antimicrobial action of minocycline microspheres versus 810-nm diode laser on human dental plaque...

Journal of Periodontology; Copyright 2013 DOI: 10.1902/jop.2013.130007

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Antimicrobial Action of Minocycline Microspheres Versus 810 nm Diode Laser on Human Dental Plaque Microcosm Biofilms

Xiaoqing Song, MD,* Tina Yaskell, BS,* Vanja Klepac-Ceraj, PhD,*† Michael C. Lynch,

DMD, PhD,‡ and Nikolaos S. Soukos, DDS, PhD*

* Applied Molecular Photomedicine Laboratory, Department of Applied Oral Sciences,

The Forsyth Institute, Cambridge, Massachusetts.

† Department of Biological Sciences, wellsley, Wellsley College, Wellsley, MA.

‡ OraPharma, Inc., 5 Walnut Grove Drive Suite 300, Horsham, Pennsylvania.

Background: The purpose of this study was to investigate the antimicrobial effects of

minocycline hydrochloride microspheres vs. infrared light at 810 nm from a diode laser on multi-species

oral biofilms in vitro. These biofilms were evolved from dental plaque inoculum (oral microcosms)

obtained from six systemically healthy human individuals with generalized chronic periodontitis.

Methods: Multi-species biofilms were derived using supra- and subgingival plaque samples from

mesiobuccal aspects of premolars and molars exhibiting probing depths in the 4-5mm range and 1-2 mm

loss of attachment. Biofilms were developed anaerobically on blood agar surfaces in 96-well plates using a

growth medium of PRAS brain heart infusion with 2% horse serum. Minocycline HCl 1 mg microspheres

were applied on biofilms on day 2 and day 5 of their development. Biofilms were also exposed on day 2

and 5 of their growth to 810 nm light for 30 seconds using power of 0.8 Watts in a continuous wave mode.

The susceptibility of microorganisms to minocycline or infrared light was evaluated by a colony forming

assay and DNA probe analysis at different time points.

Results: At all time points of survival assessment, minocycline was more effective (>2 log10 CFU

reduction) compared with light treatment (P<0.002). Microbial analysis did not reveal susceptibility of

certain dental plaque pathogens to light and was not possible following treatment with minocycline due to

lack of bacterial growth.

Conclusion: The cumulative action of minocycline microspheres on multi-species oral biofilms in

vitro led to enhanced killing of microorganisms, whereas a single exposure of light at 810 nm exhibited

minimal and non-selective antimicrobial effects.

KEYWORDS:

minocycline microspheres, infrared light, diode laser, dental plaque, biofilms, in vitro

Βiofilm-associated inflammatory diseases of the attachment apparatus around teeth in the

oral cavity, collectively known as periodontal diseases, represent a major part of the

global burden of oral disease. Gingivitis occurs in 90% of adults1, whereas periodontitis

affects over 47% of the American population aged >30 years and it is severe (>4 mm

attachment loss) in 13% of adults2. Periodontitis is also suggested as a risk factor for

coronary heart disease3, atherosclerosis

4, preterm births

5 and chronic kidney disease

6. The

primary microbial factor contributing to disease is a shift from beneficial to pathogenic

bacteria within dental plaque, and the primary immunological factor is the destructive

host inflammatory response7. Mechanical removal of periodontal biofilms (brushing,

scaling and root planing) is the method of treatment. However, residual microorganisms

recolonize the subgingival environment immediately following scaling and root planning

and form new biofilms8.


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The local use of antibacterial agents in sustained-release vehicles as an adjunct to

scaling and root planing has statistically significant effects on pocket depth reduction and

a decreased percentage of sites with bleeding on probing9. On the other hand, the use of

infrared light (805-810 nm) following scaling and root planing was reported to have

clinical benefits10, 11

and exhibit antimicrobial action12

. However, two recent systematic

reviews were not able to offer consistent evidence supportive of the efficacy of laser

treatment as an adjunct to non-surgical periodontal treatment in subjects with chronic

periodontitis13, 14

. Recently, the American Academy of Periodontology stated that there is

lack of consistent evidence to support the use of laser treatment for subgingival

debridement either alone or as an adjunct to non-surgical periodontal therapy15

.

