0099-2399/91/1712-0589/$03.00/0 JOURNAL OF ENDODONTICS Copyright 9 1991 by The American Association of Endodontists
Printed in U.S.A.
VoL 17, No. 12, DECEMBER1991
Natural Modifiers of the Inflammatory Process in the Human Dental Pulp Scott B. McClanahan, DDS, MS, Donald W. Turner, DDS, PhD, Edward J. Kaminski, PhD, Edward M. Osetek, DDS, MA, and Michael A. Heuer, DDS, MS
Concentrations of the protease inhibitors al-antitrypsin and a2-macroglobulin were determined in normal and inflamed human dental pulps. Carious pulpal exposure which is associated with polymorphonuclear leukocyte infiltration and release of lysosomal enzymes was chosen as the point of verifiable inflammatory activity in the pulp. Normal samples were collected from nondiseased third molar teeth treatment planned for extraction and inflamed human pulps were collected from teeth with deep carious lesions. One half of each sample was assayed for concentration of protease inhibitors by enzyme-linked immunosorbent assay and the remaining half was examined histologically to verify the clinical diagnosis and categorize the extent of the inflammatory process, al-Antitrypsin and o~2macroglobulin were detected in normal and inflamed human dental pulps in the nanogram per milliliter range. Statistically significant differences were found in the concentrations of a2-macroglobulin (p < 0.01) in moderate to severe inflammation versus normal pulp categories and between mildly inflamed pulps and moderate to severely inflamed pulps (p < 0.05). Although differences in concentrations of alantitrypsin were seen between inflamed and normal pulps, the differences were not satistically significant. The presence of these two protease inhibitors in the human dental pulp tissue and the increase in their concentration in acute inflammation indicates that these proteins play a role in the pathogenesis of puIpal inflammatory disease.
requirements for such processes as fibrinolysis, blood coagulation, and the inflammatory reactions involving activation of the kallikrein and complement systems (1, 2). Alpha-1-antitrypsin (A 1AT) is an acute phase plasma protein synthesized in the liver that functions as a serine protease. A IAT has a molecular size of approximately 52,000 daltons and a serum concentration of 150 to 350 mg per dl (3). Deficiency states of AIAT are seen in adult patients with chronic obstructive lung disease, emphysema, and cirrhosis in childhood liver disease (4). Human a2-macroglobulin (A2M) is a glycoprotein with a molecular size of 725,000 daltons and is a tetramer of identical 185,000-dalton subunits which are linked by disulfide bonds (5). The site of synthesis of A2M in vivo is unknown but a series of culture studies found that cells of the monocytemacrophage lineage produced A2M (6). The serum concentration has been demonstrated to be in the range of 220 mg per 100 ml (7). A2M is limited to inhibitory activity within the circulation due to its size (8). Since no deficiency states of A2M exist, it is difficult to designate a specific physiological function. A2M demonstrates inhibition of the four classes of proteases; carboxyl, thiol, serine, and metalloproteases. Since this broad specificity suggests a defensive function, A2M might be thought of as a part of the immune defense system (5). The protease inhibitors A1AT and A2M have welldescribed interactions with lysosomal enzymes. A1AT is the dominant polymorphonuclear neutrophil (PMN) elastase inhibitor, but A2M has inhibitory function as well (9-12). A 1AT and A2M have also been demonstrated to inhibit PMN collagenase ( 13, 14). Although nothing has been reported of the protease inhibitors in the dental pulp, Morand et al. (15) measured the collagenolytic and elastinolytic activity in diseased human pulps. They reported significant increases in both enzyme activities in pulps which were classified as either suppurative pulpitis or as necrotic symptomatic teeth with periapical radiolucencies. The experimental model chosen for this study was the human dental pulp and the inflammatory process initiator was dental caries. It was necessary to clinically delineate a point of early polymorphonuclear neutrophil infiltration. Numerous studies have indicated a clinical determination of
The proteases of the human body have well-known degradarive functions in digestion. Additionally, the assembly and protective functions are often at the limits of physiological processes and may potentially damage the host. Protease inhibitors are a group of plasma proteins that function by blocking the catalytic sites of the proteases. The protease inhibitors limit the action of the proteases to the biological
589
590
McClanahan et al.
PMN infiltration and acute inflammation can be made based upon carious pulpal exposure (16, 17). The purpose of this study was to determine whether A1AT and A2M could be detected in the human dental pulp at a point of verifiable inflammatory activity, i.e. at the time of the acute infiltration of PMN and associated lysosomal enzyme release in a carious pulpal exposure, and, if present, to compare these concentrations with the concentrations of A1AT and A2M in normal dental pulps. MATERIALS AND METHODS
Sample Collection Pulp samples were collected from patient volunteers, United States Navy personnel stationed at the Naval Training Center, Great Lakes, Illinois, as part of acceptable and normal therapeutic procedures. The volunteers were male and female dental patients ranging from 18 to 30 yr of age. The inflamed samples were clinically arbitrarily categorized due to carious exposures. Radiographically, all samples demonstrated deep caries closely approximating the pulp chamber with no evidence of periapical pathosis. During caries excavation, once a carious exposure occurred, the pulp chamber was unroofed and the pulps were removed with an endodontic spoon excavator. Every effort was made to gather the specimen as atraumatically as possible. Once the pulp tissue was freed, the specimen was removed to a glass slab on the bracket table. The pulp sample was bisected longitudinally with a scalpel. One half was placed in a preweighed 250-ul Eppendorf centrifuge tube and frozen immediately for enzyme-linked immunosorbent assay with long-term sample storage at -700C. The remaining half was placed in 10% Formalin for histological verification of the clinical categorization of the pulpal status. The normal pulp samples were collected from noncarious, nonperiodontally involved third molar teeth treatment planned for extraction. The teeth were extracted as atraumatically as possible and could generally be categorized as elevator or "roll-out" extractions. After extraction, a groove was cut incisogingivally on the facial and lingual surfaces of the maxillary teeth and mesial and distal surfaces of the mandibular teeth with a high-speed handpiece and water coolant. The teeth were cracked in half with a 301 elevator and the pulps gently removed with an endodontic spoon. The pulp specimen was then treated in the same manner as the inflamed pulps specimens.
