta), GR-546 35 Thessaloniki, Greece.
References 1. Weisenseel JA, Hicks L, Pelleu GB. Calcium hydroxide as an apical barrier. J Endodon 1987;13:1-5. 2. Leonardo CD, Leal JM, Filho APS. Pulpectomy: immediate root canal filling with calcium hydroxide. Oral Surg 1980;49:441-50. 3. Rotstein I, Friedman S, Katz J. Apical closure of mature molar roots with the use of calcium hydroxide. Oral Surg Oral Med Oral Pathol 1990;70: 656-60. 4. Pitt Ford TR, Rowe AHR~ A new root canal sealer based on calcium hydroxide. J Endodon 1989;15:286-9. 5. Bystrom A, Claesson R, Sundqvist G. The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Endod Dent Traumatol 1985;1: 170-5. 6. Sjogren U, Figdor D, Spangberg L, Sundqvist G. The antimicrobial effect of calcium hydroxide as a short-term intracanal dressing. Int Endod J 1991 ;24:119-25. 7. Chivian N. Root resorption. In: Cohen S, Burns RC, eds. Pathways of the pulp. 5th ed. St. Louis: Mosby Year Book, 1991:512. 8. Das S. Effect of certain dental materials on human pulp in tissue culture. Oral Surg Oral Med Oral Pathol 1981;52:76-84.
Journal of Endodontics 9. Swierenga SH, MacManus JP, Whitfield JF. Regulation by calcium of the proliferation of heart cells from young adult rats. In Vitro 1976;12:31-6. 10. Schroder U. Effects of calcium hydroxide-containing pulp-capping agents on pulp cell migration, proliferation and differentiation. J Dent Res 1985;64:541-8. 11. Torneck CD, Moe M, Howley TP. The effect of calcium hydroxide on porcine pulp fibroblasts in vitro. J Endodon 1983;9:131-6. 12. Terranova V. Extracellular matrix molecules modulate the phenotype of the resident cells: mechanism of periodontal ligament cell and endothelial cell adherence to dentin. In: Davidovitch Z, ed. Biological mechanisms of tooth eruption and root resorption. Birmingham: EBSCO Media, 1988:23-34. 13. Holland R, Pinheiro CE, de Mello W, Nery M J, de Souza V. Histochemical analysis of the dogs' dental pulp after pulp capping with calcium, barium, and strontium hydroxides. J Endodon 1982;8:444-7. 14. Hanks CT, Bergenholtz G, Kim JS. Protein synthesis in vitro, in the presence of Ca(OH)2-containing pulp capping medicaments. J Oral Pathol 1983;12:356-65. 15. Seux D, Coubte ML, Hartmann DJ, Gauthier JP, Magloire H. Odontoblast-like cytodifferentiation of human dental pulp cells in vitro in the presence of a calcium-hydroxide-containing cement. Arch Oral Biol 1991 ;36:117-28. 16. Tziafas D, Panagiotakopoulos N, Komnenou A. Immunolocalization of fibronectin during the early response of dog dental pulp ~to demineralized dentine or calcium hydroxide-containing cement. Arch Oral Bio11995;40:2331. 17. Tziafas D. Reparative dentinogenesis. A monograph on the dentinogenic potential of dental pulp. Thessaloniki: University Studio Press, 1997: 78-80. 18. Mjor IA, Dahl E, Cox CF. Healing of pulp exposures: an ultrastructural study. J Oral Pathol Med 1991 ;20:496-501.
You Might Be Interested The economics of health care is, of course, of enormous interest today. Indeed studies now evaluate the advisability of therapeutic regimens based on such measures as dollars-expended-per-years-of-life-extension-expected. Thus, a recent study of the effectiveness of intensive therapy for insulindependent diabetes mellitus concluded that the therapy might be adopted because the cost was $19,987 per quality-adjusted life year expectancy increase (JAMA 276:1409). That's the easy part. Similar data are available for, for instance, the use of Beta-blockers after myocardial infarction, angiotension-converting enzyme inhibitors for heart failure, etc. Thus, it is likely possible to calculate the c o s t a billions, surely--if these therapies were available to the total U.S. population, or to the world's population--trillions. And also, no doubt, it is possible to calculate the tax increases necessary if they were to be provided as a government service. Should they be? What if the underlying illness is clearly related to a life-style choice--smoking cigarettes, for example. Answering such questions is the hard part.