To our knowledge, there exists no data comparing, side-by-side, the antimicrobial

effects of local sustained-release agents and infrared light on dental plaque-derived

biofilms in vitro. In the present study, we investigated the antimicrobial effects of

minocycline microspheres§ - a subgingival sustained-release product containing

minocycline hydrochloride into poly(glycolide-co-D,L-lactide) - and infrared light with a

wavelength of 810 ± 20 nm from a diode laser on multi-species oral biofilms evolved in

vitro from human dental plaque inoculum (oral microcosms).

MATERIALS AND METHODS

Patients and Plaque Samples

The study protocol was approved by the Institutional Review Board (IRB) Committee

at The Forsyth Institute. Samples of dental plaque were taken from six systemically

healthy individuals at The Forsyth Institute (from October 2011 to February 2012).

Permission to collect dental plaque samples was authorized by IRB-approved informed

consent (written). All the individuals were diagnosed as having generalized chronic

periodontitis based on the periodontal classification of the American Academy of

Periodontology16

. None of the patients used antibiotics nor had undergone periodontal

treatment during 3 months prior to sampling. Dental plaque samples were taken from

supra- and subgingival mesiobuccal aspects of premolars or molars in each subject with

individual sterile Gracey curettes‖. Probing depths at these sites were in the 4-5 mm

range with 1-2 mm loss of attachment. After their removal, pooled samples (usually

obtained from 3-4 sites) were placed immediately in one vial containing 4.5 ml of pre-

reduced, anaerobically sterilized (PRAS) Ringer’s solution¶. Microorganisms were

dispersed by sonication and repeated passage through Pasteur pipettes. Aliquots were

measured in a spectrophotometer in 1 ml cuvettes (one optical density unit was

considered as approximately 109 cells/ml at 600 nm). Then each sample was used for the

development of biofilms.

Preparation of Blood Agar Culture Plates

An enriched agar medium was prepared containing 20 g/l trypticase soy agar#, 26 g/l

brain heart infusion (BHI) agar**, 10 g/l yeast extract (BBL) and 5 mg/l hemin††

. The

medium was autoclaved and cooled to 50oC. Then 5% defribinated sheep blood

‡‡, 5

mg/ml menadione††

and 10 mg/ml N-acetylmuramic acid††

were aseptically added.


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Aliquots of 150 μl of the agar mixture was dispensed into wells of 96-well microtiter

plates§§

and allowed to dry.

Development of Plaque-Derived Biofilms

For biofilm development, the plaque/BHI broth inoculum contained approximately

107 cells/ml

17. One hundred fifty μl of this inoculum (approximately 1.5 x 10

6 bacteria)

was pipetted to fill four blood agar wells in each 96-well plate. The plates were then

incubated anaerobically (80% N2, 10% H2, and 10% CO2) at 35oC. After initial

incubation of 48 hours, the liquid medium was carefully aspirated from each well and the

biofilms were replenished with fresh BHI broth.

Experimental Design

The study was carried out using dental plaque samples from 6 individuals. The dental

plaque sample from each individual was divided in two parts (Fig. 1). The first part (Fig.

1A) was used for the development of biofilms that were treated with minocycline HCL 1

mg microspheres or a single exposure to 810 nm light on day 2 of their growth. The

second part (Fig. 1B) was used for the development of biofilms that were exposed to

minocycline microspheres or a single exposure to 810 nm light on day 5 of their growth.

Six independent experiments were carried out for biofilms treated on day 2 or day 5 of

their growth. Biofilms treated at different time points of their growth (day 2 or 5) were

employed in order to investigate whether the age of the biofilm contributes to resistance

towards treatment with minocycline vs. 810 nm light.