Histopathological Examination In all, 19 inflamed pulp sample fragments and 18 normal pulp sample fragments were collected and categorized. Sample I 6 was too small to be sectioned for both immunoassay and histological examination so it was used for immunoassay pilot experiments. The fragments of specimens were embedded in paraffin, sectioned at 6 urn, stained with hematoxylin and eosin, and then examined and classified histologically as normal, i.e. without inflammatory cell infiltrate, edema, or hyperemia. The inflamed samples were subcategorized based on the extent of inflammatory infiltrate, edema, and hyperemia noted in the specimen.
Journal of Endodontics
Protein Extraction Tissue samples were removed from the -70~ freezer, allowed to thaw for 10 min, and the wet weight of the specimen was obtained. One-hundred fifty microliters of phosphate-buffered saline at pH 7.0 were added to each 250tA Eppendorfcentrifuge tube, and the samples were macerated with a glass rod. Each sample was vibrated for 5 s on a Vortex Genie Mixer. The samples were allowed to stand in the buffer at 4~ for 30 min, but every 10 min the samples were vortexed for 5 s. The samples were centrifuged for 2 minutes at 11,000 rpm equivalent to a relative centrifugal force of 9880 x g. The supernatants of the samples were then diluted with PBS and aliquots of the sample dilutions were stored at -70~ for immunoassay.
Enzyme-linked lmmunosorbent Assay A1AT and A2M concentrations in standards and pulp samples were determined using the enzyme-linked immunosorbent assay double antibody sandwich technique. The amplified substrate system as described by Cart et al. (18) was used in the assay. Fifteen normal samples and 15 inflamed samples were used for the assays. The remaining inflamed pulps were used in pilot experiments to determine methodology for protein extraction. Extracted inflamed pilot and normal sample aliquots were used in pilot experiments to ascertain dilution ranges for each of the assays. A 1AT standard dilution concentration ranged from 2.5 to 100 ng and A2M standard dilution concentration ranged from 16.7 to 800 ng. Through pilot experiments a range of sample dilutions for both A 1AT and A2M was determined as 1:100, 1:500, 1:1000, and 1:2000 for the normal samples and 1:50, 1:100, 1:500, and 1:1000 for the inflamed samples. If any sample reruns were required, the dilutions were adjusted appropriately. Each standard and sample dilution for A1AT and for A2M was run in triplicate. RESULTS
Histopathological Results The ages of the patients whose pulps were collected in the inflamed groups ranged from 18 to 20 yr, with the average age being 18.4 yr. Nineteen pulps were collected in the inflamed category and examined histologically. The inflamed samples were classified as mild (n -- 7), mild to moderate (n = 2), moderate (n = 5), or severe (n = l) based on the extent of the acute inflammatory response noted in the specimen. A subcategory which includes degenerative changes was added, i.e. mild inflammation with degeneration (n = 4). Figures 1 and 2 are photomicrographs illustrative of the inflammatory categories. Fourteen of the 19 pulps showed evidence of dystrophic calcification. Eighteen pulps were collected in the normal category and examined histologically. The range of ages for the patients in the normal group was 18 to 30 yr, with the average age being 21. l yr. Figure 3 illustrates a normal pulp sample. Two of the normal pulps had a small area of dystrophic calcification. Nine normal pulp specimens revealed areas of
Vol. 17, No. 12, December 1991
FIG 1. Mild inflammation: sample I 10, illustrating a mild PMN infiltrate, absence of edema, and hyperemia (H & E; original magnification x40).
Pulpal Inflammatory Modifiers
591
FIG 3. Normal pulp: sample N 5, illustrating intact odontoblastic layer, nerve tissue, and absence of inflammatory infiltrate, hyperemia, or edema (H & E; original magnification xl0). TABLE 1. Average protease inhibitor concentrations
FIG 2. Severe inflammation: sample I 3, illustrating a predominate PMN infiltrate, congested capillaries, extravasation of RBC's, generalized edema, and dystrophic calcification (H & E; original magnification x40).
fatty change. Because of the absence of inflammation, the pulps met the experimental criteria for normal. Three of the samples collected as clinically normal pulps were rejected because of failure to meet the specified criteria.
