Received: 22 May 2023 Revised: 1 October 2023 Accepted: 6 October 2023

DOI: 10.1002/JPER.23-0328

H UMAN RANDOMIZED CONTROL TRIAL

Suppression of subgingival bacteria by antimicrobial

photodynamic therapy using transgingival irradiation:
A randomized clinical trial

Jun-ichiro Hayashi1 Kohta Ono1 Yuki Iwamura1 Yasuyuki Sasaki1

Tasuku Ohno1 Ryoma Goto1 Eisaku Nishida1 Genta Yamamoto1
Takeshi Kikuchi1 Naoya Higuchi2 Akio Mitani1 Mitsuo Fukuda1

1 Department of Periodontology, School of

Dentistry, Aichi Gakuin University, Abstract
Nagoya, Aichi, Japan Background: Antimicrobial photodynamic therapy (aPDT) is an effective
2 Department of Endodontics, School of method for eradicating bacteria in periodontal therapy. Standard aPDT requires
Dentistry, Aichi Gakuin University,
Nagoya, Aichi, Japan the insertion of a laser tip into a periodontal pocket, in which the direction
of irradiation is limited. Therefore, we devised an aPDT method that uses a
Correspondence
transgingival near-infrared wavelength and indocyanine green-encapsulated and
Jun-ichiro Hayashi, Department of
Periodontology, School of Dentistry, Aichi chitosan-coated nanoparticles as a photosensitizer.
Gakuin University, Nagoya, Aichi Methods: Forty patients undergoing supportive periodontal therapy, who had
464-8651 Japan.
a single root tooth with a pocket of 5 mm or deeper, were used as subjects. In
Email: jun1row@dpc.agu.ac.jp
the test group, aPDT was performed by laser irradiation from outside the gingiva
Funding information using photosensitizer nanoparticles. In the control group, pseudo aPDT without
The Japan Society for the Promotion of
Science (Tokyo, Japan), Grant/Award
photosensitizer was performed by transgingival irradiation. Subgingival plaque
Numbers: 18K09612, 19K10140; The Hori was sampled from inside the pocket before, immediately after, and 1 week after
Science and Arts Foundation (Nagoya, treatment, and evaluated by colony counting and real-time polymerase chain
Japan).
reaction.
Results: There were no significant differences in age, sex, periodontal pocket
depth, and bleeding on probing between the test and control groups. Compared
with the colony count before treatment, the count in the test group was signifi-
cantly reduced immediately after treatment. The number of patients with colony
reduction to ≤50% and ≤10% was significantly higher in the test group than in the
control group. None of the participants reported pain, although one participant
reported discomfort.
Conclusion: As a bacterial control method for residual pockets in patients
undergoing supportive periodontal therapy, transgingival aPDT is a promising
treatment strategy that is not generally accompanied by pain or discomfort.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited and is not used for commercial purposes.
© 2023 The Authors. Journal of Periodontology published by Wiley Periodicals LLC on behalf of American Academy of Periodontology.

718 wileyonlinelibrary.com/journal/jper J Periodontol. 2024;95:718–728.

