Oral Health Group
Feature

Anti-inflammatory Effects of Antimicrobial Photodynamic Therapy

December 1, 2013
by Nicolas G. Loebel, PhD


INTRODUCTION
Chronic adult periodontitis is generally understood to result from the presence of subgingival Gram-negative bacterial biofilms proliferating at the junctional epithelium.1 These biofilms are formed when colonizing microbes encapsulate themselves against the root and epithelial surfaces in a matrix composed of secreted polysaccharides, proteins, and nucleic acids. Close proximity of one microbial cell to another permits information exchange through quorum sensing and plasmid DNA transfer, with resulting survival advantages conferred across the entire colony.2 Because the microbes located within the central layers of the biofilm are often in lag phase growth with low metabolic turnover, topical microbicides tend to damage or destroy only the surface microbes leaving the underlying layers intact. The crosslinked ultrastructure of the biofilm generates tenacious tissue adherence and physical exclusion of antimicrobial substances.3,4 Sedlacek and Walker5 demonstrated that killing of bacterial strains in biofilm organization required up to 250 times the concentration of antibiotic than the same strains grown planktonically. When steady-state multi-species biofilms were treated with amoxicillin/clavulanate and amoxicillin/metronidazole, up to 50 percent of the bacteria retained viability after seven days. As a result, nonsurgical treatment of periodontitis primarily relies upon mechanical debridement (scaling and root planing).6

While many patients respond well to comprehensive mechanical debridement, a sub-population of patients continues to demonstrate chronic periodontal tissue breakdown. These patients often present with predisposing risk factors7 such as smoking, diabetes, hereditary factors, systemic disease and so forth;8 local factors such as malocclusion, inadequate prosthetic preparation, and/or chronically persistent superinfection with one or more pathogenic species may also be involved.9 Scaling and root planing has been demonstrated to leave both calculus10 and bacteria11 behind in the treated area, and also to open dentinal tubules, permitting invasion by the residual periopathogens with subgingival recolonization shortly thereafter.12 In the phenotypically susceptible host, a continued inflammatory response can occur that is paralleled by a sustained increase in gingival crevicular fluid flow rate,13 as the local microvasculature reacts to proteolytic enzymes, cytokines and other pro-inflammatory factors. The impact of effective local inflammation control is reflected in systemic acute-phase inflammatory markers such as C-reactive protein (CRP, a complement activator proportional to plasma levels of IL-6, not produced in gingival tissues). Patients responding well to mechanical debridement along with reduced symptoms of local inflammation demonstrate significant reductions in circulating CRP levels. Patients who do not demonstrate CRP reduction also exhibit poor disease resolution on the standard clinical indices of bleeding on probing, increases in attachment level and reduction in pocket depth. The systemic inflammatory response (mirroring the level of subgingival infection/inflammation) may be amplified by individual susceptibility and the risk factors associated with each patient. Cigarette smoking, body mass index, age and carriage of specific polymorphisms in the IL-1A and IL-6 promoter regions are associated with increases in serum CRP, and the CRP titer has therefore assumed a significant role as a predictor for future coronary events in healthy populations.14

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ANTIMICROBIAL PHOTODYNAMIC THERAPY (aPDT)
The first studies of photodynamic therapy as an antimicrobial modality (aPDT) were published in the mid to late 1980s.15,16 Over 400 different photosensitizers are known,17 including dyes, drugs, cosmetics, chemicals and many natural substances such as hypericin (found in St. Johns wort, a perennial plant widely known as an herbal treatment for mild depression). The Periowave™ aPDT system was first introduced to the market by Ondine Biomedical Inc., Vancouver B.C. in 2006, and applications of aPDT in dentistry have grown rapidly since that time, including diagnosis and treatment of oral cancer, treatment of oral leukoplakia, treatment of oral lichen planus, and treatment of periodontal disease, gingivitis, peri-implantitis, endodontic infections, and a variety of bacterial, fungal and viral infections.18