Inhibitor Concentrations For each standard and sample dilution, the average optical density at 4 and 7 min was calculated and the change in optical density for the 3-min interval determined. The change in optical density for the standard dilutions was plotted against the concentrations of the standard dilutions, and a linear regression was run on standard dilutions to determine the standard curve for each assay. The concentration of the protease inhibitors in the samples for each assay was extrapolated from the standard curve for that particular assay.
Group
AIAT (#g/mg pulp)
A2M (#g/mg pulp)
Normal Mild Moderate to severe
0.3419 _+ 0.1949 0.2615 + 0.0984 0.4711 _+ 0.2610
1.5308 _+ 0.9963 1.5803 _+ 1.1818 5.3754 _+ 4.4091
The concentration in the sample was multiplied by the dilution factor, and the resultant concentrations were averaged. The concentration of the protease inhibitors in the sample was converted to micrograms per milliliter. The pulp samples had been weighed before the extraction of the proteins, and weight per unit volume of the pulp sample in the 0.150 ml of buffer was determined. The weight per unit volume of pulp sample in elution buffer was expressed in milligrams per milliliter. The concentration of inhibitor was divided by the weight per unit volume of pulp sample. The resultant protease inhibitor concentration was expressed as micrograms of protease inhibitor per milligram of pulp sample.
Statistical Analysis To better facilitate distribution for analysis, the inflamed pulp subcategories were reorganized into the following: (a) mild (n = 7), which included mild inflammation and mild inflammation with degeneration and (b) moderate to severe (n = 8), which included mild to moderate inflammation, moderate inflammation, and severe inflammation. The average protease inhibitor concentrations and standard deviations are illustrated in Table 1. The data revealed that the A2M distributions were not equal and variances were different between groups. To control these disturbances, the A2M data were subjected to a natural log transformation, and all subsequent analyses are on the transformed data. By using BMDP Statistical Software (Los Angeles, CA), data were subjected to a one-way multivariate analysis of
592
Journal of Endodontics
M c C l a n a h a n et el.
variance with two dependent variables (A 1AT and A2M) and three levels of a single between-subjects variable (normal pulp, pulp with mild inflammation, and pulp with moderate to severe inflammation). The overall effect of the state of the pulp was significant (F = 3.36; df = 4,52; p = 0.0160), but the effect was attributed only to the A2M-dependent variable (F = 6.76; df = 2,27; p = 0.0042). Again, using the BMDP Statistical Software, a post hoc comparison of the A2M group means was accomplished using Tukey's Studentized range method. The Tukey method was chosen instead of the Scheffe method because according to Kirk (19), the Tukey method is a more powerful test in pairwise comparisons. The moderate to severe group differed significantly from the normal group (p < 0.01) and the mild group (p < 0.05). There was no difference between the mild and normal groups. DISCUSSION The ability to detect A 1AT in normal and inflamed human dental pulps was noteworthy, but not totally surprising, because of its widespread distribution in body fluid and organ tissue. The fact that the concentration ofA 1AT did not change with an increase in inflammatory category is puzzling. The type of inflammatory reaction of the pulp to dental caries may be an explanation. The pulpal reaction to caries is of chronic inflammation until actual exposure occurs which then is followed by an infiltration of PMN at the point of exposure. Since A1AT is an acute phase protein, an increased plasma concentration follows an acute inflammatory stimulus. The reaction time from an inflammatory stimulus in the dental pulp to an increase of A1AT in plasma concentration is not known. A basic hypothesis of this study was that to have protease inhibitor activity it is necessary that proteases be present. In human dental pulp, a known point of protease activity is during PMN cellular infiltration which has been demonstrated in carious pulpal exposure. A carious exposure serves as a predictable point in time to collect inflamed specimens which then can be compared with normal pulps. Probably, the time of sample collection in this study preceded the acute phase protein production and a subsequent increase in plasma concentration of A 1AT. A2M is confined to the vascular space because of its large molecular size. The vascular congestion which is a classical sign of early inflammation provides a mechanism for an increased intravascular concentration of A2M in the pulp tissue and at the site of inflammation. In this study, A2M was observed to increase in concentration in direct relation to progressive severity of the inflammatory responses. It may be fortuitous that the A2M concentrations are increased early in the inflammatory process. Since A2M has a broader range of activity than A1AT, it inhibits all classes of proteases and is not restricted to the serine proteases. Additionally, A2M inhibits the kinin, coagulation, and fibrinolytic cascades. The inhibitor with the broadest range of activity arriving "first on the scene" would best limit the host damage as a result of the interrelated aspects of the inflammatory process. Morand et al. (15) showed an increased elastinolytic and collagenolytic activity in diseased human pulps with suppur-
ative pulpitis and necrosis. It would be most interesting to examine the concentration of A 1AT and A2M in pulps demonstrating more intense inflammatory response. Also, it would be of interest to determine whether there is indeed a lag time and ultimately an increased localized A 1AT concentration. Similar to development of emphysema in the lungs of cigarette smokers, the burden of increased protease activity in the inflamed dental pulp possibly saturates the A 1AT and A2M molecules; subsequently leading to necrosis of the affected tissues. At what point inhibitors are saturated is not known. Since the collection of inflamed samples was based on clinical parameters, there was some distribution among the histological categories. All of the inflamed specimens met the criteria of PMN infiltration, but the clinically selected criteria did not correlate exactly with morphological change for all samples. For the purpose of statistical analysis, it would have been preferable to have had an even distribution in the inflamed histological groups mild, mild to moderate, and severe to facilitate analysis rather than combine the groups. A larger sample size could have improved the distribution. The interaction of A1AT and A2M with lysosomal enzymes in conjunction with the interaction of A2M with the enzymes of the kinin, coagulation, and fibrinolytic cascades are critical to the biological balance during inflammation. The presence of these protease inhibitors in the human dental pulp tissue and the significant increase in A2M in moderate to severe acute inflammation as determined by this study indicates that these proteins play a role in the pathogenesis of pulpal inflammatory disease. This project was supported in part by Naval Medical Research and Development Command Project MR0412002-0001.
