Microbial microleakage and pulpal inflammation: A review

Browne RM, Tobias RS: Microbial microleakage and pulpal inllanimation: A review. Endod Denl Traumatol 1986; 2: 177 183. Abstract - The evidence relating microbial microleakage and pulpal inflammation is reviewed. In vitro experiments show that all current dental materials permit fluid microleakage at the material/eavily wall interface. In vivo, this fluid microleakage is accompanied by bacterial growth, unless the material has lasting antibaclcrial properties. An association does exist bclwecn the presence of bacteria at the material/cavity wall interlace and the presence of pulpal ijiflanimation. The role of the siiirai' layer in minimizing (he harmful eifects of microbial microleakage and o( c'hemical toxic ity o( restoi-ative material is reviewed, l'urthcr research sliould be aimed al rliniinating microleakage by the development of more fully adhesive materials or by inipro\'ing their atitibacterial jjrojjertics. Roger M. Browne and Rosalind S. Tobias
Deparlmenl ot Oral Pathology, Dental School, St Chad's Queeiisway, Birmingham, U. K.

Key words: microbial microieakage, pulpal inflammation, smear layer, review. Professor R. M. Browne, Departmenf of Oral Pathology, Dental School, St Chad's Queensway, Birmingham B4 6NN, U. K. Accepted for publication 20 April 1986.

U n t i l rccciUly, most stutlies have conc-ludcd that pulpal indanimation observed beneath carefully prepared experimental cavities containing filling materials is due to cheniic al irritation from the materials ( 1 4 ) . Fui-lhcr, it i,s widely held tliat, the d e e p e r ihe cavity (thai is, the It'ss tlic thirkness of residual dcnlin beneath the cavity lloor, between t h e material and the pulp cavity), the greater the a m o u n t ol' pul|ial irritation (5). Whereas there is little doubt that such a relationshi]) exists in the eonsideration ol'thermal damage generated during careless cavit)" prcjjaration, it is far less certain with toxic damage from dental materials. In the earliest studies in which the relationship between cavity d e p t h and pulpal inflammation due to different materials was reported (6 8), tlic damaging effects of e a v i t y preparation itself were not fully appreciated. I n d e e d , some subsequent studies (9) have failed to establish any relationship between residual detitin thickness and pulpal inflammation observed in ca\ i\\cs filled l)y a variety of matei'ials. Present e\'idetice suggests that none of the current d e n t a l materials provides a perfect seal witli the e a v i t y wall and there is always a microspace at the interface between the two along whieh fluids and microorganisms (an petietrate from the oral environment (10 15). Microorganisms, introduced d u r i n g cavity preparation, may also remain alt a e h e d to tlie eavity walls, perlia|)s in the smear layer, and proliferate after insertion of the material.

The effect of microbial microleakage at the material cavity/wall interface oti the dental pulp has been a lacuna in the biological testing of dental restorative matei'ials. 1 his paper reviews the evidence relating microbial microleakage and pulpal inllammalion. Microleakage Mieroleakage is the term used for the ]:)enetiation of oral fluids and microorganisms along the material ca\-it.y/wall interface following insertion of a restoration. As mentioned above, there is always a microspace at this interface, caused partly by the contraction of the mass of material during the setting proc'ess and partly because none of the currently available tuaterials adhere coiiipleteh' to the cavity walls. The size of the mierosjjaee (16) \aries from material to material and as a consequenc'e the amount of microleakage is also likely to vary. A measure oi the amount of microleakage in vitro might thus be used to ]irediet the performance of restorative materials in the oral environment, because as a clinical occurrence it remains a jjrimary source of restorative failure (17). A wide variety of methods has been used in vitro to determine the extent of microleakage around, and the marginal adaptation of dental restorations. A discussion of these is beyond the scojie of this paper, but sc\'eral comprehensive
177