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HAYASHI et al. 719

KEYWORDS
chitosan, indocyanine green, periodontitis, photodynamic therapy

1 INTRODUCTION side the gingiva to deliver light energy to the inside of

periodontal pockets (termed “transgingival irradiation”).
Gram-negative bacteria inhabit periodontal pockets, form- Since indocyanine green (ICG), a photocatalytic dye,23 is
ing a biofilm, and their virulence factors lead to further excited by near-infrared wavelengths within the biologi-
inflammation.1,2 Remission of periodontitis requires the cal window,24 we employed this dye as a photosensitizer
eradication of these pocket bacteria, and mechanical and an 810 nm diode laser as the light source. For this
debridement of the subgingival area, including scaling method, ICG-encapsulated nanoparticles (ICG-nano/c)
and root planing (SRP), has been adopted as the standard were developed.19 ICG-nano/c are coated with chitosan to
treatment.3 positively charge the nanoparticles25 and to bind to neg-
However, in recent years as the population has aged in atively charged bacterial surfaces. Our experiment using
developed countries, there are an increasing number of a gingival model showed a reduction in the number of
cases that require extra care. Mechanical debridement can- periodontal bacteria to less than one-thousandth of their
not avoid damage and is accompanied by pain at the sites original population.20 In this gingival model, a laser with
of inflamed gingiva at pocket walls. Bacteremia caused by a higher peak power of 2 W was required for tissue pene-
the damage has also been identified, which represents a tration. This power is higher than that of low-level lasers
risk factor for cardiovascular disease in older patients.4–6 used in conventional aPDT and is equivalent to high-level
Additionally, there is a risk of postoperative bleeding in laser treatment that aims at coagulation and transpiration
patients receiving anticoagulants.7 Chemotherapy target- of tissues. However, because it was applied in a non-
ing biofilms is prone to problems such as the emergence of contact defocusing manner in our method, no soft tissue
resistant bacteria and side effects.8 This problem cannot be degeneration was observed.20
avoided in the treatment of periodontal disease.9 In this study, we report a pilot clinical study conducted
In response to these challenges, laser-based antimicro- as the first step toward the clinical application of this trans-
bial therapy is attracting attention as a new option for gingival aPDT system. The objectives of this study were
controlling bacteria in periodontal pockets.10,11 Antimi- to investigate the bactericidal effect on bacteria in human
crobial photodynamic therapy (aPDT), which uses low- periodontal pockets under the irradiation conditions con-
power laser light to excite a photosensitizer that generates firmed in basic experiments, to confirm the safety of the
reactive oxygen species able to eliminate bacteria, has photosensitizer and laser used in the system, and to col-
recently been applied as a potentially effective treatment lect data necessary to determine the sample size for future
method.12–14 In the application of aPDT to treat periodon- studies.
tal disease, dyes such as methylene blue15,16 and toluidine
blue O17,18 are mainly used as the photosensitizer. For con-
ventional operation of aPDT, a photosensitizer is injected 2 MATERIALS AND METHODS
into the periodontal pocket, and then a laser tip is inserted
into the periodontal pocket followed by laser irradia- 2.1 Study design and participants
tion. However, this intra-pocket irradiation method limits
the irradiation angle, and the light may not reach com- This study was a single-blind, randomized, controlled trial
plex pockets or bone defects sufficiently because of blind for patients who had residual periodontal pockets under-
spots. Therefore, our research group has conducted basic going supportive periodontal therapy (SPT). As a result of
research to develop a new laser irradiation method.19,20 SPT, the target residual pockets were less inflamed, but
Living soft tissues contain large amounts of water and retained biofilm that required periodic removal.
oxidized hemoglobin, which strongly absorb light.21 How- Fifty outpatients undergoing SPT, who regularly vis-
ever, overlaying their absorption spectra shows a band ited Aichi Gakuin University Dental Hospital every 3 to
with a low absorption rate for both components. Light eas- 4 months, were selected by reference to previous clinical
ily penetrates soft tissues in the range 700 to 1000 nm, data during SPT. Each patient was given a detailed descrip-
called the “biological window of optical properties.”22 tion of the procedure, and after eliminating those who
We investigated a new irradiation method using this met the exclusion criteria or declined to participate, 40
property, in which light irradiation is applied from out- outpatients who provided written informed consent were
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720 HAYASHI et al.

enrolled by the investigators (Figure 1A). Participants were added to 2% hexaglycerin-condensed ricinoleate contain-
randomly assigned to the experimental (n = 20) or control ing triglyceride and n-hexane at 2 mL/min with stirring
(n = 20) group by blindly taking a card with a number from at 35◦ C. After centrifugation, the nanospheres were mixed
1 to 40 and were anonymized by issuing an identification with a 2% polyvinyl alcohol/0.5% chitosan solution to
code for each. form a chitosan coating. After further centrifugation,
This study was approved by the Nagoya City University nanosphere clusters were suspended in mannitol and
Hospital Certified Institutional Review Board under the lyophilized. The obtained nanospheres contained 5 mg/g
Clinical Trials Act of Japan (CRB4200003; approval num- of ICG and had an average size of 560 nm.
ber: 2020A002) and was conducted in accordance with the A diode laser† with a wavelength of 810 ± 20 nm was
Helsinki Declaration of 1975, as revised in 2013. After noti- used as the light source. The light was emitted at a spread
fication to the Ministry of Health, Labor and Welfare of angle of 20.49◦ C from a diffusing irradiation probe. A cus-
Japan, this study was registered with the Japan Registry tom air-cooling system was coaxially integrated into the
of Clinical Trials (jRCTs041200061), a national registry of laser system.
Japan. This study was performed from November 11, 2020
to December 22, 2021.
2.4 aPDT procedure