The assertions contained herein are those of the authors and are not to be construed as official or as reflecting the views of the Department of the Navy or the Departmentof Defense. We wish to thank Dr. Mark E. Cohen of the Naval Dental Research Institute, Great Lakes, IL, for his expertise in the statistical analysis of the data and Mr. Andrew Markow of the Department of Pathology, Northwestern University Medical and Dental Schools for histological preparation of the specimens. Dr. McClanahan is a former endodontic resident, Northwestern University Dental School, and is head, Endodontics Department, Naval Dental Clinic, Okinawa, Japan. Dr. Turner is professor and director, Division of Periodontics, Northwestern University Dental School. Dr. Kaminski is professor, Department of Pathology, Northwestern University Medical and Dental Schools. Dr. Osetek is director, Advanced Program in Endodontics, and associate dean, Clinical Affairs, Northwestern University Dental School. Dr. Heuer is senior associate dean, Academic Affairs, Northwestern University Dental School. Address requests for reprints to Dr. Scott McClanahan, P.O. Box 2554, Camp Butler, FPO Seattle, WA 98774.
References
1. Welsh KA. Unifyingconcepts among proteases. Proteases and biological control. New York: Cold Spring Harbor Laboratory, 1975:1-11. 2. HeimburgerN. Proteinase inhibitors of human plasma--their properties and control functions. Proteases and biological control. New York: Cold Spring Harbor Laboratory, 1975:367-86. 3. Brantly M, Nukiwa T, Crystal RG. Molecular basis of alpha-l-antitrypsin deficiency. Am J Med 1988;84:13-31. 4, Talamo RC. Basic and clinical aspects of the alpha-l-antitrypsin. Pediatrics 1975;56:91-9. 5. Travis J, Salvesen GS. Human plasma proteinase inhibitors. Annu Rev Biochem 1983;52:655-709. 6. Hovi T, Mosher D, Vaheri A. Cultured human monocytes synthesize and secrete alpha-2-macroglobulin.J Exp Med 1977;145:1580-9.
Pulpal Inflammatory Modifiers
Vol. 17, No. 12, December 1991 7. Adham NF, Wilding P, Mehl J, Haverback BJ. Alpha-2 macroglobulin levels in human serum. J Lab Clin Med 1969;71:271-82. 8. Travis J. Structure, function, and control of neutrophil proteinases. Am J Med 1988:84:37-42. 9. Ohlsson K. Neutral leucocyte proteases and elastase inhibited by plasma alpha-1-antitrypsin, Scand J Clin Lab Invest 1971 ;28:251-3. 10. Janoff A. Inhibition of human granulocyte elastase by serum alpha-1antitrypsin. Am Rev Respir Dis 1972;105:121-2. 11. Ohlsson K, Olsson I. The neutral proteases of human granulocytes-isolation and partial characterization of granulocyte elastase. Eur J Biochem 1974;42:519-27. 12. Ohlsson K, Olsson I. Neutral proteases of human granulocytes. II1. Interaction between human granulocyte elastase and plasma protease inhibitors. Scand J Clin Lab Invest 1974;34:349-55. 13. Ohlsson K, OIsson I. The neutral proteases of human granulocytes-isolation and partial characterization of two granulocyte collagenases. J Biochem 1973;36:473-81.
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14. Ohlsson K, Olsson I. Neutral proteases of human granulocytes. IV. Interaction between human granulocyte collagenase and plasma proteases inhibitors. J Lab Clin Med 1977;89:269-77. 15. Morand MA, Schilder H, Blondin J, Stone PJ, Franzblau C. Collagenolytic and elastinolytic activities from diseased human dental pulps. J Endodon 1981 ;7:156-60. 16. Seltzer S, Bender IB, Ziontz M The dynamics of pulp inflammation: correlations between diagnostic data and actual histologic findings in the pulp. Oral Surg 1963;16:846-871 and 16:989-977. 17. Torneck CD. Changes in the fine structure of the human dental pulp subsequent to carious exposure. J Oral Patho11977;6:82-95. 18. Carr RI, Mansour M, Sadi D, James H, Jones JV. A substrate amplification system for enzyme-linked immunoassays. Demonstration of its general applicability to ELISA systems for detecting antibodies and immune complexes. J Immunol Methods 1987;98:201-8. 19. Kirk RE. Experimental design: procedures for the behavioral scientist. Belmont, CA: Brooks Cole Publishing Co., 1968:90.
