Systemic evaluation of various haemostatic agents following local application prior to direct pulp capping

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1 Braz J Oral Sci. July-September Vol. 4 - Number 14 Systemic evaluation of various haemostatic agents following local application prior to direct pulp capping Franklin Garcia-Godoy 1 Peter E. Murray 2 1 DDS, MS, Professor and Associate Dean for Research, Nova Southeastern University, College of Dental Medicine, Fort Lauderdale, Florida. 2 PhD, Assistant Professor, Director of Oral Biology, Nova Southeastern University, College of Dental Medicine, Fort Lauderdale, Florida. Received for publication: March 11, 2005 Accepted: August 12, 2005 Abstracty Limited physiological information is available regarding the role of hemostatic agents to control the success of direct pulp capping treatment in order to help save vital pulps from pulpotomy, pulpectomy or extraction. The aim of this study was to test whether placement of various hemostatic agents would alter short-term pulp healing following direct pulp capping. Class V cavity preparations with pulp exposures were prepared in 31-nonhuman primate teeth, pulp hemorrhage was controlled by placement of epinephrine, Consepsis, sodium hypochlorite or combinations of these agents. Each exposure was then direct capped with composite resin and then observed immediately or after 13, and 28-days. Histological examination of pulp healing was conducted according to ISO standards for pulp inflammation, soft tissue organization, reactionary and reparative dentin bridge formation. Physiological measurement of heart rate and blood pressure were continuously recorded during the three operative procedures. Local pulp treatment with the various hemostatic agents did not alter systemic blood pressure or heart rate during local pulp application. All hemostatic treatments provided acceptable biocompatibility. Minor differences in pulp healing were observed, but there were no statistically significant differences between treatments (Pe 0.05). We conclude that hemostatic treatment had little effect on systemic pulp physiology or healing. Key Words: sodium hypochlorite, epinephrine, heart rate, blood pressure, healing Correspondence to: Franklin Garcia-Godoy Associate Dean for Research Nova Southeastern University, College of Dental Medicine Fort Lauderdale, FL Tel: (954) Fax: (954) godoy@nsu.nova.edu 791

2 Introduction Mechanical pulp exposure may occur during cavity preparation. However, it should be avoided because it reduces the prognosis of restorative treatment. The selection of a direct pulp capping agent to optimize pulp healing has proven controversial, although the consensus is shifting towards acceptance of composite resins, when calcium hydroxide [Ca(OH)2] was previously the only material of choice 1-3. Before pulp capping can take place, pulp lavage and hemorrhage control must be achieved. These procedures are most likely even more important to preserve pulp vitality than is the choice of a specific direct pulp capping agent, because of evidence that pulp healing is most compromised by hemorrhagic injury 4-5. The most common accepted clinical technique is simply to apply pressure to the exposure site with a sterile cotton pellet until hemorrhage subsides 6-7 sometimes the soaking of cotton pellets with sterile saline or other chemicals have been recommended While some alternatives such as electrosurgery 14, or laser irradiations 15 have proven to be of limited usefulness. hypochlorite [NaOCl] has a long history of removing diseased or infected tissues from wounds 16 and for removing diseased pulp tissue Since the 1950 s, NaOCl has demonstrated acceptable pulp biocompatibility when used as a local pulp hemostatic agent The solvent activity of a 5.25% concentration of NaOCl is limited to superficial pulp cells, leaving the subjacent pulp tissue unaffected Little is known about the biocompatible effects of other chemical agents such as epinephrine 25, 26 and commercial formulations of NaOCl agents. Consequently the aim of this study was to examine pulp healing and compare the biocompatibility of these chemicals when used as agents to control hemostasis following pulp exposure. Material and Methods Thirty one sound teeth were used from two-adult Rhesus macaca monkeys. The animals were procured and housed according to the University ethical and animal review guidelines and procedures. Prior to operative procedures, each animal was tranquillized and sedated. Teeth were scaled and polished with rubber cup and pumice paste to remove plaque and calculus. Saliva was controlled by high-speed evacuation and quadrants