For biofilms treated on day 2 of their development (Fig. 1A), each experiment

included: 4 minocycline-treated groups (3 biofilms per group; 12 biofilms total); 4 light-

treated groups (3 biofilms per group; 12 biofilms total); and 5 control groups (untreated

with minocycline microspheres or 810 nm light, 3 biofilms per group; 15 biofilms total).

For all biofilm groups, assessments of bacterial survival took place on day 4, 7, 9 and 11

of their growth (3 biofilms per time point). Assessments of bacterial survival were carried

out by calculating the mean number of colony-forming units (CFUs) per time point and

by assaying the microbial composition of biofilms using a whole genomic probe assay

(see below). Control biofilms were also assessed for their microbial composition on day 2

of their growth (baseline). Six independent experiments were carried out for biofilms

treated with minocycline microspheres or 810 nm light on day 2 of their growth (6

experiments x 39 biofilms/experiment = 234 biofilms total).

For biofilms treated on day 5 of their growth (Fig. 1B), each experiment included the

same biofilm groups as above. For all biofilm groups, assessments of bacterial survival

took place on day 7, 9, 11 and 14 of their growth. The microbial composition of control

biofilms was also assessed on day 5 of their growth (baseline). A total number of 234

biofilms in 6 independent experiments was used.

Treatment with Minocycline Microspheres

Minocycline HCL 1 mg microspheres were provided as a dry powder packaged in a

unit-dose cartridge, which was inserted in a cartridge handle to administer the product.

The cartridge tip was placed in contact with the liquid medium covering the biofilm in

each well and the thumb ring was gently pressed to express the powder while


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withdrawing the cartridge tip away from the surface of the medium. Once delivery was

complete, the thumb ring was retracted and the cartridge was removed. A fresh cartridge

was used for each biofilm. A unit-dose cartridge delivered minocycline hydrochloride

equivalent to 1 mg of minocycline free base on each biofilm. Every two days, the liquid

medium was carefully aspirated from each well and the biofilms were replenished with

fresh BHI broth. For survival assessment, adherent bacteria were scraped at different time

points (Fig. 1). Following scraping, microorganisms were dispersed in BHI broth and

aliquots were measured in a spectrophotometer in 1 ml cuvettes. Then serial dilutions

were prepared and 100 μl aliquots were spread over the surfaces of blood agar plates. The

plates were incubated anaerobically at 35ºC for 7 days. The primary endpoint for

evaluation was the mean number of colony-forming units (CFUs) per time point.

Treatment with Diode Laser

A portable Odyssey® 2.4G diode laser‖‖

emitting infrared light with a wavelength of

810 + 20 nm was used for treatment of biofilms. Before phototherapy, the growth

medium was aspirated from each well, gently. A fiber tip (with a diameter of 400 μm)

was placed 1 mm above the biofilm surface and delivered light with an output power of

0.8 W. The entire surface was exposed to light using 3-4 mm quick horizontal moves of

the fiber tip for 30 seconds in the continuous mode. In the present study, the power

setting and exposure time were 0.8 W and 30 sec, respectively, similar to those used in a

clinical setting. The instructions of the manufacturer of the diode laser were followed

concerning the parameters (power density, exposure time) and methodology for the

application of light. Exposure of biofilms to light yielded a total applied energy of 24

Joules (0.8 W for 30 sec). Biofilms were exposed to a single light exposure on day 2 and

day 5 of their growth. Every two days, the liquid medium was gently aspirated from each

well and the biofilms were replenished with fresh BHI broth. For survival assessment,

adherent bacteria were scraped at different time points (Fig. 1). Following scraping,

biofilms were replenished with fresh BHI broth, serial dilutions were prepared and 100 μl

aliquots were spread over the surfaces of blood agar plates. The plates were incubated

anaerobically at 35ºC for 7 days.

