IMPORTANCE OF BACTERIAL ENDOTOXIN ( LPS ) IN ENDODONTICS

1Chairman, Department of Endodontics, School of Dentistry of Araraquara, UNESP, Araraquara, São Paulo, Brazil. 2Graduate Student in Pediatric Dentistry, Department of Pediatric, Preventive and Social Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil. 3Chairman, Discipline of Pediatric Dentistry, Department of Pediatric, Preventive and Social Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil. 4Associate Professor, Discipline of Pediatric Dentistry, Department of Pediatric, Preventive and Social Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.


INTRODUCTION
Bacterial endotoxin (LPS) has been amply studied.In fact, interest in knowledge concerning the structure of bacterial endotoxin, its mechanism of action, and forms of inactivation in both the clinical and laboratory studies is obvious by the fact that in the past 10 years, a total of 28.100 articles have been reported on Medline (http:// www.ncbi.nln.nih.gov/PubMed).In Dentistry, much research using different in vivo and in vitro methodologies has emphasized the importance of anaerobic bacteria and endotoxin in the etiology of chronic periapical lesions 3,9,13,19,21,25,36,42,52,57,63 .However, only few articles have evaluated the effect of the presence of LPS in root canals on apical and periapical tissues 8,10,32,37,43,55 and some articles have reported the inactivation of LPS toxic properties after endodontic procedures both in vivo and in vitro 1,4,6,23,37,42,48,49,55,61,68 .

