Daniel Fine, DMD, professor and chair, Department of Oral Biology; Director of the Center for Oral Infectious Diseases, UMDNJ-New Jersey Dental School

For the past 25 years, our group has been involved in studying antimicrobial approaches to the treatment and prevention of periodontal diseases. During that time, we have had the opportunity to collaborate with industrial partners in conducting studies at various stages of product development. These studies range from developing new product formulations, to demonstrating the effectiveness and safety of new, cutting-edge products, to working on innovative and unique methods for elucidating the mechanisms of action of various products. Work by our group has also demonstrated new uses for existing products, such as applying pre-procedural rinses to prevent back spray and bacteremia during dental procedures, and thus has helped to expand the breadth of existing product claims. Our expertise in clinical microbiology, microbial genetics and periodontology has enabled us to develop in vitro, in situ and in vivo methods for assessing the effect of antimicrobial agents on microorganisms responsible for the development of periodontal diseases and their sequelae.

Periodontal disease(s) are a group of inflammatory diseases initiated by bacteria that colonize the teeth, migrate to the area below the gingiva (“gum”) and eventually overwhelm the host’s ability to defend itself from the progressing army of bacteria and their products. While bacteria initiate the disease, it is the host’s response to the bacteria that produces the tissue destruction and bone loss that characterize the clinical signs of disease. If this is left untreated, the disease will lead to a weakening of the tooth’s support and, ultimately, to tooth mobility and tooth loss. Approximately 40 to 65% of the U.S. population between the ages of 35 and 75 has some form of periodontal disease. Since bacteria initiate the disease, our approach has been to develop methods to combat the bacterial initiators at their earliest stages.

As the bacteria colonize the root surface below the gum, the gum begins to separate from the tooth, creating a deepened space, or “pocket,” with an ulcerated inner wall. This breach in the integrity of the inner lining layer can allow the colonizing bacteria and/or their products to enter the contiguous blood vessels. Thus, it should come as no surprise that current research implicates periodontal infections as risk factors for coronary heart disease and pre-term/low-birth-weight delivery, systemic conditions associated with chronic infection.

Enlarge the image Figure 1: Hydroxyapatite (HA) disk is exposed to test agent, placed in broth containing 24 hour culture of bacteria. Disk is washed and once again placed in bacteria containing broth. This can be repeated several times to determine whether the agent binds to the disk and whether it is released in a rapid but prolonged manner and retains its active form.

Bacteria that successfully colonize the oral cavity do so by adhering to oral tissues and forming a biofilm called dental plaque. This community of microorganisms becomes increasingly complex and pathogenic as it “matures.” When the bacteria adhere, they undergo phenotypic changes that may enhance their resistance to antimicrobial agents. The dense biofilm architecture and intercellular matrix also make these bacteria extremely resistant to antimicrobial agents.

In 1985, as a result of our basic research in the area of dental plaque development, our group was chosen to be involved in the effectiveness studies of the first local delivery antimicrobial product approved by the FDA for treating periodontal disease. This multi-center trial was designed to assess the efficacy of a fiber consisting of a copolymer of ethylene-vinyl-acetate and tetracycline hydrochloride. When placed into an infected periodontal pocket, this fiber provided a constant and prolonged release of tetracycline at the site of disease activity during the 10-day treatment period, reducing the bacterial challenge and promoting healing. We have subsequently been involved in three FDA studies related to antimicrobial efficacy.

Antimicrobial testing: in vitro and in vivo biofilm models

Enlarge the image Figure 2A: In vivo model. Stent placed in mouth containing the HA disks. Disks left in mouth for up to 12 hours so that plaque can accumulate. Test agent can be applied in a passive manner to determine its affect on plaque deposition on the HA disk (tooth analogue). Figure 2B: Stents removed. Disks are intact (see left stent). Disks are removed (right stent). When HA disk is removed it is placed in transport media, and plated to determine colony counts per surface area of disk. Active agent is always compared to placebo agent.

Standard in vitro antimicrobial testing is done by placing the test agent in a broth culture containing specific bacteria. Our in vitro studies utilize hydroxyapatite (HA) disks as a surrogate for the enamel tooth surface. One application of this method is to determine the substantivity of antimicrobial agents, i.e., their ability to be adsorbed by oral tissues and subsequently released in order to prolong their period of action. As can be seen in Figure 1, in these studies the HA disk is first coated with saliva, simulatingthe first step in plaque formation on enamel, and then the disk is exposed to the antimicrobial agent of interest. To determine if the agent, in fact,has been adsorbed to the saliva-coated disk and then released in an active form, the treated disk is washed and transferred to a broth containing representative oral bacteria, and the viability of the bacteria is determined after a short exposure. For in vivo testing, we have developed a method in which the HA disks are mounted in a stent that is placed in the mouths of volunteers for as long as six to 12 hours to allow for the accumulation of dental plaque. In this model (Figure 2), the test and control agents can be applied to the disk prior to plaque accumulation to determine whether plaque can be prevented from forming on the tooth analogue. Alternatively, the test agent can be passively applied to already-formed plaque to assess its antimicrobial activity. These model systems allow us to perform rapid, cost-effective analysis of new antimicrobial products to assess their efficacy in the challenging environment of the oral cavity. This approach has been utilized in the development of dentifrice and mouthrinse products by several of our industrial partners. Our industry partners include but are not limited to Colgate Palmolive, Pfizer and Warner Lambert.

Antimicrobial testing: application of biofilm models to dental practice

One example of our clinical studies leading to new uses for existing products are studies demonstrating that rinsing with an antiseptic mouth rinse prior to certain dental procedures can reduce the numbers of viable bacteria in the aerosols caused by the use of ultrasonic scaling devices. The Centers for Disease Control and Prevention has recognized these studies and uses the information from them to make pragmatic suggestions to dental practitioners. We have also demonstrated that pre-procedural use of antiseptic agents can reduce the potential for bacteremia resulting from invasive dental procedures.

Antimicrobial testing: application of biofilm models to medical practice

Genetic approaches have provided researchers with the potential to explore microbial strategies heretofore inaccessible to our research community. For the past 20 years our group has been studying Actinobacillus actinomycetemcomitans, a biofilm-forming microbe that causes a rapidly progressive form of periodontal disease affecting children. While working on Actinobacillus, Jeffrey Kaplan, PhD, a member of our group in the Oral Biology Department recently discovered a gene responsible for an enzyme that disrupts biofilm formation. Coincidentally, this enzyme was also found to disrupt biofilm formation by Streptococcus epidermidis, a commensal skin bacterium that is the primary cause of infection of indwelling catheters. Application of this enzyme to catheters prevents biofilm formation and removes existing biofilms. This discovery may potentially have an impact on indwelling catheter infections responsible for as many as 10,000 deaths and more than $11 billion in hospital costs per year. Overall, our collaborations with our industrial partners have been rewarding for both participants. However, most gratifying is the fact that these partnerships have produced experimental evidence that has been translated into clinically applicable procedures for the public.

Daniel H. Fine, DMD, is professor and chair of the Department of Oral Biology and director of the Center for Oral Infectious Diseases at NJDS. His research focuses on genetic determinants of bacterial virulence and studies of antimicrobial interactions with plaque biofilms. He has had numerous industrial and NIH sponsored grants, has organized several national research meetings, and has served as a consultant for The National Institute of Dental and Craniofacial Research, as well as consulting for industry. Dr. Fine has published more than 80 peer-reviewed scientific manuscripts and has been an invited speaker at numerous universities in the U.S. and abroad. §