Composition of Biofilms

The microbial composition of biofilms was assayed using whole genomic probe assay

in a checkerboard DNA-DNA hybridization method as described previously17, 18

. Briefly,

Tris-EDTA buffer (1.5 ml) was added to the plates and the bacterial colonies were

scraped off the surface with sterile L-shaped glass rods. The suspensions were placed into

individual Eppendorf tubes and sonicated for 10 sec to break up clumps. The optical

density (OD) of each suspension was adjusted to a final OD of 1.0, which corresponded

to approximately 109 cells. Ten μl of the suspension (10

7 cells) were removed and placed

in another Eppendorf tube with 140 μl of TE buffer and 150 μl of 0.5M NaOH. The

samples were lysed and the DNA was placed in lanes on positively charged nylon

membrane using a Minislot device‖‖

. The MiniSlot device18

allowed lysates loaded in

parallel channels to be aspirated through the membrane, depositing horizontal lanes on

the membrane surface. After fixation of the DNA to the membrane, the membrane was

placed in Miniblotter 45 apparatus¶¶

(checkerboard system with 45 channels)18

with the

lanes of DNA at perpendicular to the lanes of the device. Digoxigenin-labeled whole


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genomic DNA probes to 40 bacteria species were hybridized in individual lanes of the

Miniblotter. After hybridization, the membranes were washed at high stringency and the

DNA probes were detected using antibody to digoxigenin conjugated with alkaline

phosphatase for chemifluorescence detection. Signals were detected using AttoPhos

substrate##

and were scanned using a Storm Fluorimager***

. Computer-generated images

were analyzed to determine the fluorescence intensity associated with each sample and

probe. Two lanes in each membrane contained DNA standards with 1 ng (105

bacteria)

and 10 ng (106

bacteria) of each species. The sensitivity of the assay was adjusted to

permit detection of 104 cells of a given species by adjusting the concentration of each

DNA probe. The measured fluorescence intensities were converted to absolute counts by

comparison with the standards on the same membrane. Failure to detect a signal was

recorded as zero.

Data Analysis

For each treatment, the endpoint value was calculated CFUs following treatment

relative to the pretreatment CFU level (Control). Pairwise comparisons of treatment

effects (mean log10 CFU) were performed by Kruskal-Wallis test in STATA version

12.1†††

. DNA-DNA checkerboard data were compared using Wilcoxon rank sum test for

the following: 1) control biofilms and biofilms treated with laser light on day 2; 2)

control biofilms and biofilms treated with laser light on day 5; 3) control biofilms and

biofilms treated with laser light on day 2 vs. day 5 for individual days 7, 9, and 11. Due

to 40 comparisons of bacterial taxa, Bonferroni correction was used to determine if the

post-hoc tests were significant. To control for the type I error and thus, account for

multiple comparisons (hypotheses), Bonferroni correction was used to determine whether

the differences among taxa between the two groups were significant.

RESULTS

Treatment of Biofilms with Minocycline Microspheres or Diode Laser

Minocycline was more effective when compared to laser treatment and control for all

days tested, and regardless whether the biofilms were treated on day 2 or day 5 of their

growth (P<0.002, Kruskal-Wallis). Minocycline produced 2-3 log10 CFU reduction in

biofilms treated on day 2 (Fig. 2A) and day 5 (Fig. 2B) at all time points of survival

assessment. On day 7 and 9 of growth of biofilms treated on day 5, no colonies were

detected on plates following treatment with minocycline (Fig. 2B). In laser treated

biofilms, killing was <1 log10 (ranged between 17% and 54%) (Fig. 2A and Fig. 2B).

Checkerboard DNA-DNA hybridizations were not performed for the minocycline treated

biofilms due to lack of bacterial growth on the plates.

Microbial Analysis Following Laser Treatment

DNA probe analysis of biofilms treated on day 2 or 5 of their development

demonstrated that the composition of each was similar (Fig. 3). No significant differences

in levels of microorganisms were found in statistical comparisons between control

biofilms and biofilms that received a single exposure of infrared light up to day 11 of

their growth (P>0.05) with one exception. Three microorganisms - Cambylobacter

rectus, Cambylobacter showae and Capnocytophaga ochracea – exhibited greater growth


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in biofilms (treated on day 2) on day 11 of their development, but were not significant

after Bonferroni correction for multiple comparisons (Fig. 3).