Key attributes of aPDT include high efficacy against biofilms, avoidance of the resistance issues that plague antibiotic usage and the activation of both innate and adaptive arms of the immune system.19 In oncotherapeutic applications, PDT has been demonstrated to induce production of cytokines and chemokines that attract and activate neutrophils,20 macrophages21 and dendritic cells,22 leading, in some cases, to production of a tumor antigen-specific immune response and tumor suppression,23 cure of metastatic tumors24 and incredibly, even regression of distant established tumors.25

BASIC PHOTODYNAMICS
Figure 1 demonstrates the basic photodynamics involved in aPDT. A photosensitizer molecule, with absorption of red light photons is shown on the left figure, causing electrons to be “pumped” from the normal ground state P0 to an excited state, 1P0. The “activated” photosensitizer can then engage in many different kinds of chemical reactions which are destructive to the cell walls of microbes, such as electron transfer reactions. A second activation pathway also exists, where energy transfers from the sensitizer in a resonant process to surrounding molecular oxygen. The oxygen molecules in turn “pump” to their excited state, generating singlet oxygen — a powerful oxidizer capable of directly destroying bacteria, viruses and fungi through lethal peroxidative reactions.

Because singlet oxygen diffuses only very short distances before reacting, the effects of aPDT are highly localized. The Periowave™ photosensitizer is preferentially taken up by bacterial cell membranes over human cells, because the positively-charged (cationic) sensitizer is electrostatically attracted to the negatively-charged (anionic) bacterial cells (human cell membranes are zwitterionic, taking up far less sensitizer and therefore minimizing any damage). The destructive reactions caused by singlet oxygen are therefore selective to the stained microbes. The destructive effect is further amplified by the “PDT bystander” effect, discovered by Christensen and Moan in 1979.26 The authors demonstrated a cooperative inactivation process between cells in a given microcolony, most probably mediated by toxic photoproducts or transfer of lysosomal enzymes between confluent cells. The bystander effect causes a significant kill enhancement, important in the tough, crosslinked biofilms associated with periodontal disease. Because the various kill mechanisms primarily act outside the cell, at the level of the cell membrane, lethality is unaffected by growth phase of the organism.27

DISRUPTION OF BACTERIAL VIRULENCE FACTORS
One of the key factors differentiating aPDT from antibiotic therapy is that fact that aPDT has been shown to inactivate bacteria directly, and also the bacterial virulence factors responsible for triggering inflammation. The key virulence factors of P. gingivalis include the cytotoxin lipopolysaccharide (“bacterial endotoxin or LPS”), proteolytic enzymes and many toxic low-molecular weight compounds such as hydrogen sulfide and ammonia. The proteases in particular are thought to be responsible for the majority of periodontal tissue damage. LPS may be released from Gram-negative bacteria when the cells are killed by conventional antibiotics and antiseptics, and the resulting toxic effect can be significant enough to be lethal to a patient.28 Because aPDT destroys the LPS and other virulenc
e factors themselves, this toxic side-effect is minimized.

Studies have shown that aPDT can reduce the total proteolytic activity of P. gingivalis to zero, using only low levels of light and dilute photosensitizer solutions.29 Similar results were obtained when evaluating the Arginine-specific proteinase activity of P. gingivalis after aPDT.29 Treatment of both the underlying virulence factors as well as the causative organism brings together two of the most successful first-line approaches in the clinical management of refractory periodontal disease.

CRP REDUCTION IN VIVO
Clinical study of the Periowave™ photodisinfection system30 in 28 systemically healthy patients demonstrated significant decreases of several acute-phase inflammatory markers, including CRP, at the six month follow-up point, compared to SRP alone. Two cycles of aPDT during the same treatment session were required to generate this long-term effect. The aPDT treatment was highly effective, with zero defect sites requiring further treatment in the aPDT arm at six months. Of note, patients were selected from a cohort undergoing long term maintenance therapy for chronic adult periodontitis and had therefore received numerous mechanical debridement procedures over many years prior to receiving the aPDT treatment.