ROLE OF GRAM-NEGATIVE MICROORGANISMS AND ENDOTOXIN ON ETIOLOGY OF PERIAPICAL LESIONS
When dental pulp is exposed to the oral cavity due to caries or trauma, it is initially contaminated by predominantly aerobic and facultative microorganisms.Due mainly to the existing nutritional relationships between microorganisms together with the slow decrease of oxygen tension in root canals, a microbial shift takes place leading to a predominance of anaerobic microogranisms 60 .Since the 1980's, technical advances in microbiological culture and identification have shown that anaerobic microorganisms predominate in root canals of teeth with pulp necrosis and radiographically visible chronic periapical lesion 29,30,51,60 , especially Gram-negative 4 .This polymicrobial infection is located not only in the lumen of the root canal and dentinal tubules, but also in apical craters and the entire root canal system 29,30,58 .
Gram-negative microorganisms have different virulence factors 26 and form products and sub-products that are toxic to apical and periapical tissues.They also contain endotoxin in their cell wall 33 .
Endotoxin, present on all Gram-negative bacteria, is composed of polysaccharides (polymerized sugars), lipids (complexes containing fatty acids) and proteins.Endotoxin can be named lipopolysaccharide (LPS), emphasizing its chemical structure 46,65 .Lipid A is the region of the endotoxin molecule responsible for its toxic effects 11,27,33,34,65 .In 1993, Raetz 45 published a short review about the synthesis of lipid A and classified the endotoxins as extraordinary lipids.
Besides the chemical structure, much has been studied about the mechanism of action of endotoxins.When free to act, endotoxins do not cause cell or tissue lesions directly, but they stimulate competent cells to release chemical mediators.Researches showed that macrophages are the main target of endotoxins.Thus, endotoxins are not intrinsically venoms.Their effects depend on the host's response, as reported by Lewis Thomas, in The Lives of a Cell: "This oppressive uncontrolled and autodestructive behavior of the host is what makes endotoxin a venom."Furthermore, the same autor wrote: "Endotoxins are read by our tissues as the worst of news.When in contact with an endotoxin, our organism places all of its defenses at disposal with the idea to bombard, block and destroy all the tissue in the area.This appears to generate panic" 46 .
During endodontic treatment this is particularly significant because endotoxin (LPS) is released during multiplication or bacterial death causing a series of biological effects 4,33 , which lead to an inflammatory reaction 46 and periapical bone resorption 59,67 .
Even though the bacterial etiology of periapical lesions has already been proven since the classic study of Kakehashi, et al. 24 , few investigations have evaluated the isolated effect of LPS in contact with apical and periapical tissues 8,10,32,37,43,55 .
Among all animals, humans are the most sensitive to the effects of endotoxins 66 , which makes the knowledge of their biological effects on tissues fundamentally important.Endotoxins from vital or nonvital, whole or fragmented bacteria act on macrophages 46 , neutrophils 35 and fibroblasts 9 , leading to the release of a large number of bioactive or cytokine chemical inflammatory mediators 33 , such as tumor necrosis factor (TNF) 5,33,68 , interleukin-1 (IL-1) 31,33,68 , IL-5 31 , IL-8 31 , alpha-interferon 33 and prostaglandins 7 .Furthermore, LPS is cytotoxic 20 and acts as a potent stimulator of nitric oxide production 5,68 .
LPS also activates Hageman factor (factor XII of coagulation), has a lethal effect on animals 33 , induces fever 21 , activates the complement system 7,21,34 , thus acting in inflammatory response reactions by increasing vascular permeability, neutrophil and macrophage chemotaxis, lysozyme and lymphokine release 34 , activation of the metabolic cycle of arachidonic acid 7,33 being mitogenic for B lymphocytes 33 and causing mastocyte degranulation 18 .In infected root canals, endotoxin can contribute to an increased release of vasoactive neurotransmitter substances in the region of the nerve endings in periapical tissues, causing pain 51 .
According to Torabinejad, et al. 62 , the products of arachidonic acid metabolism and the activation of the complement system play an important role in bone resorption that is associated with periapical lesions in human teeth.
Besides causing an inflammatory reaction, LPS adheres irreversibly to mineralized tissues acting as a potent stimulator of bone resorption 52,67 , acting on the synthesis and release of cytokines that activate osteoclasts 22,23 , such as IL-1 and TNF, and stimulates the release of prostaglandin-E2 that also influences osteoclasts 48,64 .In tissue culture, Nair, et al. 36 observed stimulation of bone resorption by endotoxin, confirming the role of LPS in the pathogenesis of periapical lesions seen by others 4,10,32,52,59,67 .
Considering the discussed above, the major objective of the dental professional during treatment of root canals of permanent teeth with pulp necrosis and chronic periapical lesion should not be only bacterial death, but also the inactivation of lipid A, which is the toxic portion of endotoxin.This objective is not reached by using root canal antibacterial dressings, which only kill the bacteria remaining in the root canal system after biomechanical preparation.
Medical and dental literature have published studies that have attempted to obtain a medication or substance that inactivates bacterial endotoxin, eliminating its biologically toxic potential.Caustic soda 10,39 , polymyxin B 44 , neutrophilic enzymes 35 , lysozyme 41 , formocresol 50 , 1.25% chlorhexidine 1 , and sodium hypochlorite 6 have been tested, with no significant results.Many of these products present inherent limitations due to their high toxicity causing damaging effects when in contact with vital tissues.Thus, their routine clinical use is limited.The action of laser on periapical bacterial biofilm has also been tested 2 , however, its use is limited by the fact that there is no free access to the sites where the endotoxin is present, the root canal system of infected teeth, except when apical surgery is performed.