DISCUSSION

In the present study, the antimicrobial effects of minocycline hydrochloride microspheres

and infrared light at 810 nm from a diode laser were investigated on human dental plaque

microorganisms in the biofilm phase. The microcosm biofilm model that was employed

in this study originated directly from the whole-mixed natural dental plaque. Evidence

concerning microcosm biofilm development, characterization, structure and validation

has been reported previously17

. Microbial analysis using checkerboard DNA–DNA

hybridization demonstrated the presence of a mixed microflora reflecting the complexity

of dental plaque (Fig. 3). Bacterial growth from pooled human dental plaque on blood

agar has been demonstrated previously19

. Plaque microcosms are functional models used

to study drug delivery and targeting20

.

Exposure of oral microcosms to minocycline hydrochloride microspheres in vitro led

to almost complete killing of dental plaque species. The killing of biofilm

microorganisms ranged from 2 log10 to 100% (Fig. 2). Although biofilms were developed

using plaque obtained from different donors, biofilm variability did not reflect significant

differences in responses to either light or minocycline microspheres. Several studies have

investigated the effects of minocycline on oral biofilms. Sedlacek and Walker

investigated the effects of minocycline and other antibiotics commonly used as adjuncts

to periodontal therapy on biofilms grown on saliva-coated hydroxyapatite for 4 to 10

days21

. Treatment with tetracycline for 48 hours at different time points of biofilm growth

resulted in greater reductions of bacterial survival compared with doxycycline and

minocycline. The percentage of viable microorganisms surviving ranged from 10-30% in

young biofilms (4 hours to 5 days) to 60-95% in older biofilms (7 – 10 days). Application

of minocycline microspheres in periodontal pockets ≥5 mm of human subjects for 10

days immediately following scaling and root planing led to a statistically significant

greater reduction in proportions and numbers of the key periodontal pathogens

Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola compared

with scaling and root planing alone22

. In the same study, DNA probe analysis by

checkerboard DNA-DNA hybridization showed significant decreases in proportions of

Parvimonas micra, Eubacterium nodatum, Treponema socranskii and Prevotella

nigrescens. In contrast, increase in proportions of Actinomyces and Streptococci species

was observed. These data indicate a specific antibacterial effect of minocycline

microspheres on dental plaque bacteria and they are in disagreement with the findings of

our study as well as in contrast to the generally held concept that tetracyclines are broad-

spectrum antibiotics23

.

The goal for the use of a 810 nm diode laser in periodontal treatment is the ablation of

diseased pocket lining epithelium24

and disinfection of periodontal pockets11

. With regard

to the disinfection of periodontal pockets by lasers, there are conflicting data in the

literature. Moritz et al. have shown that the bacterial damage following irradiation

depends on the amount of energy applied with the construction of the cell wall being

crucial for the individual bacterial susceptibility to light25

. Gram-negative species showed

immediate structural damage, whereas gram-positive microorganisms required repeated


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exposure to irradiation25

. Bacterial damage by a diode laser at 810 nm is possible without

injury to periodontal tissues due to heat development26

. In our study, the power setting

and exposure time were 0.8 W and 30 sec, respectively, similar to those used in a clinical

setting. Biofilm bacteria showed resistance to diode laser light at 810 nm with maximum

killing not exceeding 54% (Fig. 2). This killing was non-selective as microbial analysis

demonstrated (Fig. 3). The limited reduction of viable counts following exposure to light

may be the result of diode laser-induced heat accumulating in the blood agar that served

as substrate for biofilm development. Light at 810 nm was not expected to induce killing

of black-pigmented bacteria (e.g. P. gingivalis, P. nigrescences, Prevotella intermedia,