Browne and Tobias reviews have appeared elsewhere (12, 13, 17, 18). In general terms, microleakage is evaluated by measuring the passage of tracer molecules along the material cavity/wall interface or demonstrating the biological consequences of this trallic by inducing recurrent caries. However, there appears to be considerable variation in the results obtained by the different methods with the same materials, itidicating that there is a need for better understanding and standardization of ihe test methods (18). The results can be briefly summarized by saying that all currently available materials exhibit microleakage in viLro, but to a different degree. As might be expected, those materials which adhere to some degree to the ctivity walls, in particular the glass ionomer cements, demonstrate the ieasl degree of microleakage. However, microleakage to tracer molecules dose not necessarily imply that microbial microleakage will also occur. Although the microspace appears to be sufliciently large to allow microbial spread with all materials, there are other factors which will also determine this, in particular the availability of nutrients, including oxygen, and the antibacterial pro]jerties of the material. These latter factors are of particular importance in vivo and may explain conflicting findings. For exatnple, in vitro tracer studies have shown tliat zinc phosphate cement allows less microleakage than zinc oxide/eugenol cement (15, 19). Yet, in vivo, mieroorganisms are rarely found at the material cavity/wall interface around zinc oxide/eugenol fillings but are usually present around zinc phosphate cement. These in vivo findings probably t-eflect the lastitig antibacterial effect of zine oxide/eugenol (20) which, despite its poor seal, prevents colonization ol' the microspace by Ijacteria. These observations support the view (18) that mo.st investigations of microleakage in vitro are of donbtlul chnical relevance. Bacterial microleakage The evaluation of the possible toxic effects of restorative materials upon the dental pulp was first undertaken in a scientific way in the 1930s. It was not until 1959 that it was first suggested that bacterial microleakage at the material cavity/wall interface may infiuence the pulpal responses observed beneath experitncntal cavities filled with different materials (21). Since 1969, a grouj) of research workers in Sweden has carried out numerous investigations into the causes of pulpal inflammation a,ssociated wilh dental restorative materials and their results suggest that bacterial irritation is the main cause of pulpal damage beneath silicate, acrylic and composite resin fillings and beneath inlays cemented with zinc phos178 phate or polycarboxylate cements (22 28). Other workers have also demonstrated large numbers of microorganisms at the material/cavity wall interface in cavities filled with silicates (29-34) and composites (34-37). The possibility that bacterial microleakage may complicate the interpretation of puljial responses observed beneath cavities filled with a variety of experimental materials is now widely recognized (5, 33, 34, 38-43). Some investigators have beeti unable lo find any direet correlation between the presence of bacteria withiti the cavity on histolfjgical sections and pulpal inflammalion (4, 44, 45) and consider that growth of bacteria on the cavity floor may be favored by, and thus a consequence of, pulpal inllanimation (44) rather than its cause. Many studies, while recognizing an association between bacterial mieroleakage and ]3ulpal inflammation, have reported cavities in which moderate to severe inflammation is present in ihe apparent absence of bacteria (42, 46, 47). Sueh observations lead to the conclusion that, whereas bacteria are an important cause of pulpal inflammation beneath cavities filled with different experimental materials, chemical toxicity also Inlays some, although probably a mueh smaller, role. One of the major probletns in evaluating any correlation between bacterial microleakage and pulpal inflammation lies in the techniques used for demonstrating bacteria. For practical experimental reasons the presence or absence of bacteria is determined from tissue sections stained by various modifications of Gram's method. It is notoriously diflicult to identify bacteria using such methods, particulary if the bacteria are Gram-negative (48, 49). Further, the incidence of baeteria at the material/cavity wall interface will be influenced not only by the number of sections examined but also by baeterial displacement during the processing of the teeth for histological assessment. Culturing samples from the cavily wall provides more reliable evidence for the presence of baeteria, but this usually precludes microscopical examination of the .same specimen (34, 50). As a consequence the variable data on any correlation between pulpal inflamuiatioii and bacterial microleakage is not unexpected. Reeently, in our laboratory using histological and cultural techniques on the same specimens, we have demonstrated a good correlation between the results obtained by both methods (51, 52). This has allowed us to evaluate the relationship between pulpal inflammalion and bacterial microleakage with greater confidence. A recent study (52) has demonstrated a clo.se correlation between the extent of pulpal inflammation and degree of bacterial mi( roleakage using a wide range of tnaterials. This ecjrrelation