2.2 Inclusion and exclusion criteria The deepest pocket of ≥5 mm probing depth in a single
root tooth of each participant was selected as the target
Participants had to meet all inclusion criteria as follows: site. First, 10 mg of ICG-nano/c was dissolved in 1 mL
(1) patients with periodontitis who had a ≥5 mm probing of phosphate-buffered saline (PBS). Then, 0.2 mL of ICG-
depth on a single rooted tooth in the SPT phase; (2) patients nano/c solution was placed in the target pocket using a
aged from 20 to 85 years old at registration; and (3) patients 1 mL syringe with a blunt needle (29 G) just before laser
who were able to participate in the study for two consecu- irradiation. In the pseudo aPDT control group, PBS solu-
tive weeks and had given their consent. For patients whose tion was used as a placebo instead of the photosensitizer.
initial visit was before 2019, periodontitis was defined as The laser (100 msec repeated pulse, peak power output
clinical attachment loss greater than 1 to 2 mm and prob- of 2 W, 50% duty cycle, average power of 1 W) irradiated
ing depth greater than 4 mm at the initial visit based on for 60 s and paused for 10 s three times from 10 mm out-
the 1999 AAP diagnostic criteria.26,27 For patients after that side of the gingiva in defocus with blowing air (2 L/min)
year, periodontal disease was defined based on the new for cooling (Figure 1B). The power density and the energy
2018 AAP-EFP classification.28 Participants who met any density were 1.46 W/cm2 and 250.38 J/cm2 , respectively.
of the following criteria were excluded from the study: (1) We previously showed that the light energy transmitted
patients who had taken antibiotics within the last three through 3 mm of soft tissue was reduced by approximately
months; (2) patients with diabetes; (3) patients who had one-third20 ; therefore, the power density in the pocket was
taken anticancer drugs within the last six months; (4) estimated to be 0.4 to 0.5 W/cm2 .
patients with a history of allergy to ICG; (5) patients with
a history of allergy to crustaceans; and (6) patients who
were pregnant, breastfeeding, or may be pregnant. Users 2.5 Study protocol
of anticancer drugs were excluded because of their poten-
tial to affect immune responses. Patients with crustacean The time schedule and flow diagram for this study are
allergies were also excluded because chitosan is made from shown in Figure 1. On the day of case enrollment (Visit 0),
crustacean chitin. after assessment of eligibility according to the selection cri-
teria, informed consent was obtained. Then, probing depth
and bleeding on probing at the test site were recorded by
2.3 aPDT system the calibrated examiners.
On the day of the procedure (Visit 1), subgingival plaque
ICG-nano/c was prepared by the emulsion solvent diffu- samples were taken from the test site using three paper
sion method in oil,25 according to a previously reported points‡ placed into the pocket for 10 s. One was used
protocol.19 Briefly, a solution of 5 mg of ICG,* polylactic for a colony count and the others were used for bacterial
acid glycolate, and Span80 in acetone and methanol was
† LIGHTSURGE SQUARE, Osada, Tokyo, Japan
* Ophthagreen, Santen Pharmaceutical, Osaka, Japan ‡ Absorbent Paper Points #45, Zipperer, Johnson City, TN
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HAYASHI et al. 721

F I G U R E 1 Study design and aPDT procedure. (A) Study flow diagram. Initially, 50 SPT patients were approached, but 10 were excluded
after assessment of eligibility at Visit 0. Enrolled participants were allocated randomly into a control group and a test group of 20 each, and
then received intervention at Visit 1. Bacterial samples were collected immediately after treatment at Visit 1. After a 1-week observation
period, follow-up examination and bacterial sampling were performed at Visit 2. (B) Schematic drawing of transgingival irradiation. A
chitosan-coated ICG photosensitizer was injected into the periodontal pocket. Then, an infrared diode laser (810 nm wavelength) with a
defusing irradiation probe was used for defocused irradiation 10 mm from the gingiva with air-blow cooling at 2 W peak power and a 50% duty
cycle (100 ms repeated pulse, 1.46 W/cm2 , 250.38 J/cm2 ). Three 60 s irradiations were performed with 10 s pauses in between. aPDT,
antimicrobial photodynamic therapy; ICG, indocyanine green; ICG-nano/c, ICG-encapsulated nanoparticles; PCR, polymerase chain
reaction; SPT, supportive periodontal therapy
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722 HAYASHI et al.