Prevotella melaninogenica) within biofilms. The endogenous porphyrins of these

microorganisms absorb light mainly in the blue and red region of the electromagnetic

spectrum27

. In a clinical setting, heat development may lead to elimination of periodontal

microorganisms following penetration of light at 810 nm into the gingival tissue and its

absorption by endogenous chromophores (e.g. hemoglobin, melanin) and water. Diode

laser light at 805 nm eliminated pathogens in periodontal pockets, such as

Aggregatibacter actinomycetemcomitans and P. gingivalis10, 11

. Another in vivo study

demonstrated the killing effect of light (1064 nm) from a Neodymium:Yttrium-

Aluminum-Garnet (Nd:YAG) laser on P. gingivalis, Prevotella intermedia and A.

actinomycetemcomitans, however recolonization occurred on the irradiated tooth surfaces

seven days following laser treatment28

. Similar findings were reported with P. gingivalis,

T. forsythia, and T. denticola ten weeks following the use of Nd:YAG laser in

conjunction to scaling and root planing versus scaling and root planing alone29

. Fontana

et al.30

reported considerable reduction of microorganisms (e.g. Prevotella, Pseudomonas

and Fusobacterium species) in periodontal pockets of rats following exposure to light for

9 seconds from a 810 nm diode laser operating in the range of 400-1,200 mW. Recently,

Cappuyns et al. compared the clinical and antimicrobial effects of photodynamic therapy

(synergism of light and a photoactive agent), subgingival diode laser treatment with a

wavelength of 810 nm (power output of 1 W and exposure time of 60 sec) and scaling

and root planing in a randomized, split-mouth controlled clinical trial in patients with

residual pockets previously treated for periodontal disease non-surgically31

. Although the

suppression of P. gingivalis, T. forsythia and T. denticola was significantly greater at 14

days following photodynamic therapy and scaling and root planing compared with diode

laser treatment, the differences were no longer significant at 2 months. The same authors

reported no significant differences among photodynamic therapy, 810 nm diode laser

therapy and conventional deep scaling and root planing on the expression of

inflammatory mediators32

.

Local drug delivery in adjunct to scaling and root planing appears to offer additional

benefits to periodontal pocket reduction compared with scaling and root planing alone33

.

On the other hand, the effectiveness of diode lasers on the elimination of pathogenic

periodontal microorganisms has been reported with conflicting results. This study shows

that the cumulative action of minocycline microspheres on multi-species oral biofilms

evolved in vitro from human dental plaque inoculum leads to enhanced killing of

microorganisms, whereas a single exposure of light at 810 nm exhibits minimal and non-

selective antimicrobial effects. However, there are limitations to our study related to the

absence of all relevant processes that occur because of the interaction with the more


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complex system of gingival tissue. In case of laser treatment, whether our findings can be

confirmed remains to be elucidated in large clinical studies.

ACKNOWLEDGEMENTS:

This study was supported by a grant from OraPharma, Inc., 5 Walnut Grove Drive Suite 300, Horsham,

Pennsylvania. Michael C. Lynch, DMD, PhD is currently Director, Clinical Strategy and Research, Oral

Care Johnson & Johnson Consumer & Personal Products Worldwide, Division of Johnson & Johnson, 185

Tabor Road, K2-204 Morris Plains, NJ 07950

CONFLICT OF INTEREST STATEMENT

Authors at The Forsyth Institute report no conflicts of interest related to this study.

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Correspondence: Nikolaos S. Soukos, DDS, PhD, Applied Molecular Photomedicine

Laboratory, The Forsyth Institute, 245 First Street, Cambridge, MA 02142, USA. Tel:

617/892-8467; e-mail: [email protected].

Submitted January 8, 2013; accepted for publication April 22, 2013.


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Figure 1.

Experimental design. Biofilms developed from the same dental plaque inoculum were treated with

minocycline microspheres or a single exposure to 810 nm light on day 2 (A) and day 5 (B) of their growth

in two separate experiments. Assessments of bacterial survival took place on day 4, 7, 9 and 11 for

biofilms treated on day 2 of their development (A), whereas biofilms treated on day 5 were assessed for

survival on day 7, 9, 11 and 14 of their growth (B).

Figure 2.