The Way it Was Caught in the Jaws of the Amalgam Controversy Double Jeopardy For endodontists, amalgam has been the backbone of practice for many years in the area of root-end fills and core build-ups. However, it has been of concern to some practitioners because the placement of a root-end amalgam in a tooth with a metallic post may lead to the generation of a galvanic couple. When present, these couples have the potential to generate significant amounts of electrical current (Austin et al., Oral Surg 1982;54:79). The possibility of setting up an intraoral current of this nature was recognized in the early 1800s (Brockway, Am J Dent Sci 1844;5172; Mackall, Am J Dent Sci 1839;1:86. "Everyone has heard of the popular experiment, of placing silver on the top and zinc under the tongue, and making the edges touch to produce the peculiar metallic taste, caused by the galvanic action of the metals; but it is not generally known, even among the profession, that a constant galvanic action is kept up in the mouth, when more than one kind of metal is used in filling the teeth." While this problem could be the source of a failure in surgical treatment, the uses of amalgam in contemporary endodontics are slowly fading in light of controversial studies and the introduction of new materials. Recently though, the news media (60 Minutes) visited with ADA Fifth District Trustee Herber Simmons concerning the contemporary use of amalgam by dentists and the concerns expressed by recent Swedish and Canadian animal studies. Throughout the interview, as briefly printed in the ADA News (Berry, 1990;1:5), dentistry is placed in a defensive posture by the media, even though, as studies have pointed out, amalgam has never been linked to any systemic disease. This same situation occurred in 1839, when the editor of the American Journal of Dental Science expressed his own concern on the perceived impropriety of using dissimilar metals in the patient's mouth, even to the point of exposing the names of the professionals who chose to render this course of treatment. 'I understand there are those in this city, who use the Crawcour Succedaneum and Amalgam of Silver and Mercury; our object being to correct errors in dental practice, we hope all such will discontinue its use, in justice both to the profession and the public; and also to save us the painful necessity of exposing their names."
James L. Gutmann
Printed in U.S.A.
VoL 17, No. 12, DECEMBER1991
Natural Modifiers of the Inflammatory Process in the Human Dental Pulp Scott B. McClanahan, DDS, MS, Donald W. Turner, DDS, PhD, Edward J. Kaminski, PhD, Edward M. Osetek, DDS, MA, and Michael A. Heuer, DDS, MS
Concentrations of the protease inhibitors al-antitrypsin and a2-macroglobulin were determined in normal and inflamed human dental pulps. Carious pulpal exposure which is associated with polymorphonuclear leukocyte infiltration and release of lysosomal enzymes was chosen as the point of verifiable inflammatory activity in the pulp. Normal samples were collected from nondiseased third molar teeth treatment planned for extraction and inflamed human pulps were collected from teeth with deep carious lesions. One half of each sample was assayed for concentration of protease inhibitors by enzyme-linked immunosorbent assay and the remaining half was examined histologically to verify the clinical diagnosis and categorize the extent of the inflammatory process, al-Antitrypsin and o~2macroglobulin were detected in normal and inflamed human dental pulps in the nanogram per milliliter range. Statistically significant differences were found in the concentrations of a2-macroglobulin (p < 0.01) in moderate to severe inflammation versus normal pulp categories and between mildly inflamed pulps and moderate to severely inflamed pulps (p < 0.05). Although differences in concentrations of alantitrypsin were seen between inflamed and normal pulps, the differences were not satistically significant. The presence of these two protease inhibitors in the human dental pulp tissue and the increase in their concentration in acute inflammation indicates that these proteins play a role in the pathogenesis of puIpal inflammatory disease.
requirements for such processes as fibrinolysis, blood coagulation, and the inflammatory reactions involving activation of the kallikrein and complement systems (1, 2). Alpha-1-antitrypsin (A 1AT) is an acute phase plasma protein synthesized in the liver that functions as a serine protease. A IAT has a molecular size of approximately 52,000 daltons and a serum concentration of 150 to 350 mg per dl (3). Deficiency states of AIAT are seen in adult patients with chronic obstructive lung disease, emphysema, and cirrhosis in childhood liver disease (4). Human a2-macroglobulin (A2M) is a glycoprotein with a molecular size of 725,000 daltons and is a tetramer of identical 185,000-dalton subunits which are linked by disulfide bonds (5). The site of synthesis of A2M in vivo is unknown but a series of culture studies found that cells of the monocytemacrophage lineage produced A2M (6). The serum concentration has been demonstrated to be in the range of 220 mg per 100 ml (7). A2M is limited to inhibitory activity within the circulation due to its size (8). Since no deficiency states of A2M exist, it is difficult to designate a specific physiological function. A2M demonstrates inhibition of the four classes of proteases; carboxyl, thiol, serine, and metalloproteases. Since this broad specificity suggests a defensive function, A2M might be thought of as a part of the immune defense system (5). The protease inhibitors A1AT and A2M have welldescribed interactions with lysosomal enzymes. A1AT is the dominant polymorphonuclear neutrophil (PMN) elastase inhibitor, but A2M has inhibitory function as well (9-12). A 1AT and A2M have also been demonstrated to inhibit PMN collagenase ( 13, 14). Although nothing has been reported of the protease inhibitors in the dental pulp, Morand et al. (15) measured the collagenolytic and elastinolytic activity in diseased human pulps. They reported significant increases in both enzyme activities in pulps which were classified as either suppurative pulpitis or as necrotic symptomatic teeth with periapical radiolucencies. The experimental model chosen for this study was the human dental pulp and the inflammatory process initiator was dental caries. It was necessary to clinically delineate a point of early polymorphonuclear neutrophil infiltration. Numerous studies have indicated a clinical determination of
The proteases of the human body have well-known degradarive functions in digestion. Additionally, the assembly and protective functions are often at the limits of physiological processes and may potentially damage the host. Protease inhibitors are a group of plasma proteins that function by blocking the catalytic sites of the proteases. The protease inhibitors limit the action of the proteases to the biological
589
590
McClanahan et al.