of teeth were isolated with sterile cotton rolls. Class V cavities were prepared with a #330 carbide bur at ultra-high speed under water spray coolant and the pulps intentionally exposed with the same bur; a new bur was used for every fourth tooth. Hemorrhage was controlled using a cotton pellet containing one of several agents; a commercial NaOCl-product,, or epinephrine (Table 1), each applied with light pressure for 30-seconds or until hemorrhage was clinically controlled. The cavities were gently rinsed with sterile water and the haemostatic agent reapplied if bleeding continued. Each pulp exposure was direct pulp capped by placing a thin layer of UltraBlend Plus only over the exposed pulp and light cured for 20-secs (Ultradent Inc., South Jordan, Utah). The enamel and dentin walls were etched with a 35% phosphoric acid for 15-secs, rinsed with sterile water for 10-secs and gently air dispersed; not dried. The bonding adhesive (PQ- 1 ) was evenly applied to the prepared cavity surfaces for 20-secs, without disturbing the pulp, and air thinned using a vacuum tip until the surface presented a glossy appearance. The adhesive was light-cured for 10-secs to avoid thermal damage to the underlying pulp tissue. Composite resin (Amelogen ) was placed to the axial floor and light cured for 20-secs. A second layer was placed, contoured and finished to the cavosurface margin, followed by etching, rinsing and sealing with (Perma-seal ) to ensure a complete seal of the restoration. The standardized methods and procedures used in this study are congruent with those described elsewhere The heart rate and blood pressure of the animals was continuously monitored and recorded during the three operative procedures: during cavity preparation, haemostatic, and the restorative phase. Table 1 -Tooth numbers, extraction interval, hemostatic agent formulations. Hemostatic agent Extracted [Days] Number of Teeth Hypochorite a 0 3 b 0 3 Hypochorite product c 13 3 Hypochorite product c + b 28 2 Epinephrine d + b 28 2 Hypochorite a 28 6 Hypochorite product c 28 6 Epinephrine d 28 6 a 5.25% NaOCl solution, b Chlorhexidine, Astra, Westborough, MA c Ultradent, South Jordan, Utah d 2% Xylocaine with epinephrine / 1:100,

3 Table 2 - Histological valuation criteria Inflammatory cell response The pulp contained few inflammatory cells, or an absence of inflammatory cells associated with the pulp beneath the cut tubules of the cavity floor. The pulp had localized inflammatory cell lesions predominated by polymorphonuclear leukocytes, or mononuclear lymphocytes. The pulp had polymorphonuclear leukocyte lesions involving more than one third of the coronal pulp. Severe The pulp tissue is largely necrotic, following chronic inflammatory cell injury. Soft tissue organization Normal Exposure cell loss Pulp cell loss No tissue injury, disruption, or loss of cell survival. A superficial loss of cell survival at the site of injury. An extensive loss of cell survival involving more than the superficial pulp. Reactionary dentin No secretion of reactionary dentine at the site of injury. A thin zone (d 10 µm) of dentine secretion by primary odontoblasts. A thick zone (e 10 µm) of dentine secretion by primary odontoblasts. Reparative dentin Bacterial staining Severe No secretion of reparative dentin by odontoblastoid cells at the site of injury. A thin zone (d 10 µm) of dentin secretion by odontoblastoid cells. A thick zone (e 10 µm) of dentin secretion by odontoblastoid cells to bridge the site of exposure. Absence of bacterial staining in any section. A positive bacterial staining reaction seen along the cavity walls. A positive bacterial staining reaction seen within the cut dentinal tubules of the cavity preparation. A positive bacterial staining reaction seen within the dental pulp. Serial sections of 7µm thickness stained with hematoxylin and eosin, were examined by light microscopy, and histological analysis was conducted according to International Organization for Standardization (ISO) Technical Report 7405 usage test guidelines 31. The inflammatory response of each pulp was categorized according to ISO guidelines and published criteria The categories of pulp inflammation and other histological assessment criteria are shown in Table 2. Bacterial contamination of each restoration was assessed using McKay s stain 32 to detect for the presence of Gram positive and Gram negative microorganisms. Data were analyzed using analysis of variance (ANOVA) statistical procedures, and Chi-square (X 2 ) at a confidence level of 95% (STAT view software, SAS Inc.). These statistical analyses are reportedly among the most versatile and conservative of