Microleakage
There is evidence that restorative materials may not adequately bond to or seal enamel or dentin. In this case, bacteria, food debris, or saliva may be drawn into the gap between the restoration and the tooth by capillary action. This effect has been termed microle- akage, and its influence on pulpal irritation has been extensively studied. Several early studies reported that various dental restorative materials irritated pulp tissue in animal tests. However, several other studies hypothesized that the products of microleak- age, not the restorative materials, caused the irrita- tion. Subsequently, numerous studies suggested that bacteria present under restorations and in dentinal
tubules might be responsible for pulpal irritation.
Other studies showed that bacteria or bacterial prod- ucts such as lipopolysaccharides could cause pulp irritation within hours of being applied to dentin.
Finally, a classic animal study shed light on the roles of restorative materials and microleakage on pulpal irritation. Amalgam, composite, zinc phos- phate cement, and silicate cement were used as restorative materials in class 5 cavity preparations in monkey teeth. The materials were placed directly on pulp tissues. Half of the restorations were sur- face sealed with ZOE cement. Although some irrita- tion was evident in all restorations at 7 days, after 21 days, the sealed restorations showed less pulpal irritation than those not sealed, presumably because microleakage had been eliminated. Only zinc phos- phate cement elicited a long-term inflammatory response. Furthermore, the sealed teeth exhibited a much higher rate of new dentin formation, termed dentin bridging, under the material. Only amalgam seemed to prevent bridging. This study suggests that microleakage plays a significant role in pulpal irritation, but that the materials can also alter normal pulpal and dentinal repair.
Recently, the concept of nanoleakage has been put forward. Like microleakage, nanoleakage refers to the leakage of saliva, bacteria, or material com- ponents through the interface between a material and tooth structure. However, nanoleakage refers specifically to dentin bonding, and may occur between mineralized dentin and a bonded mate- rial in the very small spaces of demineralized col- lagen matrix into which the bonded material did not penetrate. Thus nanoleakage can occur even when the overall bond between the material and dentin is intact. It is not known how significant a role nanoleakage plays in the biological response to materials, but it is suspected of contributing to the hydrolytic degradation of the dentin-material bond, leading ultimately to much more serious microleakage.
The full biological effects of restorative materials on the pulp are still not clear. Restorative materials may directly affect pulpal tissues, or may play an auxiliary role by causing sublethal changes in pulpal cells that make them more susceptible to bacteria or neutrophils. It is clear, however, that the design of tests measuring pulpal irritation to materials must include provisions for eliminating bacteria, bacterial products, and other microleakage. Furthermore, the role of dentin in mitigating the effects of microleak- age remains to be fully understood. Recent research has focused on the effects that resin components have on the ability of odontoblasts to form repara- tive dentin. Other research has established the rates at which these components traverse the dentin (see the next section on dentin bonding).
Although it is true that the majority of studies in this arena have in the past focused on the damag- ing effects of materials on the cells of the pulp and dentin, more recent evidence suggests that there are potentially beneficial effects that may derive from these interactions. Subtoxic exposure to certain den- tal materials, such as acid etchants, bonding resins, liners and bases, cements, and restorative materi- als, may solubilize molecules sequestered within the dentin during tooth development. These mol- ecules include growth factors and other proteins and enzymes capable of stimulating existing odon- toblasts or signaling undifferentiated cells to migrate to the site and begin the process of dentinal regen- eration. These events may occur whether or not the material produces a sealed margin with the tooth, and are likely moderated by the health of the tooth in terms of the level of inflammation and the presence of bacterial infection. The exciting aspect of acquir- ing this new knowledge is the potential to design dental materials capable of initiating the tooth repair process in a systematic rather than random way. This is discussed at greater length in Chapter 16, Tissue Engineering.
Dentin Bonding
Traditionally, bond strengths to enamel have been higher than those to dentin. Bonding to dentin has proved more difficult because of its composition (being both organic and inorganic), wetness, and lower mineral content. The wettability of demineral- ized dentin collagen matrix has also been problem- atic. Because the dentinal tubules and their resident odontoblasts are extensions of the pulp, bonding to dentin also involves biocompatibility issues.
After being cut, such as in a cavity preparation, the dentin surface that remains is covered by a 1- to 2-μm layer of organic and inorganic debris. This layer has been named the smear layer (Fig. 6.8). In addition to covering the surface of the dentin, the smear layer debris is also deposited into the tubules to form dentinal plugs. The smear layer and dentinal plugs, which appear impermeable when viewed by electron microscopy, reduce the flow of fluid (con- vective transport) significantly. However, research has shown that diffusion of molecules as large as albumin (66 kDa) will occur through the smear layer.