ROLE OF CALCIUM HYDROXIDE IN THE INACTIVATION OF BACTERIAL ENDOTOXIN
The first reference 40 to the introduction of calcium hydroxide in dentistry was in 1838.However, its clinical use progressed only after the studies by Hermann 16 in 1920.Calcium hydroxide, which has a highly alkaline pH, has been used in numerous different clinical situations, i.e., direct pulp protection, pulpotomy in permanent or deciduous teeth, root canal dressing in the treatment of permanent teeth with incomplete rhizogenesis, filling sealer in root canals, root perforations, dental resorptions, and antiseptic intracanal dressing 56 .This ample use has been attributed to its antibacterial activity 12,29,30 , biocompatibility 15,17,38,54 , hygroscopic property, ability to reduce periapical exudate 16 , and its capacity to induce mineralization 28,54 and to dissolve necrotic tissue remnants after biomechanical preparation that can act as bacterial substrate 14 leading to the stimulation of apical and periapical repair of teeth with chronic lesions.
Currently, one of the concerns of Endodontics is the treatment of teeth with pulp necrosis and periapical lesion because treatment failure is higher than in cases without periapical lesion.In teeth with chronic periapical lesion, there is a greater prevalence of Gram-negative anaerobic bacteria disseminated throughout the root canal system (dentinal tubules, apical craters and cementum lacunae), including apical bacterial biofilm.Because these areas are not reached by instrumentation, the use of an rootcanal dressing is recommended to aid in the elimination of these bacteria and increase the possibility of clinical success 25,28,38,63 .
According to Leonardo, et al. 30 , teeth with and without radiographically visible periapical lesion are different pathological entities requiring different treatment.In the first case, they recommend the use of an root canal dressing between treatment sessions, because the success of treatment in cases with a periapical lesion is directly related to the elimination of bacteria, products and subproducts from the root canal system.The procedures and medicaments used in root canal treatment should not only lead to bacterial death, but also to the inactivation of bacterial endotoxin.
Because of the lack of information concerning the effect of intracanal dressings on residual LPS that may adhere to mineralized tissues 8 , Safavi and Nichols 48 evaluated in vitro the effect of calcium hydroxide on bacterial LPS.They concluded that calcium hydroxide hydrolyzes the highly toxic lipid A molecule that is responsible for the damaging effects of endotoxin.In a later study, Safavi and Nichols 49 concluded that calcium hydroxide transforms lipid A into fatty acids and amino sugars which are atoxic components.These results were confirmed in recent studies by Barthel, et al. 4 and Olsen, et al. 42 who reported that calcium hydroxide detoxifies bacterial LPS in vitro.
In 2002, Nelson-Filho, et al. 37 carried out an in vivo study to evaluate radiographically the effect of endotoxin plus calcium hydroxide on apical and periapical tissues of dog's teeth.They observed that the endotoxin caused the formation of periapical lesions after 30 days and that calcium hydroxide inactivated bacterial LPS.Silva, et al. 55 analyzed histopathologically apical and periapical tissues of dog teeth in which the root canals were filled with bacterial LPS and calcium hydroxide.They reported that LPS caused the formation of periapical lesions and that calcium hydroxide detoxified this endotoxin in vivo.
More recently, Tanomaru, et al. 61 evaluated the effect of biomechanical preparation using different irrigating solutions and a calcium hydroxide-based root canal dressing in dog teeth containing endotoxin.Biomechanical preparation with only irrigating solutions did not inactivate the endotoxin, however, the same treatment associated with the use of the calcium hydroxide root canal dressing (Calen ® , SS White Artigos Dentários Ltda -RJ -Brasil) was effective in the inactivation of the toxic effects of this endotoxin.With the objective of evaluating the production of TNF-α, IL-1 and nitrite in cultures of human monocytes incubated with different concentrations of LPS and associated with the calcium hydroxide-based paste (Calen ® ) or pure calcium hydroxide, Zuccolotto 68 showed that calcium hydroxide was capable of inactivating LPS.
Jiang, et al. 23 also evaluated the direct effects of LPS on osteoclastogenesis and the capacity of calcium hydroxide to inhibit the formation of osteoclasts stimulated by endotoxin.They reported that calcium hydroxide significantly reduced osteoclast differentiation.
This new knowledge has revolutionized concepts about root canal dressings, indicating calcium hydroxide as not only the medicament most indicated, but fundamentally the only one currently capable of inactivating the endotoxin present in the root canal system of teeth with pulp necrosis and chronic periapical lesion.

CONCLUSIONS
-Bacterial endotoxin (LPS), which is a component of Gram-negative cell wall, is present in all teeth with pulp necrosis and radiographically visible chronic periapical lesion.It plays fundamental role in the genesis and maintenance of periapical lesions due to the induction of inflammation and bone resorption; -Calcium hydroxide inactivates the toxic effects of bacterial endotoxin, in vitro and in vivo, and is currently the only clinically effective medicament for inactivation of endotoxin.