MIcroleakaga aod pulpal inflammation

w a s statistically highly significant in our animal model, the ferret, and also in humans. ff bacterial microleakage is a significant factor in t h e cause of pulpal inflammation, it would follow t h a t experiments designed to reduce or prevent microleakage should result in a substantial reduction in innOammation. A reduction or elimination ol'pulpal inflammation has, in fact, been observed beneath cavities filled with silicates, composites and amalg a m when bacterial microleakage is reduced or prevented by surface placement in the cavity ol an antibacterial cement (31, 44, 53 55). Zinc oxide/ eugenol-based cements are cITeetive at jDreventing microleakage in the above way. Although the test rnaterial still remains in contact with the cavity floor, a tnarkcd reduction in pulpal inflammation l i a s been consistently reported. At the same time, f e w or no bacteria are observed at the material cavity/wall interlace, suggesting that it is the red u c t i o n in tnicrobial population whic h is the signific a n t factor. These results indicate that a re-assessr n e n t is required of conventional in vivo methods ol testing toxic properties of dcutal restorative materials and provide further evidence to support the views of several workers (34, 39-41, 43, 56) that bacterial contaminatioti must be considered when assessing the pulpal response to restorative dental iTiaterials. Consequently, it would seem that the v'alich'ty of studies to evaluate the pulpal compatabil i t y of dental restorative materials in which bacterial niieroleakage has not been considered is questionaV:)le. U is worth noting that, prior to the last decade, f e w studies of pulpal response beneath cavities filled w'ith experimental materials gave reference to the s e a r c h for the presenee of baeteria at the material/ eiivity wall interface. As mentioned earlier, there are two possible ways in which bacteria at the cavity wall interface may a r i s e . Some consider that tliey originate from bact e r i a which were adherent to the cavity walls at the e n d of cavity preparation, possibly within the smear l a y e r (27), and therefore recommend that, clinically, a microbicidal cavity cleanser should be applied to remove grinding debris and eliminate these bact e r i a prior to insertion of the restoration (57 59). Hovvever, as bacteria have not always been demons t r a t e d on freshly cut dentin surfaces (48, 49, 60), t h e rationale fbr applying an antibacterial cavity cleanser is questionable (46). O t h e r s consider that the microorganisms pene t r a t e by microleakage fi'om the oral ctivironment a l o n g the material cavity/wall interface after the mii-tcrial has been inserted (61). Our own studies (33, 51, 55, 62 64) indicate an increasing incidence of bacteria on the floor of cavities with time up to 28 days, supporting the hypothesis that they gain access by microleakage. lntrtlier, the absence of bacteria from the cavity floor beneath materials, over the oral surfaec of which an antibacterial cement has been placed, would support this view (31, 44, 53 55). Clinically, it could therefore be argued that the most widely accepted lining materials, zinc oxide/eugenol- and calcium hydroxide-based materials, are efl'ectivc in protecting tlie pulp, tiot because they prevent chemical Irritation from the overlying filling material, but rather because their antibacterial properties minimize the sur\ival of microorganisms that reach the cavity floor by tnicroleakage (65). Both zinc oxide/eugenol (20) and calcium hydroxide (66, 67) materials have been demonstrated to possess antibacterial effects. It should be realized that absence of stainable or cultured bacteria on the cavity floor may not preclude bacterial infiuences on the pulp. Bacterial products from saliva, denial plaque or tooth surface deposits may, without simultaneous bacterial invasion, gain access to the exposed dentinal tubules via microleakage. In some respects bacteria may act as a tracer substance demonstrating possible pathways fbr a variety of iheir loxic products (46). It has been demonstrated that an inflammatory response occurs in the dental pulp when the products of oral bacteria are applied to freshly cut dentin (68). Similar results were obtained in experimentally immunized monkeys, following presentation to the cut dentin surface of the immunogen (69, 70). Certain oral bacteria and/or their products have the ability to activate neutrophils in the dental pulp (71). Certainly, the patterti ofneutrophil accummulatiou at the pulpo-dentinal jvuiction (so displacing the degenerating odontoblast layer pulpally) commonly observed beneath the llooi' oi' cax'ities containitig tnicroorganistns at the material cavity/wall interlace, is very suggestive of strong chemotactic attraction (33) by the bacteria or iheir products. Progressive accumulation of neutrophils in this way eventually results in suppuration which may be diffuse or localized as a micro-abscess (72). The microspace at the material cavity/wall interface arises shortly after the insertion of a material, partly as a consequence of volutne changes in the material during the setting process and partly because the materials do not effectively adhere to the cavily walls. Over longer periods, probably greater than 1 mouth, it is suggested [36) ihal ihe proliferation of l)acteria may be allowed as a consequence of deterioration of the margitis of the restoration by mechanical influences and temperature ehanges in the oral environment. In a recent study (73) where greater bacterial microleakage was demonstrated around cavities m teeth in functional occlusion than in similar cavities in unopposed teeth, it was concluded that functional stress upon composite restorations was the most important factor in determi178