TA B L E 1 Demographic of the study participants

Measurement in SPT phase (Visit 1) Control group (n = 20) Test group (n = 20) p value
Sex (male/female) 10/10 11/9 0.749b
Age (years old) (mean ± SD) 66.4 ± 13.19 65.80 ± 12.08 0.882c
Probing depth (mm)a (mean±SD) 5.40 ± 0.82 5.95 ± 1.76 0.213c
(Maximum, mm) 8 10
Bleeding on probing (+/–)a 13/7 14/6 0.736b
Periodontitis classification at initial
visitd Control group (n = 20) Test group (n = 20)
Stage III/Grade A 1 2
Stage III/B 15 8
Stage III/C 0 1
Stage IV/B 4 4
Stage IV/C 0 5
Abbreviation: SPT, supportive periodontal therapy.
a
Evaluated at the sampling site.
b
Chi-square test.
c
Student’s t-test.
d
Since each participant’s initial visit was prior to the proposal of the new classification, the stage and grade were determined from the clinical data at that time.

analysis using real-time polymerase chain reaction (PCR). 2.7 Bacterial colony counts and
aPDT or pseudo aPDT was then performed on the target real-time PCR
site by dentists trained in the irradiation method. After
the aPDT procedure, bacterial plaque was sampled using The paper point for colony counting was agitated in 1 mL
a single paper point for a colony count. of PBS solution. The solution was serially diluted and
After a 1-week observation period, sampling with three seeded onto blood agar medium. After 10 days of incuba-
paper points (for colony counts and real-time PCR) was tion under anaerobic conditions at 37◦ C, the number of
performed at the follow-up (Visit 2) for both groups as growing colonies was counted. The other paper point was
described above, followed by usual ultrasonic cleaning of used for bacterial analysis by real-time PCR.§ The bacte-
the pockets and surfaces of the teeth as a posttreatment rial populations were estimated semi-quantitatively for 28
procedure. oral bacterial species including the red complex bacteria,
Porphyromonas gingivalis, Tannerella forsythia, and Tre-
ponema denticola (see Supplementary Table in the online
2.6 Demographic characteristics of the Journal of Periodontology).
participants

At Visit 0, 50 candidates were approached, of whom 10 2.8 Outcome assessments

were excluded prior to the allocation on Visit 1. The demo-
graphics of the remaining study participants are shown The primary outcome of this pilot study was set as the
in Table 1. The 40 included subjects, 19 males (38 to 85 change in the number of viable bacteria before and imme-
years old) and 21 females (47 to 80 years old), were ran- diately after aPDT in the test group to evaluate the
domized and allocated to the test (n = 20) and control bactericidal efficacy of the new method. Secondary out-
(n = 20) groups. The groups were sex- and age-matched. comes were set as (1) the change in bacterial colony counts
The mean probing depth of the target site was comparable before and immediately after treatment in the control
between the groups, being 5.95 ± 1.76 mm (maximum of group, (2) the reduction in bacterial colony counts from
10 mm) for the test group and 5.40 ± 0.82 mm (maximum before to 1 week after treatment for the control and test
of 8 mm) for the control group. There was no significant groups, and (3) the change in bacterial flora from before
difference in the number of participants who were positive to 1 week after treatment for both groups.
for bleeding on probing between the two groups. Staging Safety outcomes were evaluated immediately after treat-
and grading classification of periodontitis at initial patient ment at Visit 1 and before sampling at Visit 2. These
visits are shown in Table 1; 27 were Stage III and 13 were
Stage IV. § Saliva-Check Lab, GC Corporation, Tokyo, Japan
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HAYASHI et al. 723

included the presence of anaphylaxis or any other allergic

14.65 ± 30.69
Mean ± SD

9.02 ± 24.84
reactions after ICG nanoparticle injection, the presence of

2.29 ± 7.513
abnormalities in the mucosa of the irradiated field during
and after irradiation, and pain in response to the proce-
dure rated with a visual analog scale (VAS) ranging from
0 to 100. The patients were interviewed and gross exami-

1.205
14.73
IQR
nation was performed to detect any abnormalities during

5.35
the 1-week observation period.