David Wiley
Printed in U.S.A. VOL. 25, No. 8, AUGUST1999
Formation of Crystals on the Surface of Calcium Hydroxide-Containing Materials In Vitro Dimitrios Tziafas, DDS, PhD, and Nikolaos Economides, DDS, MSc, PhD
The purpose of the present study was to examine the surface of calcium hydroxide-containing materials when treated in different in vitro conditions. Five calcium hydroxide-containing materials (Dycal, Nu-Cap, Life, Sealapex, and Apexit) and two control calcium hydroxide-free materials (Roth 811 and AH26) were tested. The materials were placed onto Teflon discs or root dentin samples; maintained in distilled water or phosphate-buffered saline, or culture medium supplemented or not supplemented with fetal calf serum; incubated at 37°C in humidified atmosphere containing or not containing 5% CO2; and examined by scanning electron microscope. The results demonstrated precipitation of simple crystal units or organized crystalline structures in the calcium hydroxidecontaining specimens treated in all experimental conditions, except those maintained in distilled water without 5% CO 2. X-ray elemental microanalysis of the different crystalline structures showed one or two peaks corresponding to calcium or calcium and phosphorus. These data indicate that the crystals formed by reactions of calcium ions released from the calcium hydroxide-containing materials with the environmental ions might modify the material surface, especially in the presence of substrate adhesion molecules, such as fibronectin. This modification might play an important role in the regulation of cell adhesion and the initiation of new matrix synthesis.
generally accepted, the exact mechanism of its action is not yet fully understood (7). The biological effect of calcium hydroxide-containing materials, either as capping agents or as root canal sealers, has been correlated with the release of calcium and hydroxyl ions from the calcium hydroxide molecule. The mitogenic activity of calcium ions has been previously demonstrated (8, 9). Calcium ions also have a favorable role in cell migration, differentiation, and mineralization (8, 10, 11). On the other hand, the hydroxyl ions maintain a local state of tissue alkalinity, which creates a favorable environment for cell division and matrix formation (1 l). The surface of calcium hydroxide-containing material is important to the adjacent tissue response. Extracellular matrix molecules absorbed onto the surface can provide positional signals able to modulate cell activity (12). Preliminary experiments have shown that calcium ions are released from the calcium hydroxide-containing materials and form anorganic precipitations when exposed to pulp tissue in vivo (13) or to cell culture in vitro (14, 15). However, the effect of these precipitants onto the surface structure of these materials is still unknown. The purpose of the present study was to evaluate the surface of calcium hydroxide-containing materials when treated in different in vitro conditions without cells.
M A T E R I A L S AND M E T H O D S Five calcium hydroxide-containing materials, three pulp capping liners (Dycal, Caulk Lab, Milford, DE, Life, Kerr/Sybron, Torino, Italy; and Nu-Cap, Coe Co., Chicago, IL), two root canal sealers (Sealapex, Kerr/Sybron, Torino, Italy, and Apexit, Vivadent/Ivoclaar, Schaan, Liechtenstein), and two control calcium hydroxide-free materials, root canal sealers (Roth 811, Roth International, Chicago, IL; and AH26, DeTrey/Dentsply, Konstanz, Germany) were tested. The materials were mixed according to the manufacturer's instructions and placed either onto Teflon discs of 10 mm in diameter, or onto root dentin slices of 7 mm in diameter that were previously treated with 5% EDTA solution for 1 h. Three specimens of each material were prepared and incubated for 10 days at 37°C in a humidified atmosphere containing or not containing 5% CO> and then placed in 5 ml of the following solutions: 1. Distilled water 2. Phosphate-buffered saline (PBS; pH 7.2) supplemented or not supplemented with fetal calf serum (FCS; Gibco, Glasgow, UK)
Calcium hydroxide has been established for the last 3 decades as the most popular material for restricting bacterial contamination and providing the appropriate chemical stimulation for tissue healing in various endodontic situations where formation of calcific barriers are necessary. It is the usual dental material for direct pulp capping, pulpotomy, apical closure, root fractures, or resorption therapeutic procedures (1-3). It has also been introduced as an intracanal medication and root canal sealer in endodontic therapy (4-6). Although the beneficial properties of calcium hydroxide are 539
540
Tziafas and Economides
FIG 1. No crystal formation on AH26 surface (original magnification x500).