Browne and Tobias

nhig microleakage. It is probable that this faclor has not been suHiciently considered in designing in vivo studies of the pulpal eflects of experimental materials. It might, for examjile, be of significance in explaining the greater incidence of bacterial microleakage in cavities in ferrets' teeth than in human teeth around the same materials (42). Finally, the importance of bacterial contatnination in determining the response of the dental pulp exposed directly to a variety of materials has been demonstrated in gnotobiotic animals (40, 43, 5G, 74). Comparison of the pul])al response to the same material in conventional and germ-free animals demonstrated a much greater inflammatory response in the conventional animals. Indeed, in the germ-free animals, few materials eaused significant change. to remove the smear layer, ihus greatly inereasing the i^ermeability of the dentin, sliould lead to greater |3ulpal irritation. Weak organic acids, such a.s phos]3horic atid citric, have been widely advocated as eavity (Onditiouing agents prior tf) insertion of cotnposite restorations. These conditioners dissolve the smear layer so exposing and unlilocking the peripheral ends of the dentinal tubules. Ihis results in a greatly increased permeability of the dentin (58, 80, 83). Several studies have shown that the pulpal inflammation beneath cavities so treated is greater tlian that beneath cavities in which no conditioner was ap|)liecl when the same material was used (8487). It has usually been considered that this greater inflammation was a consequence of the added chemical toxicity of the acid conditioners; it is more probably due to the greater (permeability to bacterial toxins, or indeed to the toxic components of the material. Although it is unusual to liiid bacteria within the dentinal tul)ules beneath eonventional cavities, it is not uneonunon in those that have been treated with cavity conditioners (87). Cuiri'ent evidence suggests therefore that the smear layer acts as a so-called natural cavity hner (88) and is biologically advantageous to the pulp. However, it also interferes with the adhesion of most lining, eement and filling materials to the cavity walls (75), with the notable exception of the glass ionomer cetnetits (89, 90). 1 he glass ionomer cements have been shown to bond directly with dental tissues (89, 91, 92), which sliould minimize the likelihood of bacterial mieroleakage. However, even with these materials bacteria have been demonstrated histologically at the material/cavity wall interface (43, 63, 87, 93), although in general the im]3ression is that they are jjresent in smaller numbers than with other filling materials. On the other hand, as composite restcjrations retjuire an etehed surface to obtain mechanical bonding, dilute organic acids or ehelating agents are usually used for this purpose. These have the effect nl removing the smear layer and thus opening up Ihe dentinal tubules to a variable degree. Fhe extent of this ell'ect vaiic^s accoiding to the nature and concentration of Ihe solution, the duration of its aj^plieation and whether it is applied in vivo or in vitro (94, 95). It appears that tlie ehelating agent l'^D'IA is the least damaging when ttsed tnider clinieal conditions. I'hcre is, con.scquently, a cotiflict between the biological needs for the smear layer in jjrotec ting the ]3ulp and the mechanical needs for bonding of, in l^artic ular, composite materials. It is not surprising, therefore, that opinions differ as to whether the layer should be removed prior to cavity restoration (96). However, even after etching, most composites still