multiple comparison tests 33. Results Blood pressure, heart rate and pulp histology Table 3. Statistical correlations between haemostatic agents and variables Statistic Comparison between materials and: P value ANOVA Blood pressure ANOVA Heart rate X 2 Inflammation X 2 Soft tissue organization X 2 Reactionary dentin X 2 Reparative dentin bridge A comparison between the hemostatic agents demonstrated very little systemic differences between their effects on heart rate or blood pressure, suggesting that pulp hemorrhage control does not place a strain on the whole body systemic system, or do they present a serious threat to the individuals health during operative procedures, During the different stages of operative procedures there was little change in heart rate (P = ) or blood pressure (P = ) (Figures 1 and 2). Furthermore, there were few 793

4 Heart rate (Beats per minute) Braz J Oral Sci. 4(14): Phase of treatment Epinephrine Epinephrine/ / Hemostatic agent product Phase of treatment Cavity preparation Hemostasis Restorative Mean blood pressure (mm mercury) product Epinephrine Epinephrine/ Hemostatic agent / Cavity preparation Hemostasis Restorative Fig. 1 - Heart rate during operative procedures. Fig. 2 - Blood pressure during operative procedures. Inflammatory cell response Severe Soft tissue organization Pulp cell loss Exposure cell loss Normal Reactionary dentin Reparative dentin a. (0 days) b. (0 days) c. product (13 days) d. product (13 days) e. product + (13 days) f. Epinephrine + g. Epinephrine h. (0 days) Fig. 3 - Histological assessments of hemostatic treatments prior to pulp capping. differences in heart rate (P = 0.994) or blood pressure (P = ) between hemostatic treatments (Table 3, Figures 1 and 2). With some agents hemorrhage control was difficult to achieve, necessitating repeated re-applications, sometimes with alternative agents. Nevertheless, hemostatic control was always accomplished prior to pulp capping with the adhesive resin. Generally the histology showed that each hemostatic agent permits pulp healing against the composite resin, passing the ISO 7405 requirements for clinical testing. There were few differences between the agents (Table 2) in terms of inflammation (X 2, P = ), reactionary dentin (X 2, P = 0.988), reparative dentin (X 2, P = ) and tissue 794

5 organization (X 2, P = ). No bacterial microorganisms were observed in any of the teeth stained with McKay s stain. Immediate capping pulp histology Six capped pulps showed no inflammatory activity, reactionary or reparative dentin secretion or injury of cells, deeper than the superficial layer. However some extravasted red blood cells and operative debris were associated with pulp exposure. Histology of pulp capping after 13-days with NaOCl Three exposed pulps were treated with a NaOCl-product; only one-pulp showed some reactionary and reparative dentin formation associated with the composite resin capping material. The two remaining pulps had severe inflammation and one was proceeding to necrosis, no reactionary or reparative dentin was observed in these teeth. These pulps also showed the greatest mean loss in cells in comparison with other pulps (Figure 3). Histology of pulp capping after 28-days with NaOClproduct Twenty-two exposed pulps were treated with hemostatic agents and combinations of agents. Following the use of NaOCl-product, reactionary and reparative dentin was observed in all six-treated pulps, reparative dentin was observed subjacent to the capping material in four pulps, and in two pulps reparative dentin was observed in the deeper pulp. This level and positioning of reparative dentin secretion is not as complete as with other treatments after 28-days (Figure 3). The incidence of new dentin secretion (100%), contrasts with that observed in a third of teeth after 13-days (33%). Over the same time difference, the mean level of pulp inflammation reduced and tissue organization injury was not as severe (Figure 3). Histology of pulp capping after 28-days with NaOClproduct and Both of the 2 capped pulps showed normal tissue organization, no inflammation, and some reactionary and reparative dentin secretion at the exposure site (Figure 3). Histology of pulp capping after 28 days with epinephrine and Both of the 2 capped pulps showed low levels of pulp inflammation, near normal tissue organization, with some reactionary and reparative dentin secretion at the exposure site (Figure 3). Histology of pulp capping after 28-days with epinephrine The four capped pulps showed low levels of pulp inflammation, near normal tissue organization, with some reactionary and reparative dentin secretion at the exposure