Numerous studies have shown that removing the smear layer improves the strength of the bond between dentin and restorative materials with con- temporary dentin-bonding agents. A variety of agents have been used to remove the smear layer, including acids, chelating agents such as ethylene- diaminetetraacetic acid (EDTA), sodium hypochlo- rite, and proteolytic enzymes. Removing the smear layer increases the wetness of the dentin and requires that the bonding agent be able to wet dentin and
displace dentinal fluid. The precise mechanism by which bonding occurs remains unclear. However, it appears that the most successful bonding agents are able to penetrate into the layer of collagen fibrils that remains after acid etching removes the mineral com- ponent. There, they create a hybrid layer of resin and collagen in intimate contact with dentin and dentinal tubules. The strength of the collagen itself has also been shown to be important to bond strengths.
From the standpoint of biocompatibility, the removal of the smear layer may pose a threat to the pulp for three reasons. First, its removal juxta- poses resin materials and dentin without a barrier, and therefore increases the risk that these materials can diffuse and cause pulpal irritation. Second, the removal of the smear layer makes any microleakage more significant because a significant barrier to the diffusion of bacteria or bacterial products toward the pulp is removed. Third, the acids used to remove the smear layer are a potential source of irritation themselves. Nevertheless, removal of the smear
layer is now a routine procedure because superior bond strengths are achieved.
Numerous acids, including phosphoric, hydro- chloric, citric, and lactic acids, have been used to remove the smear layer. The effect of these acids on pulp tissues depends on a number of factors, includ- ing thickness of dentin between the restoration and the pulp, strength of the acid, and degree of etching.
Most studies have shown that dentin is a very effi- cient buffer of protons, and that most of the acid never reaches the pulp if sufficient dentin remains. A den- tin thickness of 0.5 mm has proved adequate in this regard. Citric or lactic acids are not as well buffered, probably because these weak acids do not dissociate as efficiently. Usage tests that have studied the effects of acids have shown that phosphoric, pyruvic, and citric acids produce moderate pulpal inflammatory responses, but this resolves after 8 weeks. Recent research has shown that in most cases the penetra- tion depth of acid into the dentin is less than 100 μm.
However, the possibility of adverse effects of these acids cannot be ruled out, because odontoblastic pro- cesses in the tubules may be affected even though the acids do not reach the pulp itself.
The more positive aspect of this dissolution of the dentin by acids used in dentin-bonding agents may be the release of potentially bioactive molecules entrapped during development. Significant research has shown that extracted dentin matrix proteins, which contain a large variety of phosphorylated and nonphosphorylated proteins, proteoglycans, metal- loproteinases, and a variety of growth factors, may be released from intact dentin by basic and acidic chemicals, including acid etchants used in dental adhesives. Studies have shown that many of these molecules may serve as cell signaling agents to recruit undifferentiated cells or as direct stimulants to upregulate the production of extracellular matrix as a step in the process of dentin remineralization.
The specific role for each of the molecules in this pro- cess is not currently known, nor is it known what the desirable concentration of a specific protein or com- bination of molecules is to produce optimal results.
However, the fact that these naturally present mol- ecules may be released by routine dental restorative procedures and serve as participants in the repair process provides an opportunity for the develop- ment and design of future materials.
Bonding Agents
There have been a number of studies of biocompatibil- ity of dentin-bonding systems. Many of these reagents are cytotoxic to cells in vitro if tested alone. However, when placed on dentin and rinsed with water between applications of subsequent reagents as pre- scribed by the manufacturer, cytotoxicity is reduced.
Longer-term in vitro studies suggest, however, that S
P T
FIG. 6.8 Scanning electron micrograph of cut dentin.
When a dentin surface is cut with a bur, a layer of debris, called the smear layer (S), remains on the surface. The smear layer consists of organic and inorganic debris that covers the dentinal surface and the tubules (T). Often, the debris fills the distal part of the tubules in a smear plug (P). (From Brännström M. Dentin and Pulp in Restorative Dentistry.
London: Wolfe Medical Publications; 1982.)
components of the bonding agents may penetrate up to 0.5 mm of dentin and cause significant suppression of cellular metabolism for up to 4 weeks after applica- tion. This suggests that residual unbound constituents may cause adverse reactions.