PMN infiltration and acute inflammation can be made based upon carious pulpal exposure (16, 17). The purpose of this study was to determine whether A1AT and A2M could be detected in the human dental pulp at a point of verifiable inflammatory activity, i.e. at the time of the acute infiltration of PMN and associated lysosomal enzyme release in a carious pulpal exposure, and, if present, to compare these concentrations with the concentrations of A1AT and A2M in normal dental pulps. MATERIALS AND METHODS
Sample Collection Pulp samples were collected from patient volunteers, United States Navy personnel stationed at the Naval Training Center, Great Lakes, Illinois, as part of acceptable and normal therapeutic procedures. The volunteers were male and female dental patients ranging from 18 to 30 yr of age. The inflamed samples were clinically arbitrarily categorized due to carious exposures. Radiographically, all samples demonstrated deep caries closely approximating the pulp chamber with no evidence of periapical pathosis. During caries excavation, once a carious exposure occurred, the pulp chamber was unroofed and the pulps were removed with an endodontic spoon excavator. Every effort was made to gather the specimen as atraumatically as possible. Once the pulp tissue was freed, the specimen was removed to a glass slab on the bracket table. The pulp sample was bisected longitudinally with a scalpel. One half was placed in a preweighed 250-ul Eppendorf centrifuge tube and frozen immediately for enzyme-linked immunosorbent assay with long-term sample storage at -700C. The remaining half was placed in 10% Formalin for histological verification of the clinical categorization of the pulpal status. The normal pulp samples were collected from noncarious, nonperiodontally involved third molar teeth treatment planned for extraction. The teeth were extracted as atraumatically as possible and could generally be categorized as elevator or "roll-out" extractions. After extraction, a groove was cut incisogingivally on the facial and lingual surfaces of the maxillary teeth and mesial and distal surfaces of the mandibular teeth with a high-speed handpiece and water coolant. The teeth were cracked in half with a 301 elevator and the pulps gently removed with an endodontic spoon. The pulp specimen was then treated in the same manner as the inflamed pulps specimens.
Histopathological Examination In all, 19 inflamed pulp sample fragments and 18 normal pulp sample fragments were collected and categorized. Sample I 6 was too small to be sectioned for both immunoassay and histological examination so it was used for immunoassay pilot experiments. The fragments of specimens were embedded in paraffin, sectioned at 6 urn, stained with hematoxylin and eosin, and then examined and classified histologically as normal, i.e. without inflammatory cell infiltrate, edema, or hyperemia. The inflamed samples were subcategorized based on the extent of inflammatory infiltrate, edema, and hyperemia noted in the specimen.
Journal of Endodontics
Protein Extraction Tissue samples were removed from the -70~ freezer, allowed to thaw for 10 min, and the wet weight of the specimen was obtained. One-hundred fifty microliters of phosphate-buffered saline at pH 7.0 were added to each 250tA Eppendorfcentrifuge tube, and the samples were macerated with a glass rod. Each sample was vibrated for 5 s on a Vortex Genie Mixer. The samples were allowed to stand in the buffer at 4~ for 30 min, but every 10 min the samples were vortexed for 5 s. The samples were centrifuged for 2 minutes at 11,000 rpm equivalent to a relative centrifugal force of 9880 x g. The supernatants of the samples were then diluted with PBS and aliquots of the sample dilutions were stored at -70~ for immunoassay.
Enzyme-linked lmmunosorbent Assay A1AT and A2M concentrations in standards and pulp samples were determined using the enzyme-linked immunosorbent assay double antibody sandwich technique. The amplified substrate system as described by Cart et al. (18) was used in the assay. Fifteen normal samples and 15 inflamed samples were used for the assays. The remaining inflamed pulps were used in pilot experiments to determine methodology for protein extraction. Extracted inflamed pilot and normal sample aliquots were used in pilot experiments to ascertain dilution ranges for each of the assays. A 1AT standard dilution concentration ranged from 2.5 to 100 ng and A2M standard dilution concentration ranged from 16.7 to 800 ng. Through pilot experiments a range of sample dilutions for both A 1AT and A2M was determined as 1:100, 1:500, 1:1000, and 1:2000 for the normal samples and 1:50, 1:100, 1:500, and 1:1000 for the inflamed samples. If any sample reruns were required, the dilutions were adjusted appropriately. Each standard and sample dilution for A1AT and for A2M was run in triplicate. RESULTS
Histopathological Results The ages of the patients whose pulps were collected in the inflamed groups ranged from 18 to 20 yr, with the average age being 18.4 yr. Nineteen pulps were collected in the inflamed category and examined histologically. The inflamed samples were classified as mild (n -- 7), mild to moderate (n = 2), moderate (n = 5), or severe (n = l) based on the extent of the acute inflammatory response noted in the specimen. A subcategory which includes degenerative changes was added, i.e. mild inflammation with degeneration (n = 4). Figures 1 and 2 are photomicrographs illustrative of the inflammatory categories. Fourteen of the 19 pulps showed evidence of dystrophic calcification. Eighteen pulps were collected in the normal category and examined histologically. The range of ages for the patients in the normal group was 18 to 30 yr, with the average age being 21. l yr. Figure 3 illustrates a normal pulp sample. Two of the normal pulps had a small area of dystrophic calcification. Nine normal pulp specimens revealed areas of
Vol. 17, No. 12, December 1991
FIG 1. Mild inflammation: sample I 10, illustrating a mild PMN infiltrate, absence of edema, and hyperemia (H & E; original magnification x40).