Journal of Endodontics
FIG 2. A calcium hydroxide-containing material placed on Teflon discs in distilled water and cultivated for 10 days without 5% CO2. Cubic or parallelepipedic crystals have been deposited onto the surface of Sealapex (original magnification x 1000).
3. Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented or not supplemented with FCS and penicillin (100 IU/ml). The specimens were dehydrated in the room environment and grated alcohols, coated with evaporating carbon, and examined under a scanning electron microscope (JEOL JSM-840 A, Tokyo, Japan). X-ray microprobe analysis associated with the scanning electron microscopic examination was performed lbr chemical analysis of the specimens. RESULTS
Control Materials The surface of the calcium hydroxide-free endodontic materials (AH26 and Roth 811) did not exhibit any change in the surface morphology and chemical composition throughout all experimental conditions. No crystal formation or any other type of anorganic deposition was observed in any of the specimens (Fig. 1).
Calcium Hydroxide-Containing Materials Specimens with calcium hydroxide-containing materials placed in the control condition (distilled water without CO 2) revealed the typical surface structure of freshly mixed materials (Fig. 2). Anorganic depositions were not found in this condition. In contrast, all specimens with calcium hydroxide-containing materials in all experimental conditions demonstrated precipitation of simple crystal units or formation of crystalline structures on their surfaces. The morphology, organization, and chemical composition of the crystals depended on both: the material and experimental condition. Differences between specimens dehydrated in room atmosphere or alcohols were not noticed. In all specimens of calcium hydroxide-containing materials treated in distilled water in the presence of CO 2, similar large crystal units of parallelepipedic or rhombic form, ranging from 20 to 30/xm in diameter and intergr0wn of crystalline structures, were observed (Fig. 3). When the materials had been placed onto dentin slices, the crystals were mainly found around them. The X-ray microanalysis of the crystals showed only one peak corresponding to calcium. Specimens treated with PBS or DMEM, in the presence or
FIG 3. Calcium hydroxide-containing material on Teflon disc with 5% CO2. Cubic crystals deposited in Apexit (original magnification ×80).
absence of CO 2, exhibited formation of crystals differing in shape and site. The structures were ranged from single rod-shaped or parallelepipedic crystals to more complicated oblong forms with 5 to 40/xm in length. The most characteristic variety in the form of the deposited crystals was found in Sealapex specimens (Fig. 4). In contrast, Dycal exhibited numerous oblong crystalline structures 1 to 3 /xm in width and 20 to 40/zm in length (Fig. 5). The X-ray microanalysis of the microcrystals revealed two peaks corresponding to calcium and phosphorous. However, the peak intensity of phosphorous was higher in Dycal than in Sealapex. In general, the most complicated forms of crystalline structures were seen when fetal calf serum had been added in the culture medium (Fig. 6). DISCUSSION These experiments clearly showed that cultivation of the five calcium hydroxide-containing materials tested herein under different environmental conditions induced formation of crystalline structures onto the material surfaces. The same culture conditions had no effect on the surface morphology of the two calcium hydroxide-free endodontic materials used in this study as controls: Roth 811 and AH26. The size, shape, and composition of the
Vol. 25, No. 8, August 1999
FIG 4. Calcium hydroxide-containing materials placed on Teflon discs with no PBS supplement with FCS and cultivated for 10 days with 5% CO2. Synthetic crystalline structures have been growth onto the surface of Sealapex (original magnification ×950).