Hydroxyethyl methacrylate (HEMA), a hydro- philic resin contained in several bonding systems, is at least 100 times less cytotoxic in tissue culture than bisphenol A-glycidyl methacrylate (Bis-GMA).
Studies using long-term in vitro systems have shown, however, that adverse effects of resins occur at much lower concentrations (by a factor of 100 or more) when exposure times are increased to 4 to 6 weeks. Many cytotoxic effects of resin components are reduced significantly by the presence of a den- tin barrier. However, if the dentin in the floor of the cavity preparation is thin (<0.1 mm), there is some evidence that HEMA is cytotoxic in vivo. Further, studies have shown that HEMA is capable of dif- fusing through dentin, presumably via the dentinal tubules, even in opposition to an outward flow of fluid driven by normal pulpal pressure. What effect HEMA may then have on the pulp cells in situ is not known, but HEMA has been shown to stimulate the expression of growth factors in mouse odontoblast- like cells.
Other studies have established the in vitro cyto- toxicity of most of the common resins in bonding agents, such as Bis-GMA, triethylene glycol dimeth- acrylate, and urethane dimethacrylate (UDMA).
Combinations of HEMA and other resins found in dentin-bonding agents may act synergistically to cause cytotoxic effects in vitro. There have been very few clinical studies of diffusion of hydrophilic and hydrophobic resin components through dentin.
These studies indicated that some diffusion of these components occurs in vivo as well. Interestingly, there has been one report that some resin compo- nents enhance the growth of oral bacteria. If substan- tiated, this would cause concern about the ability of resin-based materials to increase plaque formation.
Finally, studies have also shown that the release of matrix metalloproteinases (MMPs) from dentin by virtue of its interaction with the acid components in dentin adhesives may cause degradation of the adhesive bond by enzymatic action on the exposed collagen within the hybrid layer. The application of an MMP inhibitor, such as chlorhexidine, has been shown to minimize this effect, and has been recom- mended for maintaining the clinical durability of the dentin bond. However, the overall effect of chlorhex- idine on pulp cells has yet to be determined.
Resin-Based Materials
Resin-based materials have been used as dental cements and restorative materials. Because they are a combination of organic and inorganic phases, these
materials are called resin composites. In vitro, freshly set chemically cured and light-cured resins often cause moderate cytotoxic reactions in cultured cells over 24 to 72 hours of exposure, although several newer systems seem to have minimal toxicity. The cytotoxicity is significantly reduced 24 to 48 hours after setting and by the presence of a dentin barrier.
Several studies have shown that some materials are persistently cytotoxic in vitro even up to 4 weeks, whereas others gradually improve, and a few newer systems show little toxicity even initially. In all cases, cytotoxicity is thought to be mediated by resin com- ponents released from the materials. Evidence indi- cates that the light-cured resins are less cytotoxic than chemically cured systems, but this effect is highly dependent on the curing efficiency of the light and the type of resin system. In vivo, usage tests have been used to assess the biological response to resin composites. The pulpal inflammatory response to chemically and light-activated resin composites was low to moderate after 3 days when they were placed in cavities with approximately 0.5 mm of remaining dentin. Any reaction diminished as the postoperative periods increased to 5 to 8 weeks and was accom- panied by an increase in reparative dentin (Fig. 6.9).
With a protective liner or a bonding agent, the reac- tion of the pulp to resin composite materials is mini- mal. The longer-term effects of resins placed directly on pulpal tissue are not known, but are suspected to be less favorable.
Amalgam and Casting Alloys
Biocompatibility of amalgam as a dental restor- ative material is thought to be determined largely FIG. 6.9 Light micrograph of the dentinal and pulpal response to unlined composite at 5 to 8 weeks in a mon- key. The primary dentin is the lighter layer seen at the top.
The tubules are evident. Secondary dentin is occurring (the dark, wide middle layer), and it is closely approximated by intact odontoblasts in the pulp. Few inflammatory cells are present. The response seen in this micrograph is indicative of a favorable response to the material. (Courtesy A.K. Avery, Ann Arbor, Michigan.)
by the corrosion products released while in ser- vice. Amalgam is a complex metallic material com- posed of multiple phases, and its corrosion, in turn, depends on the type of amalgam, whether it contains the tin-mercury γ2 phase, and its composition. In cell culture screening tests, free or nonreacted mercury from amalgam is toxic. With the addition of copper, amalgams become toxic to cells in culture, but low- copper amalgam that has set for 24 hours does not inhibit cell growth.