Maximum
Test group

0.001
2.9 Sample size calculation

0.232
132.00

110.00
34.00
Because this was a pilot study, the sample size was
designed to estimate the mean change in colony counts

Median
of the test groups with a certain degree of accuracy. To

0.68
0.22
calculate the sample size, colony count data for P. gingi-

1.21
valis after SRP in SPT patients were considered according
to Kargas et al.29 Assuming a correlation coefficient of 0.3

Minimum
for the colony counts before and after treatment, the stan-
dard deviation (SD) of the change in colony counts can be

0.00

0.02
0.01
estimated to be 21.3, based on the data of Kargas et al.29
Assuming that the SD of the change in colony counts
in this study would also be 21.3, the number of subjects

14.66 ± 42.96
Mean ± SD
required was calculated to be 18.2. Considering dropouts

1.88 ± 3.40
1.57 ± 3.90
and other factors, the number of patients in the test group
was set at 20. The control group was also assumed to

The p values were obtained by Wilcoxon signed-rank test used for evaluation difference of CFU in each group.
require the same number of subjects.
Comparison of viable colony count before and after treatment in each group

IQR

9.89
1.73
1.37

2.10 Statistical analysis

Control group
Maximum

Statistical analysis of the obtained data in this study was

0.294

0.24
performed using a statistical processing program.** For the
193.00
13.80
17.80

primary endpoint, the mean and SD were calculated, and

the normality of the distribution was analyzed using the
Shapiro–Wilk test for bacterial evaluation. The Wilcoxon
Median

signed rank test was used to evaluate the difference in

Abbreviations: CFU, colony forming unit; IQR, interquartile range.
0.40

0.69
0.35

colony counts before and after treatment for the control

and test groups. The Mann–Whitney U-test was used for
statistical analysis of comparisons between the control and
Before treatment versus immediately after treatment.
Minimum

test groups.
Before treatment versus 1 week after treatment.
0.003

0.015
0.01

3 RESULTS
Immediately after treatment

3.1 Primary outcome

One week after treatment
Viable CFU (×105 )

The mean viable bacterial colony count before treatment

Before treatment

in the test group was 14.65 ± 30.69 × 105 colony-forming

units (CFU), and that immediately after treatment was
TA B L E 2

2.29 ± 7.5 × 105 (Table 2). The median CFU before and
b
p valuea

p value

** SPSS Statistics Ver. 28, IBM company, Armonk, NY

b
a
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724 HAYASHI et al.

immediately after treatment were 1.21 × 105 and 0.22 × 105 ,

p valuea
respectively. The interquartile range of the test group

0.004
before treatment was 14.73 × 105 . The viable colony counts

0.6
did not show a normal distribution. Therefore, nonpara-
metric analysis was applied for comparison. The difference

Mean ± SD

18.29 ± 4.53
in bacterial colony counts before and immediately after

1.18 ± 3.37
treatment for the test group was significant.

3.2 Secondary outcomes

IQR

3.20
0.38
3.2.1 Comparison of viable bacterial colony
counts

Test group
Maximum
The results of the colony counts are summarized in Table 2.

19.75
14.51
There was no significant difference in bacterial colony
counts before and immediately after treatment for the

Median
control group, despite a significant decrease in the test
group. Additionally, the viable colony counts 1 week after

0.48
0.13
treatment in both groups showed no significant difference
compared with the colony counts before treatment.

Minimum
Table 3 shows the ratio of the colony counts of individual
cases immediately after or 1 week after treatment to those

0.00

0.00
before treatment, and these ratios were compared between
the control and test groups. Regarding the ratio of CFUs
immediately after treatment to before treatment, a signif-

79.59 ± 274.56
Mean ± SD
1.67 ± 2.67
icant difference was found between the two groups. This
indicated that our aPDT method reduced bacteria in the
periodontal pockets. By contrast, there was no significant
difference between the control and test groups in the ratio
of CFUs 1 week after treatment to those before treatment. 23.95
IQR

The numbers of patients who showed a decrease in the

1.61

Mann-Whitney U-tests were used for comparisons between the control and test groups.
viable colony count immediately after treatment are com-
Control group
Viable colony count ratio of after versus before treatment

pared in Table 4. The number of patients who had a CFU

Maximum

ratio (immediately after treatment/before treatment) of

1266.67

≤0.5 or ≤0.1 was significantly larger in the test group than

10.50

in the control group. However, this difference was not sta-

Abbreviations: CFU, colony forming unit; IQR, interquartile range.

tistically significant when the threshold was ≤0.01. This

means that more patients in the test group showed a reduc-
Median

tion in the number of colonies to less than 50% or 10% after

0.64

1.41

treatment compared with the control group.