Survival fraction of microorganisms in plaque-derived biofilms at different time points following their

treatment with minocycline microspheres or with a single exposure 810 nm light on day 2 (A) and 5 (B) of

their growth. Each bar represents the mean values ± standard deviations of the means from six patients

(data from each patient were representative of three independent biofilms). The control group represents

survival fraction of 100%. Minocycline produced >2 log10 CFU reduction in biofilms (A & B) at all time

points of survival assessment and was more effective when compared to laser treatment (P<0.002). On day

7 and 9 of growth of biofilms treated on day 5 (B), killing was 100% following treatment with minocycline.

Figure 3.

Profiles of mean DNA counts of 40 microorganisms in control (black bars) and laser treated (white bars)

biofilms on day 11 of their growth. Biofilms treated with light on day 2 (left) or 5 (right) of their

development showed similar compositions. Each bar represents the mean DNA count (x105) of values

obtained from six individuals. Error bars denote the standard error of the mean. Differences in levels of

microorganisms between control and treated biofilms were not significant (P>0.05)

§ Arestin®, OraPharma, Horsham, PA

‖ Pearson Dental, Sylmar, CA

¶ Anaerobe System Morgan Hill, CA

# BBL, Cockeysville, MD

** Difco Laboratories, Detroit, MI

†† Sigma Chemical Co., St. Louis, MO

‡‡ Northeast Labs, Waterville, ME

§§ NUNC, Rochester, NY

‖‖ Ivoclar Vivadent, Inc., Buffalo, NY, USA

¶¶ Immunetics, Cambridge, MA, USA

## Amersham Life Science, Arlington Heights, IL, USA

*** Molecular Dynamics, Sunnyvale, CA, USA

††† StataCorp, College Station, TX, USA

1 1

Control group (No minocycline/No light)

Minocycline group

Light group

Minocycline application

Light application

DAY 0 DAY 2 DAY 4 DAY 7 DAY 9 DAY 11

Survival assessment

Control group (No minocycline/No light)

Minocycline group

Light group

Minocycline application

Light application

DAY 0 DAY 5 DAY 7 DAY 9 DAY 11 DAY 14

Survival assessment

FIGURE 1

Sample of dental plaque obtained from an individual

Biofilms treated on DAY 2

of their growth

Biofilms treated on DAY 5

of their growth

(A)

(B)

1 2

102

4 7 9 11

7 9 11 14

Days

% o

f sur

vivo

rs

% o

f sur

vivo

rsA

B Minocycline Light at 810 nm

Day 2

Day 5

102

101

100

10-1

10-2

101

100

10-1

10-2

FIGURE 2

1 3

Actinomyces gerencseriae Actinomyces israelii Actinomyces naeslundii 1 Actinomyces naeslundii 2 Actinomyces odontolyticus Aggregatibacter actinomycetemcomitans Campylobacter gracilis Campylobacter rectus Campylobacter showae Capnocytophaga gingivalis Capnocytophaga ochracea Capnocytophaga sputigena Eikenella corrodens Eubacterium nodatum Eubacterium saburreum Fusobacterium nucleatum ss nucleatum Fusobacterium nucleatum ss polymorphum Fusobacterium nucleatum ss vincentii Fusobacterium periodonticum Gemella morbillorum Leptotrichia buccalis Neisseria mucosae Parvimonas micra Porphyromonas gingivalis Prevotella intermedia Prevotella melaninogenica Prevotella nigrescens Propionibacterium acnes Selenomonas noxia Streptococcus anginosus Streptococcus constellatus Streptococcus gordonii Streptococcus intermedius Streptococcus mitis Streptococcus oralis Streptococcus sanguinis Tannerella forsythia Treponema denticola Treponema socranskii Veillonella parvula

0 5 10 15 2051015 20

Biofilm treated with light on Day 2

0

Mean counts (x105) Mean counts (x105)

Biofilm treated with light on Day 5

laser treatedcontrol

FIGURE 3

antimicrobial action of minocycline microspheres versus 810-nm diode laser on human dental plaque...

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