Pulpal Inflammatory Modifiers
591
FIG 3. Normal pulp: sample N 5, illustrating intact odontoblastic layer, nerve tissue, and absence of inflammatory infiltrate, hyperemia, or edema (H & E; original magnification xl0). TABLE 1. Average protease inhibitor concentrations
FIG 2. Severe inflammation: sample I 3, illustrating a predominate PMN infiltrate, congested capillaries, extravasation of RBC's, generalized edema, and dystrophic calcification (H & E; original magnification x40).
fatty change. Because of the absence of inflammation, the pulps met the experimental criteria for normal. Three of the samples collected as clinically normal pulps were rejected because of failure to meet the specified criteria.
Inhibitor Concentrations For each standard and sample dilution, the average optical density at 4 and 7 min was calculated and the change in optical density for the 3-min interval determined. The change in optical density for the standard dilutions was plotted against the concentrations of the standard dilutions, and a linear regression was run on standard dilutions to determine the standard curve for each assay. The concentration of the protease inhibitors in the samples for each assay was extrapolated from the standard curve for that particular assay.
Group
AIAT (#g/mg pulp)
A2M (#g/mg pulp)
Normal Mild Moderate to severe
0.3419 _+ 0.1949 0.2615 + 0.0984 0.4711 _+ 0.2610
1.5308 _+ 0.9963 1.5803 _+ 1.1818 5.3754 _+ 4.4091
The concentration in the sample was multiplied by the dilution factor, and the resultant concentrations were averaged. The concentration of the protease inhibitors in the sample was converted to micrograms per milliliter. The pulp samples had been weighed before the extraction of the proteins, and weight per unit volume of the pulp sample in the 0.150 ml of buffer was determined. The weight per unit volume of pulp sample in elution buffer was expressed in milligrams per milliliter. The concentration of inhibitor was divided by the weight per unit volume of pulp sample. The resultant protease inhibitor concentration was expressed as micrograms of protease inhibitor per milligram of pulp sample.
Statistical Analysis To better facilitate distribution for analysis, the inflamed pulp subcategories were reorganized into the following: (a) mild (n = 7), which included mild inflammation and mild inflammation with degeneration and (b) moderate to severe (n = 8), which included mild to moderate inflammation, moderate inflammation, and severe inflammation. The average protease inhibitor concentrations and standard deviations are illustrated in Table 1. The data revealed that the A2M distributions were not equal and variances were different between groups. To control these disturbances, the A2M data were subjected to a natural log transformation, and all subsequent analyses are on the transformed data. By using BMDP Statistical Software (Los Angeles, CA), data were subjected to a one-way multivariate analysis of
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M c C l a n a h a n et el.
variance with two dependent variables (A 1AT and A2M) and three levels of a single between-subjects variable (normal pulp, pulp with mild inflammation, and pulp with moderate to severe inflammation). The overall effect of the state of the pulp was significant (F = 3.36; df = 4,52; p = 0.0160), but the effect was attributed only to the A2M-dependent variable (F = 6.76; df = 2,27; p = 0.0042). Again, using the BMDP Statistical Software, a post hoc comparison of the A2M group means was accomplished using Tukey's Studentized range method. The Tukey method was chosen instead of the Scheffe method because according to Kirk (19), the Tukey method is a more powerful test in pairwise comparisons. The moderate to severe group differed significantly from the normal group (p < 0.01) and the mild group (p < 0.05). There was no difference between the mild and normal groups. DISCUSSION The ability to detect A 1AT in normal and inflamed human dental pulps was noteworthy, but not totally surprising, because of its widespread distribution in body fluid and organ tissue. The fact that the concentration ofA 1AT did not change with an increase in inflammatory category is puzzling. The type of inflammatory reaction of the pulp to dental caries may be an explanation. The pulpal reaction to caries is of chronic inflammation until actual exposure occurs which then is followed by an infiltration of PMN at the point of exposure. Since A1AT is an acute phase protein, an increased plasma concentration follows an acute inflammatory stimulus. The reaction time from an inflammatory stimulus in the dental pulp to an increase of A1AT in plasma concentration is not known. A basic hypothesis of this study was that to have protease inhibitor activity it is necessary that proteases be present. In human dental pulp, a known point of protease activity is during PMN cellular infiltration which has been demonstrated in carious pulpal exposure. A carious exposure serves as a predictable point in time to collect inflamed specimens which then can be compared with normal pulps. Probably, the time of sample collection in this study preceded the acute phase protein production and a subsequent increase in plasma concentration of A 1AT. A2M is confined to the vascular space because of its large molecular size. The vascular congestion which is a classical sign of early inflammation provides a mechanism for an increased intravascular concentration of A2M in the pulp tissue and at the site of inflammation. In this study, A2M was observed to increase in concentration in direct relation to progressive severity of the inflammatory responses. It may be fortuitous that the A2M concentrations are increased early in the inflammatory process. Since A2M has a broader range of activity than A1AT, it inhibits all classes of proteases and is not restricted to the serine proteases. Additionally, A2M inhibits the kinin, coagulation, and fibrinolytic cascades. The inhibitor with the broadest range of activity arriving "first on the scene" would best limit the host damage as a result of the interrelated aspects of the inflammatory process. Morand et al. (15) showed an increased elastinolytic and collagenolytic activity in diseased human pulps with suppur-