FIG 5. Calcium hydroxide-containing materials placed on Teflon discs in DMEM supplemented with FCS and cultivated for 10 days with 5% CO2. Simple crystals or synthetic forms of crystalline structures have been deposited onto the surface of Sealapex (original magnification × 1500).
simple crystal units or the complex crystalline structures depended mainly on the environmental condition rather than the material itself, whereas the number of precipitants was related to the material used. In general, Sealapex showed a variety in the form of deposited crystals, whereas on the Dycal surface, we found the most organized forms of crystalline structures. The more simple form of crystals was found after treatment of calcium hydroxide-containing materials in distilled water in the presence of CO 2. Similar treatment in the absence of CO 2 did not show any change on the material's surface. This fact indicates that the previous precipitations had been formed by reaction of the calcium ions released from the setting materials with the CO 2. The cubic shape of the crystals, as well as the presence of only one peak corresponding to calcium in their microanalysis, confirmed the calcium carbonate nature of these crystals. Groups of calcium phosphate crystals were found on calcium hydroxide-containing materials treated in PBS or DMEM solutions. Microanalysis of their surface morphology also showed phosphorus-free crystals when CO 2 had been added in the culture chamber. The most complicated forms of crystalline structures
Crystal Formation onto Calcium Hydroxide Materials
541
FIG 6. Calcium hydroxide-containing materials placed on Teflon discs in DMEM supplemented with FCS and cultivated for 10 days with 5% CO2. Simple crystals or synthetic forms on the surface of Apexit (original magnification ×1000).
were found in the presence of FCS, a very common supplement in the culture media. It seems reasonable to suggest that the adhesive effect of the substrate adhesive molecule fibronectin contained in the FCS might result in formation of the complex crystalline structures. Strong interaction between crystals and fibronectin has been previously demonstrated (15, 16). Formation of calcium carbonate or calcium phosphate crystals on the surface of the calcium hydroxide-containing materials has been previously noticed in studies either in vitro (14, 15) or in vivo (13, 16). However, this is the first systematic analysis of surface morphology of calcium hydroxide-containing materials in various in vitro conditions without cells. The mechanism by which calcium hydroxide-containing materials stimulate tissue repair by hard tissue formation during the vital pulp therapy or in other endodontic situations remains an open question. Cell adhesion onto a mechanical support leading to polarized secretion of extracellular matrix has been recognized as a critical step in hard tissue formation during pulp or periodontal tissue wound healing (12, 17, 18). In vitro (14) or in vivo (16) observations have shown that fibronectin-mediated pulp cell attachment onto microcrystals can initiate differentiation into hard tissue-forming cells. Accumulation of endogenous growth factors onto the fibronectin-rich crystal surface can change the materialtissue interface into a dynamic biologically active substrate able to stimulate specific expression in the interacting cells (17). Therefore, it seems possible to hypothesize a role for crystals formed after application of calcium hydroxide-containing materials onto a wound surface in the regulation of cell adhesion and initiation of polarized matrix deposition. Synergistic effects of calcium and hydroxyl ions on cell proliferation, migration, or matrix synthesis of course cannot be excluded. In conclusion, present experiments indicate that various forms of crystals can be formed by the reaction of calcium ions released from the calcium hydroxide-containing materials with the free ions in the surrounding tissue fluids. Organization and chemical composition of the material-tissue interface did not remain constant, but depended basically on the environmental conditions. The possible role of crystals and their molecular interactions with extracellular matrix molecules formed after placement of the calcium hydroxide-containing materials at endodontic wound healing situations remains to be further investigated.
542
Tziafas and Econornides
Dr. Tziafas is assistant professor and Dr. Economides is a research fellow, Department of Endodontology, Dental School, Aristotle University Thessaloniki, Thessaloniki, Greece. Address requests for reprints to Dr. Nikolaos Economides, 8 Agiou Georgiou Square (Rotonta), GR-546 35 Thessaloniki, Greece.