site (Figure 3). However, in contrast to the other treatments, reparative dentin secretion was generally not associated with the capping material (Figure 3). Histology of pulp capping after 28-days with NaOCl Six pulps were treated with NaOCl, two pulps showed each category of inflammation; none, slight and moderate. Therefore, the mean category of inflammation was slight (Figure 3). Tissue organization was largely normal, with a slight level of reactionary dentin and reparative dentin secretion at the site of exposure in most pulps (Figure 3). Discussion The histological observations from this study provide some biological insight into several issues that deal with direct pulp capping with adhesive systems. Each of the different hemorrhage control agents is acceptable to pass ISO biocompatibility usage screening requirements for clinical evaluation. However, because a few differences in pulp healing were observed between different hemostatic treatments, this suggests a role for the importance of the capping materials. A good reason for the lack of serious pulp inflammation and healing defects, was the lack of bacterial staining in any of these sections, this avoids the bacterial stimulus responsible for the most serious pulp inflammation and healing complications 10, These observations highlight the importance of sealing restorations along the enamel-resin margins to provide pulp protection and to attempt to improve the prognosis of operative treatment. Moreover, sealing of the restoration interface appears a more important factor than placement of any one particular direct capping agent; recently it has been demonstrated that reactionary and reparative dentin secretion is a natural healing response to pulp injury not a specific response to any specific Ca(OH) 2 agent, as originally assumed 37. The use of composite resin materials for direct pulp capping remains highly controversial, some investigators report serious pulp necrosis 38, while others report excellent healing 10, The lack of consensus is reflected in some reviews that have been indecisive over the most appropriate selection of pulp capping materials, failing to completely recommend composite resins, resinmodified glass ionomers or the traditional use of calcium hydroxide. There were few differences between the physiological and histological effects of the hemostatic treatments. However, NaOCl appeared to be the most effective agent in removing the cavity biofilm and to control hemorrhage, enabling the pulp capping materials to be placed most easily. However, it was not possible to measure the ease of use of NaOCl in comparison with the other treatments, because it is subjective and likely influenced by the level of operator experience. This ISO usage study showed that each tested 795

6 treatment regimen provides the necessary clinical requirements for placement of the composite resin material to accomplish direct pulp capping. This study has demonstrated that mechanically exposed pulps will heal naturally following proper haemostatic treatment and capping with composite resin, provided bacterial leakage can be prevented and maintained. However, this study can only be used as a guide to help address the short-term performance of these pulp-capping treatment regimens, unfortunately many questions remain to be answered over the longer term. The use of non-human primates as part of ISO preclinical testing 31 has shown that the cleansing of biofilm from all cavity surfaces, followed by sealing against exudates and leakage will mediate high levels of reparative dentin against direct adhesive capping, as observed in this study. Clearly, the dental pulp maintains its physiological vitality when bacteria and their toxic components are excluded from gaining access to the vital tissues of the dentin and pulp. The key to accomplishing improvements in the prognosis of direct pulp capped teeth requires further investigation of methods to control hemorrhage and preservation of pulp vitality. References 1. Hafez, AA, Kopel HM, Cox CF. Pulpotomy reconsidered: An application of an adhesive system to pulpotomized permanent primate pulps. Quintessence Int 2000; 21: Mjor IA. Pulp-dentin biology in restorative dentistry. Part 7: The exposed pulp. Quintessence Int 2002; 33: Murray PE, Smith AJ. Saving pulps a biological basis. Prim Dent Care 2002;9: Dummer PM, Hicks R, Huws D. Clinical signs and symptoms in pulp disease. Int Endod J 1980; 13: Matsuo T, Nakanishi T, Shimizu H, Ebisu S. A clinical study of direct pulp capping applied to carious-exposed pulps. J Endod 1996; 22: Hunter FA. Saving pulps. 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