ative pulpitis and necrosis. It would be most interesting to examine the concentration of A 1AT and A2M in pulps demonstrating more intense inflammatory response. Also, it would be of interest to determine whether there is indeed a lag time and ultimately an increased localized A 1AT concentration. Similar to development of emphysema in the lungs of cigarette smokers, the burden of increased protease activity in the inflamed dental pulp possibly saturates the A 1AT and A2M molecules; subsequently leading to necrosis of the affected tissues. At what point inhibitors are saturated is not known. Since the collection of inflamed samples was based on clinical parameters, there was some distribution among the histological categories. All of the inflamed specimens met the criteria of PMN infiltration, but the clinically selected criteria did not correlate exactly with morphological change for all samples. For the purpose of statistical analysis, it would have been preferable to have had an even distribution in the inflamed histological groups mild, mild to moderate, and severe to facilitate analysis rather than combine the groups. A larger sample size could have improved the distribution. The interaction of A1AT and A2M with lysosomal enzymes in conjunction with the interaction of A2M with the enzymes of the kinin, coagulation, and fibrinolytic cascades are critical to the biological balance during inflammation. The presence of these protease inhibitors in the human dental pulp tissue and the significant increase in A2M in moderate to severe acute inflammation as determined by this study indicates that these proteins play a role in the pathogenesis of pulpal inflammatory disease. This project was supported in part by Naval Medical Research and Development Command Project MR0412002-0001.
The assertions contained herein are those of the authors and are not to be construed as official or as reflecting the views of the Department of the Navy or the Departmentof Defense. We wish to thank Dr. Mark E. Cohen of the Naval Dental Research Institute, Great Lakes, IL, for his expertise in the statistical analysis of the data and Mr. Andrew Markow of the Department of Pathology, Northwestern University Medical and Dental Schools for histological preparation of the specimens. Dr. McClanahan is a former endodontic resident, Northwestern University Dental School, and is head, Endodontics Department, Naval Dental Clinic, Okinawa, Japan. Dr. Turner is professor and director, Division of Periodontics, Northwestern University Dental School. Dr. Kaminski is professor, Department of Pathology, Northwestern University Medical and Dental Schools. Dr. Osetek is director, Advanced Program in Endodontics, and associate dean, Clinical Affairs, Northwestern University Dental School. Dr. Heuer is senior associate dean, Academic Affairs, Northwestern University Dental School. Address requests for reprints to Dr. Scott McClanahan, P.O. Box 2554, Camp Butler, FPO Seattle, WA 98774.
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The Way it Was Caught in the Jaws of the Amalgam Controversy Double Jeopardy For endodontists, amalgam has been the backbone of practice for many years in the area of root-end fills and core build-ups. However, it has been of concern to some practitioners because the placement of a root-end amalgam in a tooth with a metallic post may lead to the generation of a galvanic couple. When present, these couples have the potential to generate significant amounts of electrical current (Austin et al., Oral Surg 1982;54:79). The possibility of setting up an intraoral current of this nature was recognized in the early 1800s (Brockway, Am J Dent Sci 1844;5172; Mackall, Am J Dent Sci 1839;1:86. "Everyone has heard of the popular experiment, of placing silver on the top and zinc under the tongue, and making the edges touch to produce the peculiar metallic taste, caused by the galvanic action of the metals; but it is not generally known, even among the profession, that a constant galvanic action is kept up in the mouth, when more than one kind of metal is used in filling the teeth." While this problem could be the source of a failure in surgical treatment, the uses of amalgam in contemporary endodontics are slowly fading in light of controversial studies and the introduction of new materials. Recently though, the news media (60 Minutes) visited with ADA Fifth District Trustee Herber Simmons concerning the contemporary use of amalgam by dentists and the concerns expressed by recent Swedish and Canadian animal studies. Throughout the interview, as briefly printed in the ADA News (Berry, 1990;1:5), dentistry is placed in a defensive posture by the media, even though, as studies have pointed out, amalgam has never been linked to any systemic disease. This same situation occurred in 1839, when the editor of the American Journal of Dental Science expressed his own concern on the perceived impropriety of using dissimilar metals in the patient's mouth, even to the point of exposing the names of the professionals who chose to render this course of treatment. 'I understand there are those in this city, who use the Crawcour Succedaneum and Amalgam of Silver and Mercury; our object being to correct errors in dental practice, we hope all such will discontinue its use, in justice both to the profession and the public; and also to save us the painful necessity of exposing their names."
James L. Gutmann
