____ Remember such striking photographs? Some were shown in 1992 in two successive issues of our "Phoenix Without Ashes" ^1,2 while others were published both in Europe^3,4 and in the U.S.^5,6. All vividly show the effect of microorganisms on both adhesives and stainless steel attachments. The finding struck a cord even with the National Association of Corrosion Engineers who asked for an article on it. At that time, we suggested to stop such attacks by "immunizing" the adhesives by the addition of antimicrobial agents such as quaternary ammonium salts. Maquat (a commercial bactericide made by Mason Chemicals, Chicago, IL, described to be a 80% solution of N-alkyl dimethyl benzyl ammonium chloride), has been found by us to inhibit the growth of Pseudomonas aeruginosa when added to the adhesive1. It took, however, six years till the first commercial antimicrobial acrylic resin (containing a similar quaternary ammonium salt described as "benzalkonium chloride") was launched both in Europe and the US by an Italian company (Sintodent, Bandini, Rome).
Short history
___ In 1867, the Scott surgeon Joseph Lister published his fundamental article in Lancet (where he paid tribute to Louis Pasteur) on the use of carbolic acid (phenol) to kill germs. Nine years after Lister's publication, Samuel Gross, a reputed leader of the American surgeons, wrote "Little if any faith is placed by any enlightened or experienced surgeon on this side of the Atlantic in the so-called carbolic treatment of Professor Lister"⁸.
____Soon, European surgeons recognized the part played by bacteria in infections and "Listerism" started to be widely applied. In dentistry, the action of the microorganisms accumulating on composites has been analyzed relatively late. Far from contributing only to periodontal disease and secondary caries, these were found to cause the deterioration of the material ^9-11. Believed till recently to be due to the enzymes found in saliva¹², the alteration of some restorative composites and glass ionomers has been successfully proven and then duplicated in vitro using Pseudomonas aeruginosa^13,11. To protect from infection those handling alginate impressions14 as well those wearing prostheses^15,16 or elastic linings covering the periodontal surgical sites¹⁷, antimicrobial agents have been added to the corresponding products.
____The agents used (quaternary ammonium salts and chlorhexidine derivatives) were found to be effective in stopping the attack if added up to 0.5% in weight.
____In orthodontics, both the phenomenon and the procedure seem to have been ignored. During the years, in several conferences on the adhesive attacks at meetings held both in the U.S. and abroad addressed to specialized clinicians, each time the attendees were caught by surprise viewing slides showing the effects of microorganisms on acrylic composites and stainless steel.
The recently launched antimicrobial resin
___ The new commercially released self-curing acrylic resin acrylic has been optimized to avoid gingival inflammations while relining or creating temporary bridges. According to the manufacturer, such inflammations usually take place during the preparation phase of abutment teeth and are due to the accumulation of plaque bacteria found on the margins of temporary prosthesis. The invention is protected by an international patent and has obtained the CE (European quality mark) certification⁷. The related studies have been presented in Italy^{18, 19}, US²⁰ and in 1998 in France²¹.The thrust of the research performed by the staff of the University of Ancona has been to demonstrate first that the new formulation presents indeed an antimicrobial activity²¹, and that the modifications brought to a traditional product does not affect its thermal or mechanical properties^19,20. No growth of bacteria (among which Streptococcus mutans) was observed on the specimens containing benzalkonium chloride, while bacteria grew on all the control samples²¹. The mechanical properties of the antimicrobial resin, such as hardness, tensile strength and compression were practically unaffected²⁰: the exothermal effect resulting from the resin polymerization did not exceed the limit threshold of 40^oC ¹⁹ above which pulpal damage may occur.
____Quaternary ammonium salts (or quats) are usually made from tertiary amines and alkyl chlorides, in the presence of enough sodium hydroxide to capture the Cl- ions. As secondary and tertiary aromatic amines are too weak bases, only the primary aromatic amines can be alkylated:
C₆H₅-CH₂-NH₂+3CH₃Cl+2NaOH = [C₆H₅-CH₂-N+(CH₃)₃]Cl- + 2NaCl+ 2H₂0
____Benzalkonium chloride is a generic name for mixtures known as Benirol; BTC; Capitol; Cequartyl; Drapolene; Drapolex; Enuclen; Germinol, Germitol; Paralkan; Rodalon; Zephiran chloride. Structurally, the mixture of alkyl-dimethyl-benzyl-ammonium chlorides has the following general formula, in which R represents a mixture of alkyls from C₈H₁₇ to C₁₈H₃₇²². White, amorphous powders with a bitter taste, quats can have various solubilities according to the length of the radical R. Their toxicity is similar to that of stannous (Sn²⁺) fluoride, a common ingredient in tooth pastes (LD₅₀ orally in rats: 400mg/kg²²). This allows their use as pharmaceutical preservatives and antiseptics.