Minimum

3.2.2 Comparison of copy numbers by

0.04

real-time PCR
0.01
One week after/before

Table 5 shows a summary of bacterial copy number results

obtained by real-time PCR. The ratio of the total copy num-
ber 1 week after treatment to that before treatment in each
after/before
Immediately

participant showed no significant difference between the

CFU ratio
TA B L E 3

control and test groups.

The Gram-negative rate ratio of each participant was cal-
culated by dividing the Gram-negative rate for 1 week after
a
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HAYASHI et al. 725

Red complex rate was calculated before and at 1 week after treatment using the following formula: Red complex copy number/total copy number. The red complex rate ratio was calculated by dividing the posttreatment
Gram-negative rate was calculated before and at 1 week after treatment using the following formula: Gram-negative copy number/total copy number. The Gram-negative rate ratio was calculated by dividing the rate
T A B L E 4 Comparison of the number of patients who showed

p valuec
a viable colony count ratio below each threshold immediately after

0.414
0.776
treatment

1
Threshold Number of patients Pearson’s
(viable Control Test chi-square

Mean ± SD
colonies) (n = 20) (n = 20) value p valuea

2.26 ± 6.60

1.47 ± 2.80

1.15 ± 1.01
≤0.5 8 16 6.67 0.01
≤0.1 1 9 8.53 0.003
≤0.01 1 4 2.08 1.51
a
The p values were indicated as asymptotic significance probability of

IQR

1.03
1.26

1.17
Pearson’s chi-square test.

Maximum
by the Gram-negative ratio before treatment. Comparing
the Gram-negative rate ratio between the control and test

30.17

12.87

7.95
groups, no significant difference was detected.
There was also no significant difference in the red
complex rate ratio, which was calculated in the same

Median
manner as the Gram-negative rate, when comparing the

0.727

0.82

0.74
control and test groups. Data for each species are summa-
rized in the Supplementary Table in the online Journal of
Periodontology.

Test group
Minimum
0.02

0.02

0.01
3.3 Adverse effects and safety

No allergic reactions to ICG nanoparticles were observed.

Mean ± SD

1.62 ± 2.66

4.10 ± 1.01
1.06 ± 1.83

All patients marked 0 on the VAS regarding pain and there

was no thermal degeneration, such as wounds or burns
in the gingiva, in response to laser irradiation, but a few
patients reported a dry mouth as a result of the air blow. No
adverse effects were noted during the 1-week observation
IQR
0.84

2.84
1.73

period after treatment.

Mann-Whitney U-test were used for comparison between control and test group.
Maximum

Abbreviations: IQR, interquartile range; PCR, polymerase chain reaction.

4 DISCUSSION
9.24
7.95

7.95

The basic theory of aPDT with transgingival irradiation

was proposed in our initial in vitro study,20 which was then
Median

followed by a clinical study by another research group.30

Summary of real-time PCR results

for 1 week after treatment by the rate before treatment.

0.49

0.38

However, bacteriological evaluation of the method had not

0.7

been performed, and this study is the first clinical trial

Control group

to assess the effects on the bacterial population. In addi-

Minimum

tion, there are no clinical reports regarding aPDT using

chitosan-coated ICG nanoparticles, although some clinical
0.00
0.01

0.01

trials to evaluate the effects of aPDT with an ICG solu-

rate by the pretreatment rate.

tion have been conducted.31–33 The purpose of this study

is to confirm the actual bactericidal effect of transgingival
Red complex rateb
number ratio
Ratio (1 week

Gram-negative

irradiation, not to reduce the pocket depth. Therefore, all

after/before)

ratea ratio

participants were recruited during the SPT/maintenance

TA B L E 5

Total copy

phase after receiving non-surgical and/or surgical treat-

ratio

ments, and aPDT was performed on the stable pockets

remaining at that time.
b
a

c
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726 HAYASHI et al.