HOST CYTOKINE INACTIVATION
Periowave™ aPDT was demonstrated to directly inactivate pro-inflammatory cytokines31 in a well-validated ELISA assay based on E-selectin expression in human umbilical vein endothelial cells (HUVECs). Live cultures of P. gingivalis were simultaneously evaluated for their protease activity using a fluorescent assay. Results demonstrated that key pro-inflammatory cytokines could be completely inactivated over 60 seconds of therapy, while 4-log10 (99.99 percent) inactivation of P. gingivalis was simultaneously achieved. A comparison between bacterial kill and protease inhibition for both the aPDT treatment and 350μg/ml minocycline (a semisynthetic tetracycline derivative commonly used to treat periodontal infections and suppress protease) was carried out and is reproduced by permission in Figure 2. The results demonstrated vastly superior protease inhibition using aPDT, without damaging the cidal nature of the antibiotic, and a synergistic amplification of protease inhibition when both modalities were used simultaneously.

INNATE IMMUNITY INDUCTION (aPDT “VACCINATION”)
In an important and recent development19, researchers demonstrated that aPDT can confer protective benefit against future infections by induction of a protective innate immune response against bacterial pathogens. The effect was dose-responsive with light activation, reaching a peak at 50 J/cm2, the optical fluence utilized in the Periowave™ system along with the same phenothiazinium sensitizer. Animals who had received optimal aPDT treatment a day prior to infection were able to immediately localize and then suppress virulent MRSA inoculations, whereas control animals were unable to do so. The researchers showed that the aPDT regimen activated the entire sequential cascade of the accumulation, migration and activation of neutrophils into the infection site, and that the procedure did not harm the neutrophils themselves. Continuing this elegant work, the researchers prepared antibodies against the factors responsible for chemotactically attracting neutrophils to infection sites, and showed that this blockade eliminated the protective effect. These findings indicated that aPDT can strongly activate innate host defense mechanisms against microbial infection, and leads to the speculation that patients treated with aPDT may become less and less susceptible to future infections, independent of the microorganism involved. In fact this effect may have been noted many years ago, in studies evaluating the effect of aPDT on orolabial herpes simplex outbreaks. It was noted in this study that recurrent outbreaks never occurred at the same site as prior treatment.32

CONCLUSION
Clinical control of the predominantly Gram-negative biofilms in chronic adult periodontitis is often difficult. Mechanical debridement leaves behind many periopathogens, access to deep pockets, furcations and peri-implant lesions is anatomically restricted, and chemotherapeutic agents such as antibiotics may be displaced by gingival crevicular fluid or bound to protein fractions, reducing their efficacy. Chronically persistent infections with one or more pathogenic species often results. These pathogens upregulate the production of proteolytic enzymes, cytokines and other pro-inflammatory factors in order to sustain their continued growth, resulting in inflammation with widespread impact on the systemic physiology. Acute-phase inflammatory markers such as CRP, potentially predictive of future coronary events, may be amplified by individual susceptibility and the risk factors associated with each periodontal patient.

Antimicrobial PDT has been shown to rapidly and effectively kill the microorganisms located in periodontal biofilms, and to destroy the virulence factors produced by these pathogens. The approach can directly inhibit LPS, reducing the risk of cytotoxic shock, and can substantially reduce protease activity, minimizing the risk of further tissue damage. High-level suppression of pro-inflammatory cytokines restores neutrophil chemotaxis and activation. Long term (six month) suppression of serum CRP has been demonstrated after Periowave™ aPDT, linking reduction in periodontal inflammation to reduced coronary risk. A local vaccine-like effect has been recently discovered, where aPDT treatment induces a potent innate immune response capable of blocking disease recurrence at the same site. The combination of potent antimicrobial capability, inflammation suppression and innate immunity induction provides a clinically useful adjunct to SRP in the treatment of periodontal diseases. OH


Dr. Loebel is the Chief Technology Officer and President of Ondine Biomedical Inc., manufacturer of the Periowave™ photodisinfection system)

Oral Health welcomes this original article.

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