Possible applications in dentistry.
Prostheses. At a conference concerning the effect of microbes on adhesives, a listener removed and showed us his prosthesis which was affected by some "strange phenomenon" he could only now understand. Indeed, the resemblance between the shape and structure of the affected zone with these exhibited by microbe-attacked adhesives found on used direct bonding brackets is obvious, Fig. 3. In our opinion, all acrylic prostheses and not only the temporary bridges should be made of antimicrobials-containing acrylates.
Restoratives. As many restoratives are based today on acrylic composites, having thus a matrix which is vulnerable, the addition of antimicrobials will lengthen considerably the life of the restorations. Indeed, a composite which after years of wear did not properly fill the cavity was removed and a portion was presented to us for examination. Seen under magnification, it showed both the familiar yellowish discoloration and the specific surface alterations (pores and partly depolymerized structure) found on the microbe-attacked adhesives used in direct bonding, Fig. 4.
____We strongly believe that if acrylic composites used as restoratives would be added with tiny amounts of adequate antimicrobials, their life will be significantly increased. Microbes penetrate and seep through micro-cracks along the interface composite-dentin, consuming both. The microcracks are gradually enlarged, allowing more and more microbes to enter and develop. After a while, the restoration becomes lose and has to be replaced, and the cycle continues till the tooth involved ends under a crown.
Applications in orthodontics.
____Six years ago, we revealed the microbial attacks on adhesive and on steel direct- bonding attachments. To stop these, we used Maquat from Mason Chemical, Chicago, a quat belonging to the same family as the one used by Sintodent. Unfortunately, no orthodontic sealants or adhesives containing antimicrobial agents are yet available. This is regrettable, as the examination of debonded direct bonding brackets shows that while bracket bases are seldom attacked, typical alterations of the adhesive are the norm rather than the exception. Those due to aerobic microorganisms are easy to observe, exhibiting dark spots with definite contours. ____This type of attack is less common than that due to anaerobic microorganisms which generate a rather subtle, but more deleterious change of the adhesive at its interface with the enamel. This translates in a weakening of the neck of the adhesive sheaths (or tags) which determine bond strength²³. If the thickness of these necks is substantially diminished, the depth of the sheaths (or tags) becomes unimportant as shown in Fig. 5, leading to bond failure as sketched in Fig. 6.
____This explains why some attachments, which have withstood wear for years, debond just before the treatment's end. This is not due to an excessive stress inflicted upon the attachments, but to the continuous degradation of the adhesive caused by microorganisms. To evidence the alteration produced by anaerobes, staining agents such as methylene blue can be used. In Fig. 7 are shown two views of the same batch of used brackets, one before staining and the other after immersion in a 2% ethanol solution of methylene blue.
The bases of stainless steel brackets are likewise, but not often, it is true, attacked by iron-consuming bacteria. As this attack occurs simultaneously with an alteration of the adhesive, as shown in Fig. 8, this leads to a weakening of both the bond and the attachment.
____Before publishing our findings, Professor T. M. Graber, editor-in-chief of the American Journal of Orthodontics and Dentofac. Orthopedics, was asked for advice if these should be patented or not. In his letter to us dated April 15, 1992, he considered the reported "microorganisms effect to be very important", and added "Knowing the trials and tribulations of getting a patent and having it stolen right away anyway, I would urge you to publish rather than patent, and wait and have it taken from you in any event... If you could write an article about some other views showing the corrosion in the crevices made by the plaque, the undercuts on the bonding material, etc., I believe that this would be of value because I don't think that many of our clinicians understand what is happening".
____Following his advice, we didn't apply for a patent, but presented the manuscript for publication in American J. Orthodontics⁵. While getting and especially enforcing a patent on antibacterial orthodontic adhesives may have been be quite difficult, the usefulness of such products is indubitable.
Aren't we too slow in accepting the obvious ?