The primary outcome of this study showed a signifi- The 2 W peak power used in this study is higher than
cant reduction in bacterial counts in the test group when that used in conventional aPDT. Although this power is
comparing before and immediately after treatment. Addi- generally beyond the range of low-level laser treatment,
tionally, a significantly higher number of patients with a we previously showed that the temperature rise in soft
reduction in the colony count to ≤50% or ≤10% by aPDT tissue was limited to 2.7◦ C and no thermal degeneration
was detected in the test group than in the control group. of soft tissue was observed when air cooling and defo-
These results indicate that transgingival aPDT using ICG- cused intermittent irradiation were performed under the
nano/c had a certain bactericidal effect. However, it was same conditions as those used in this study.20 High-level
not as high as assumed from the results of our initial laser therapy often uses diode lasers at 1 to 2 W peak
experiments.20 In the initial in vitro studies, we observed power to achieve thermal degeneration of soft tissue. In
a bactericidal effect of more than 99% (˗2 log reduction; these methods, the laser probe has a very small diameter
reduced to ≤1%) against P. gingivalis planktonic cells.25 tip and the energy is concentrated at the focus, resulting
However, as shown in Table 3, the ratio of the median num- in extremely high energy density, which reaches 700 to
ber of bacteria immediately after treatment to that before 1000 W/cm2 for either contact or non-contact irradiation
treatment was 0.13, which is only an approximate ˗1 log methods.44,45 However, the laser probe used in this study
reduction. Additionally, only four cases in the test group has a flat end, which diffuses light. Therefore, the energy
had colony counts reduced to ≤1% immediately after treat- density becomes very low, being only 1.46 W/cm2 at the
ment. The bactericidal effect revealed by these results was gingival surface. This may be the reason why no thermal
low compared with previous clinical studies using intra- degeneration was observed on the gingival surface.
pocket irradiation. Pinheiro et al. reported that aPDT with One of the limitations of this study was the large vari-
a low-intensity diode and toluidine blue showed a 95.90% ance in the bacterial count data. This was the main reason
reduction in the number of bacteria in the periodontal for the lack of a normal distribution and the requirement
pocket.12 In comparison with non-aPDT methods, the bac- for nonparametric analysis. This large variance may be due
tericidal effect of the present method may be insufficient, to enumerating bacteria in living bodies and the inherent
considering that SRP shows a −5 log reduction of each variability in the sampling using paper points. Another
bacterial species.29 limitation was that there was no evaluation of clinical
To explain these results, the extracellular matrix of parameters. Periodontal pockets in SPT patients maintain
the biofilm may have affected bactericidal performance. a stable probing depth and show minimal inflammation
Lavaee et al. reported that the bactericidal effect of a-PDT even after a series of periodontal treatments. Therefore,
using methylene blue conjugated to gold nanoparticles was it is not expected that aPDT would alter these pockets.
attenuated in biofilms compared with planktonic cells.34 However, bacteria in the pocket need to be removed peri-
Similar observations were also reported for aPDT with odically, and the aPDT method tested in this study may be
ICG.35,36 Another possible explanation could be the effect a safe and useful option for this purpose.
of bleeding. Blood contains high levels of hemoglobin,
which absorbs the energy of the laser,37–39 so the energy
transmitted through the gingiva might be further atten- 5 CONCLUSION
uated. Additionally, ICG is known to be degraded by
light,40,41 and is also decomposed by self-generated singlet The results of this study confirm that aPDT with transgin-
oxygen during photosensitization.42 Therefore, the ICG gival irradiation using a near-infrared laser reduces bacte-
injected into the pocket may have lost its function through ria in human periodontal pockets. It was also shown that
these processes of energy absorbance or ICG degradation. chitosan-coated ICG nanoparticles function as an effective
The real-time PCR results also showed no significant photosensitizer for transgingival irradiation. However, the
difference between the control and experimental groups bactericidal effect was not as high as expected and its effect
in terms of the ratio of bacterial numbers before and 1 was very short-term. No laser-induced thermal denatura-
week after treatment. This may be a result of the bacte- tion was observed on inspection of the gingiva, and there
ricidal effect being less than expected, and the bacterial were no unexpected adverse effects. This study was a pilot
counts recovering during the 1 week period. We previously study and focused on bacteriological evaluation and sam-
showed that the bacterial flora in periodontal pockets after ple size estimation for a future larger study, including a
SRP differed from that before the procedure up to day comparison of clinical parameters.
21 but recovered to normal levels at day 60.43 Therefore,
a longer irradiation time and more frequent insertion of AU T H O R CO N T R I B U T I O N S
the photosensitizer are needed to be effective against the Mitsuo Fukuda, Jun-ichiro Hayashi, Tasuku Ohno, Ryoma
biofilm in the pocket. Goto, Eisaku Nishida, Genta Yamamoto, and Takeshi
 19433670, 2024, 8, Downloaded from https://aap.onlinelibrary.wiley.com/doi/10.1002/JPER.23-0328 by Cochrane Peru, Wiley Online Library on [14/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
HAYASHI et al. 727