References 1. Weisenseel JA, Hicks L, Pelleu GB. Calcium hydroxide as an apical barrier. J Endodon 1987;13:1-5. 2. Leonardo CD, Leal JM, Filho APS. Pulpectomy: immediate root canal filling with calcium hydroxide. Oral Surg 1980;49:441-50. 3. Rotstein I, Friedman S, Katz J. Apical closure of mature molar roots with the use of calcium hydroxide. Oral Surg Oral Med Oral Pathol 1990;70: 656-60. 4. Pitt Ford TR, Rowe AHR~ A new root canal sealer based on calcium hydroxide. J Endodon 1989;15:286-9. 5. Bystrom A, Claesson R, Sundqvist G. The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Endod Dent Traumatol 1985;1: 170-5. 6. Sjogren U, Figdor D, Spangberg L, Sundqvist G. The antimicrobial effect of calcium hydroxide as a short-term intracanal dressing. Int Endod J 1991 ;24:119-25. 7. Chivian N. Root resorption. In: Cohen S, Burns RC, eds. Pathways of the pulp. 5th ed. St. Louis: Mosby Year Book, 1991:512. 8. Das S. Effect of certain dental materials on human pulp in tissue culture. Oral Surg Oral Med Oral Pathol 1981;52:76-84.
Journal of Endodontics 9. Swierenga SH, MacManus JP, Whitfield JF. Regulation by calcium of the proliferation of heart cells from young adult rats. In Vitro 1976;12:31-6. 10. Schroder U. Effects of calcium hydroxide-containing pulp-capping agents on pulp cell migration, proliferation and differentiation. J Dent Res 1985;64:541-8. 11. Torneck CD, Moe M, Howley TP. The effect of calcium hydroxide on porcine pulp fibroblasts in vitro. J Endodon 1983;9:131-6. 12. Terranova V. Extracellular matrix molecules modulate the phenotype of the resident cells: mechanism of periodontal ligament cell and endothelial cell adherence to dentin. In: Davidovitch Z, ed. Biological mechanisms of tooth eruption and root resorption. Birmingham: EBSCO Media, 1988:23-34. 13. Holland R, Pinheiro CE, de Mello W, Nery M J, de Souza V. Histochemical analysis of the dogs' dental pulp after pulp capping with calcium, barium, and strontium hydroxides. J Endodon 1982;8:444-7. 14. Hanks CT, Bergenholtz G, Kim JS. Protein synthesis in vitro, in the presence of Ca(OH)2-containing pulp capping medicaments. J Oral Pathol 1983;12:356-65. 15. Seux D, Coubte ML, Hartmann DJ, Gauthier JP, Magloire H. Odontoblast-like cytodifferentiation of human dental pulp cells in vitro in the presence of a calcium-hydroxide-containing cement. Arch Oral Biol 1991 ;36:117-28. 16. Tziafas D, Panagiotakopoulos N, Komnenou A. Immunolocalization of fibronectin during the early response of dog dental pulp ~to demineralized dentine or calcium hydroxide-containing cement. Arch Oral Bio11995;40:2331. 17. Tziafas D. Reparative dentinogenesis. A monograph on the dentinogenic potential of dental pulp. Thessaloniki: University Studio Press, 1997: 78-80. 18. Mjor IA, Dahl E, Cox CF. Healing of pulp exposures: an ultrastructural study. J Oral Pathol Med 1991 ;20:496-501.
You Might Be Interested The economics of health care is, of course, of enormous interest today. Indeed studies now evaluate the advisability of therapeutic regimens based on such measures as dollars-expended-per-years-of-life-extension-expected. Thus, a recent study of the effectiveness of intensive therapy for insulindependent diabetes mellitus concluded that the therapy might be adopted because the cost was $19,987 per quality-adjusted life year expectancy increase (JAMA 276:1409). That's the easy part. Similar data are available for, for instance, the use of Beta-blockers after myocardial infarction, angiotension-converting enzyme inhibitors for heart failure, etc. Thus, it is likely possible to calculate the c o s t a billions, surely--if these therapies were available to the total U.S. population, or to the world's population--trillions. And also, no doubt, it is possible to calculate the tax increases necessary if they were to be provided as a government service. Should they be? What if the underlying illness is clearly related to a life-style choice--smoking cigarettes, for example. Answering such questions is the hard part.
David Wiley