Smear layer
Ifljacterial cause pulpal inflamniation during the first few weeks after insertion of a rnaterial in a test cavity, then their toxins must be able to reach the pulp cells in order to damage them. However, the surface of freshly cut denlin is covered by a smear layer. The smear layer is a mic rothin gelatinous layer which covers the dentinal walls after cavity preparation, .so obstructing the dentinal tubules. It is present regardless of instruments used (75) but the quality and quantity of the smear layer is influenced by the o]x'rating conditions (76). Smear layers are composed of organic and inorganic jjartides 0.5 15 |a,m in size (77). The organic component is thought to consist of heat-coagulated dentin proteins, saliva, blood and microorganisms, whereas the inorganic component is derived from tootli minerals and, possibly, inorganic contaminants (78). The presence of the smear layer provides a physical barrier to baeterial penetration as well as the diffusion of a wide range of substances in vitro (79, 80) and, in vivo, prevents the out-fiow of dentinal fluid onto the cut surface of vital dentin by the hydrostatic pressure of the pulp (80, 81). How ell'ective it is in vivo is not clear, so that although baeteria may l)e present at the material cavily/wall interlace, they may not be able to efl'eetively damage the jjulp. As indicated previously, bacterial products are able to cause |3ul]) damage when applied to freshly cut dentine in vivo, so that it ajjpears that the smear layer is not completely impermeable, and this is supported by in vitro studies (80, 82). It is likely that it is the penetration of toxins through the denlin which is the mechanism by which bacteria cause damage, at. least dtjring th(; first lew weeks after insertion of the material. It follows, therefore, that any procedure designed

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Microleakage and pulpal intlammation

exhibit some microleakage. An alternative to the creation of a leakproof margin for restorative materials is to reduce the permeability of the dentin around them. It has been demonstrated that oxalate salts are effective in decreasing the permeability of t h e smear layer on human dentin (97). It is thought t h a t the original smear layer is removed by treatnrient with oxalate solutions and rej^laced by an acid-resistant layer of oxalate crystals. This layer is rich in calcium and carboxylate groups and might thus be helpful in chemieal bonding. A further alternative: is to develo]3 restoi'ative materials with more persisting antibacterial projjerties s o that the microleakage is not so significant clinically. Fhe addition of chlorhcxidene to a polycarboxy l a t e cement, a glass ionomer cement and a composite material (98, 99) has been demonstrated to significantly increase their antiljacterial properties a n d further studies along these lines would be helpful. It is clear that the smear layer plays an imjjortant r o l e in minitnizing the harmful eifects of microbial microleakage and of chemical toxicity of restorative materials. The quest for new materials should ami a t ]jreserving this layer and so take advantage ol its benelic ial eflcets (88). Compromise may be possible, in which the biological integrity of the pulp and d e n t i n is preserved by developing unique chemical formulations compatible with adhesive biomaterials (76). eliminating mieroleakage by the development of more fully adhesive materials or by impro\-ing their antibacterial properties. Acknowledgments. The Dc Trey Di\'isioii. Dentsply Limited, ai'e thanked for their financial support. The authors would also like to thank ihe technical and secretarial staff from within the Department of Oral Pathology for their competent assistance.

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Conclusion
T h i s review has attenij^ted to bring together the evidence that microleakage is an important factor in determining pulpal inflammation beneath recently p r e p a r e d cavities. Largely IVoni /// vitro experiments, it is apparent that all currently available materials p e r m i t fluid microleakage at the inaterial/ca\ ity w^all interface. Frotii in vivo exjiierimeuts, it appears t h a t this fluid mici-oleakage is ac-eonipanied by baeterial growth, unless the niaterial placed in the cavi t y has pc~rsisting antibactei ial properties. There is accummulating evidence that au association exists between the presetice of bacteria at the material/ c a v i t y wall interface and tlie prc\senee of pulpal inflatnmation and that this association is one ol c a u s e and effect. However, the association is not absolute and it is probable that some materials exert chemical toxicity, but that this is of much less imp o r t a n c e than has bccti traditionally thought. It is prcjbable that the sueeessful clinical usage of zinc cjxiclc/eugenol- atid calcittm liydroxide-based materials as liners is a cousec|uence of their antibacterial propetties rather than providing an imperm e a b l e barrier to the chemical toxieity of the overl y i n g filling, further researcli should be aimed at

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