Kikuchi recruited participants. Kohta Ono, Yuki Iwamura, 6. Priyamvara A, Dey AK, Bandyopadhyay D, et al. Periodon-
Yasuyuki Sasaki, Naoya Higuhci, and Jun-ichiro Hayashi tal inflammation and the risk of cardiovascular disease. Curr
prepared the photosensitizer. Jun-ichiro Hayashi, Kohta Atheroscler Rep. 2020;22:28.
7. Pototski M, Amenábar JM. Dental management of patients
Ono, and Mitsuo Fukuda performed the aPDT. Jun-ichiro
receiving anticoagulation or antiplatelet treatment. J Oral Sci.
Hayashi, Akio Mitani, and Mitsuo Fukuda contributed
2007;49:253-258.
to the conception and design of the work. Kohta Ono, 8. Quirynen M, Teughels W, Van Steenberghe D. Microbial shifts
Jun-ichiro Hayashi, and Mitsuo Fukuda collected and ana- after subgingival debridement and formation of bacterial resis-
lyzed the data. Jun-ichiro Hayashi, Tasuku Ohno, Ryoma tance when combined with local or systemic antimicrobials.
Goto, Eisaku Nishida, Genta Yamamoto, Takeshi Kikuchi, Oral Dis. 2003;9(Suppl 1):30-37.
Akio Mitani, and Mitsuo Fukuda prepared the manuscript. 9. Rams TE, Degener JE, Van Winkelhoff AJ. Antibiotic resis-
All authors gave their final approval and agreed to be tance in human chronic periodontitis microbiota. J Periodontol.
2014;85:160-169.
accountable for all aspects of the work.
10. Noguchi T, Sanaoka A, Fukuda M, Suzuki S, Aoki T. Com-
bined effects of Nd:YAG laser irradiation with local antibiotic
AC K N OW L E D G M E N T S application into periodontal pockets. J Int Acad Periodontol.
We thank Professor Hiromasa Yamamoto for his guidance 2005;7:8-15.
on the preparation of nanoparticles in this research. We 11. Theodoro LH, Marcantonio RAC, Wainwright M, Garcia VG.
LASER in periodontal treatment: is it an effective treatment or
also thank Dr. Aiji Sato for his kind advice on the applica-
science fiction? Braz Oral Res. 2021;35:e099.
tion for CRB clinical trial approval and proper monitoring 12. Pinheiro SL, Donegá JM, Seabra LM, et al. Capacity of photody-
of this trial. We thank Edanz for editing a draft of this namic therapy for microbial reduction in periodontal pockets.
manuscript. This study was supported by Grants-in-Aid Lasers Med Sci. 2010;25:87-91.
for Scientific Research from the Ministry of Education, 13. Kikuchi T, Mogi M, Okabe I, et al. Adjunctive application of
Culture, Sports, Science and Technology of Japan (Tokyo, antimicrobial photodynamic therapy in nonsurgical periodontal
Japan), Grant Numbers 18K09612 and 19K10140. This study treatment: a review of literature. Int J Mol Sci. 2015;16:24111-
24126.
was also supported by a donation from The Hori Science
14. Moro MG, De Carvalho VF, Godoy-Miranda BA, Kassa CT,
and Arts Foundation (Nagoya, Japan).
Horliana ACRT, Prates RA. Efficacy of antimicrobial photody-
namic therapy (aPDT) for nonsurgical treatment of periodontal
C O N F L I C T O F I N T E R E S T S TAT E M E N T disease: a systematic review. Lasers Med Sci. 2021;36:1573-1590.
The authors declare that they do not have any conflicts of 15. Tortamano ACAC, Anselmo GG, Kassa CT, et al. Antimicrobial
photodynamic therapy mediated by methylene blue in surfac-
interest.
tant vehicle on periodontopathogens. Photodiagnosis Photodyn
Ther. 2020;31:101784.
ORCID 16. Derikvand N, Ghasemi SS, Safiaghdam H, Piriaei H,
Jun-ichiro Hayashi https://orcid.org/0000-0001-9742- Chiniforush N. Antimicrobial photodynamic therapy with
5184 diode laser and methylene blue as an adjunct to scaling and
root planing: a clinical trial. Photodiagnosis Photodyn Ther.
Takeshi Kikuchi https://orcid.org/0000-0003-0310-5302
2020;31:101818.
17. Theodoro LH, Silva SP, Pires JR, et al. Clinical and microbiologi-
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