Implantation tests show that traditional low-copper amalgams were well tolerated, but the more modern high-copper amalgams caused severe reactions when in direct contact with tissue. Unreacted mercury or cop- per leaching out from these high-copper alloys has usu- ally been the constituent leading to adverse response.
An in vitro study of the effects of particulate amalgams and their individual phases on macrophages showed that all particles except γ2 are effectively phagocytized by macrophages. Cell damage was seen in treated cultures exposed to particulate γ1, the silver-mercury matrix phase of amalgams. In usage tests, the response of the pulp to amalgam in shallow cavities or in deeper but lined cavities is minimal, and amalgam rarely causes irreversible damage to the pulp. However, pain results from using amalgams in deep, unlined cavity preparations (0.5 mm or less remaining dentin), with an inflammatory response occurring after 3 days.
This pain may be related to the high thermal and electrical conductivity of the material, which is sig- nificantly mitigated by the presence of a barrier of remaining dentin or an insulating material. Thus in cavities with less than 0.5 to 1.0 mm of dentin remain- ing in the floor, a base should be placed on the floor of the cavity preparation for two reasons. First, the transfer of hot and cold stimuli, primarily from food and drink, through the amalgam may be substantial.
Second, margins of newly placed amalgam restora- tions show significant microleakage. Marginal leak- age of salivary and microbial products is probably enhanced by the natural daily thermal cycle in the oral cavity, which may expand and contract the mar- ginal gap leading to a percolation of fluids. Although long-term sealing of the margins occurs through the buildup of corrosion products, the timeframe over which this occurs is somewhat a function of the com- position of the amalgam, being longer for the high- copper amalgams in use today.
Usage tests reported that after 3 days, the pulpal response to high-copper amalgams appears similar to that elicited by low-copper amalgams in deep, unlined cavities. At 5 weeks they provoked only slight pulpal response. At 8 weeks the inflammatory response was reduced. Bacterial tests on the high- copper amalgam pellets have revealed little inhibi- tory effect on serotypes of Streptococcus mutans, thus suggesting that metallic elements were not released
in amounts necessary to kill these microorganisms.
Although the high-copper amalgams seem biologi- cally acceptable in usage tests, liners are suggested for all deep cavities. Again, this may be related more to a need for thermal and electrical insulation than a concern over toxicity. Further, the diffusion of released metallic elements into the tooth structure produces discoloration, and may be minimized by the presence of an intervening liner. There are also reports of inflammatory reactions of the dentin and pulp, similar to the reactions to many other restor- ative materials. Mercury has been found in the lysosomes of macrophages and fibroblasts in some patients with lesions.
Cast alloys have been used for single restora- tions, fixed partial dentures, ceramic-metal crowns, and removable partial dentures. The gold content in these alloys ranges from 0 wt% to 85 wt%. These alloys contain several other noble and nonnoble metals that may have an adverse effect on cells if they are released from the alloys. However, metal ions released from these materials are most likely in contact with gingival and mucosal tissues, whereas the pulp is more likely to be affected by the cement retaining the restoration.
Glass Ionomers
Glass ionomer has been used as a cement (luting agent), liner, base, and restorative material. Light- cured ionomer systems use HEMA or other mono- mers or oligomers as additives or as pendant chains on the polyacrylic acid main chain. In screening tests, freshly prepared ionomer is mildly cytotoxic, but this effect is reduced over time. The fluoride release from these materials, which may have some therapeutic value, has been implicated in this cytotoxicity in vitro.
Some researchers have reported that certain systems are more cytotoxic than others, and though the rea- sons for this are not clear, presumably it is related to the composition of the glasses used in the material, which may contain aluminum, calcium, manganese, zinc, strontium, and other metallic elements.
The overall pulpal biocompatibility of glass iono- mer materials has been attributed to the weak acidic nature of the polyacrylic acid, as well as to its high molecular weight. Thus polyacrylic acid is unable to diffuse through dentin due to its large size. In usage tests the pulp reaction to these cements is mild, and histological studies show that any inflammatory infiltrate from ionomer is minimal or absent after 1 month. There have been several reports of pulpal hyperalgesia for short periods (days) after placing glass ionomers in cervical cavities. This effect is prob- ably the result of increased dentin permeability after acid etching. In any case, glass ionomer has not been shown to be well tolerated when placed directly upon living pulp tissue as a direct pulp-capping agent.