____Our mouths have the less enviable distinction to host an incredibly abundant microbial flora which comprises species which are both typical and "opportunist" (found there only by chance). Recently, at least two companies have launched antimicrobial tooth pastes (Cool Mint ListerineR by Warner Lambert Consumer Health Care, and BiotheneR by Laclede Professional Products). Road signs abound in S. Florida boasting that "there are more germs on your teeth than:... "on the gum stuck to that guy's shoe", "in the back of your car", or "on the bottom of your trash can", etc. (see further). In industry, the addition of antimicrobial ingredients is compulsory even in chlorinated polymers such as polyvinyl chloride. Indeed, despite its high content in chlorine, PVC is readily attacked by various microbes and fungi. Applications of polymers containing anti microbials include flooring, garden hose, pool liners, wall coverings. "Without antimicrobials, polymers can experience surface growths, cause allergic reactions, unpleasant odors, staining, embrittlement and premature product failure"²⁴.
____While some bactericides used in coatings and films are safe, nonirritating and non-sensitizing at the recommended use levels (such as Nuodex 100-VT²⁵, a quaternary ammonium salt similar to these used in our experiments as in Sintodent), most antimicrobials used in industry are by far more potent. Thus, products such Micro-Check 11 (Ferro Corporation and Vinyzene IT (Morton International) contain isothiazoline (i.e. a compound containing sulfur), while 10-16-oxybisphenoxy arsine, known as OBPA (Akzo Chemicals), contains arsenic. The recommended loading levels range between 0.6% and 3%. Most antimicrobials are carried in plasticizers, which are highly mobile and migrate throughout the end product: if a significant leaching occurs, the product is left unprotected. In the US, under the Federal Insecticide, Fungicide and Rodenticide Act, manufacturers of antimicrobial additives must pass stringent tests to prove efficacy data. Isn't it strange that the plastics we are surrounded with are protected, while these subjected to by far stronger microbial attacks, these occurring in the favorable warm, moist and rich in food environment which is the human mouth, are not ?
____Since decades it is now known that stainless steels are attacked by microbes. There are many bacteria which need iron in their metabolisms, as we do it for our blood haemoglobin. Some accelerate the oxidation of Fe to Fe²⁺ and then to Fe³⁺, depleting in oxygen the metal surface. Stainless steels, which are precisely protected against corrosion by a thin oxide layer (Cr₂O₃) can then be attacked like any common steel. Other commonly encountered microorganisms are sulfate reducers, such as Desulfovibrio desulfuricans, sulfur oxidixers, such as Thiobaccilus Ferrooxidans, Beggiatoa and Thiotrix or slime forming such as Aerobacter, Flavobacterium and some of the Pseudomonas species²⁶.

____Among the microorganisms associated with iron, Ferribaccilus and Gallionella are believed to be true iron bacteria, while Crenotrix and Leptotrix are facultative autotrophs. As all of these are found in water supplies, stainless steel pipes and tanks are found quite often to be perforated. Thus, on a basic stainless steel used both in bracket manufacture and tank construction, AISI 304 , sulfate reducers are causing conical, while iron bacteria produce both conical and spherical pits²⁶. (See similar pits on the bracket base shown in Fig. 8). Before understanding the phenomena involved, attempts to replace common steel with stainless steel in various constructions destined to be in contact with natural water have led to even greater problems ^{26, 27}.
Is it feasible to add antimicrobials to adhesives ?
___The answer is undoubtedly yes. The polymerization initiation system of the self-curing adhesives is chemically compatible, already containing as accelerators aromatic amines related to the quaternary ammonium salts used as antibacterial both by us and the manufacturers of Sintodent. In addition, the amount needed is practically insignificant, when compared with some 70% inert filler commonly used. As the additive doesn't have to exceed 1%, it barely influences mechanical properties. The substituting alkyl radicals control its solubility.
____Interestingly enough, the tertiary amines used as accelerators [N, N-dimethyl-p-toluidine, N, N-bis (2 hydroxy-ethyl)-p-toluidine] do not seem to exhibit an antibacterial activity: our experiments using the first amine showed that the growth of Pseudomonas aeruginosa incubated for 48 h at 100^oF was not deterred within a week⁵.
Conclusion
___ Sooner or later, the manufacturers of all acrylic prostheses and baseplates, as well as orthodontic adhesives and sealants, will follow the lead of Bandini, the Italian manufacturer who launched the first commercial antimicrobial acrylic resin destined for oral use. Extensively used in the plastics and coatings industry, where the exposure to microbes is not as great, antimicrobials should be widely used to help acrylic matrices to withstand microbial attacks, and in the case of orthodontics, reduce premature debondings and protect baseplates.
____More important, the addition of antimicrobials to restoratives will considerably prolong the life of restorations, perhaps completely avoiding their repeated replacement.

References
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