This document summarizes a study that investigated the effects of LED light irradiation on wound healing of free gingival grafts in rats. The study found that LED light irradiation at 10-20 J/cm2 promoted fibroblast viability and wound closure in vitro, and accelerated wound healing in vivo by promoting re-epithelialization, reducing inflammation and sequestrum formation, and increasing collagen deposition. LED light facilitated healing, likely by reducing reactive oxygen species and promoting angiogenesis and tissue remodeling, though additional research is still needed to validate the optimal LED conditions for clinical use.

2. Light-Emitting Diode Irradiation
Promotes Donor Site Wound Healing
of the Free Gingival Graft
Chen-Ying Wang et al
JOP 2015.
Shilpa Shivanand
III MDS

3. Introduction
• To restore mucogingival deformity, a variety of surgical
techniques, including free gingival graft (FGG), subepithelial
connective tissue (CT) graft, allogenic dermal matrix, tissue
engineering construct with allogenic cells, have been
developed to increase the keratinized gingiva.
Nabers JM 1966, Sallum AW 2006, McGuire MK et al 2008
• The FGG, one of the most classic approaches developed a
half-century ago, remains the most successful and predictable
technique.
Seol YJ et al 2002

4. • The major complication of FGG is the postoperative morbidity
associated with the open palatal wound, including acute pain,
excessive hemorrhage, and bone exposure.
Griffin TG et al 2006
• These complications are not resolved until the completion of
wound epithelialization, which usually takes 2 to 4 weeks.
• To accelerate the healing process and to reduce the incidence
of complications, chemotherapeutic agents, including
antibiotics and mouthrinse, hemostatic agents, ozonides and
low-intensity pulse ultrasound were introduced.
Kaigler D et al 2011, Rees TD et al 1999

5. LLLT
• Recently, lower-level laser therapy (LLLT) was used to
accelerate wound healing.
Conian MJ et al 1996
• LLLT is defined as the light source with an energy density <100
J/cm2; therefore, the temperature change can be neglected to
minimize tissue damage.
• By activating mitochondrial photo-acceptor molecule
cytochrome c oxidase, LLLT can enhance coagulation,
angiogenesis, collagen deposition, and fibroblastic
proliferation.
Hoffman M et al 2012, Lim WB et al 2011

6. • Recently, by irradiating with a 10-J/cm2 gallium aluminum
arsenide laser, the healing of palatal mucoperiosteal wounds
was promoted, which was presumably associated with
increasing the mitogenesis of fibroblasts.
Firat ET et al 2014
WHY LED… ?
• Compared to lasers, within similar range of wavelengths and
light energy, the light-emitting diode (LED) has demonstrated
comparable biologic effects and is considered an economic
alternative to lasers.
de Sousa AP et al 2013

7. MATERIALS AND METHODS
LED Device
• LED light irradiation was delivered by directly emitting 660 ±
25 nm and 3.5 mW/cm² visible red light on the culture plates
or gingival wound to achieve an energy density of 5, 10, or 20
J/cm2 (6, 12, or 24 minutes of irradiation) within the tested
region, as described previously.

8. In Vitro Study Design and Assessments
• GFs were harvested from the palatal mucosa of rats during
the preparation of the palatal wounds.
• GFs were isolated and cultured at a density of 4 × 103 cells
per well in 96-well plates for mitogenesis and cytotoxicity, and
they were given daily 0, 5, 10, or 20 J/cm2 LED light
irradiations for 24 and 72 hours.

9. • Cellular viability was evaluated using a cell proliferation assay.
• Cytotoxicity was evaluated using a cytotoxicity detection kit.
• Wound closure was evaluated using a scratch wound created
by a 200 mL pipette tip at the density of 1.2 *105 cells per
well in 12-well plates, and the images were acquired at 40x
using a digital image acquisition system.
• Specimens were evaluated 6 and 12 hours after 0, 5, 10, or 20
J/cm2 LED light irradiations were administered.

10. Animal Model
• A total of 72male rats (250 to 300 g) were used.
• In each rat, bilateral 5 * 1.5 mm2 palatal wounds parallel to
the gingival margin of maxillary molars with 1 mm distance
were created using a double-bladed scalpel (1.5 mm
interblade distance) with two number-15 surgical blades and
the LED light irradiation was started immediately after the
wound creation.

11. • Animals were divided equally into three groups: 1) the control
(Ctrl) group (no LED light irradiation); 2) the low-dose (LD) group
(daily 10 J/cm2 LED light irradiation); and 3) the high-dose (HD)
group (daily 20 J/cm2 LED light irradiation).
• Equal numbers of animals were euthanized after 4, 7, 14, and 28
days of LED treatment (n = six per group per time point), and
bilateral maxillae were harvested.
• Tissue within the palatal wound in one randomly selected side of
the maxillae was excised to evaluate the gene expression levels,
and the other side of the maxillae was fixed in 10%
formaldehyde for histologic examination.

12. Histologic Examinations
• After fixation, the maxillae were decalcified with 12.5% EDTA
and embedded in paraffin.
• Three sections (5 mm) from the cross-sectional plane at the
midwound area were selected.
• One was stained with hematoxylin and eosin, one was stained
with Masson’s trichrome for the assessment of collagen
deposition, and the other one was stained
immunohistochemically using a commercialized staining kit
with a polyclonal anti–tumor necrosis factor (TNF)-a antibody
(1:200 dilution).
• All of the images were acquired using a digital image
acquisition system.

13. • Wound healing was assessed by the fraction of inflammatory
cells/total cells, status of re-epithelialization and wound
closure, scar elevation index (SEI; an index to express the
degree of scar hypertrophy in the healing process), the
fraction of collagen deposition/lamina propria, and the
fraction of TNFa-positive area/lamina propria using an image
analysis software.

14. PCR Analysis
• The RNA harvested from the tissue was reverse transcribed to
cDNA using a cDNA synthesis kit and the mRNA levels of
genes were analyzed quantitatively with a PCR system and
sequence-specific gene expression assays for GAPDH
(housekeeping gene), heme oxygenase-1 (HO-1; marker of
dissociating reactive oxygen species [ROS]), receptor for
advanced glycation end products (RAGE), TNF-a, fibronectin
(extracellular matrix [ECM] protein for cell attachment),
periostin (matricellular protein essential for periodontal tissue
integrity), and Type I collagen (major ECM protein in the
periodontium).

15. Statistical Analyses
• The sample size was determined by the power analysis based
on the results from a previous study and the assumption of
80% power, a = 0.05, and normal distribution and equivalent
variance of the samples.
• Two-way ANOVA followed by post hoc tests were used to
compare the difference between the Ctrl and LED light–
irradiated groups at each time point.
• The data are presented as the mean ± SD of the
measurements, and P <0.05 was considered statistically
significant.

16. RESULTS
In Vitro Assessments
• Cellular viability was promoted significantly in the specimens
treated with 5 (P <0.001) and 20 (P <0.05) J/cm2 LED light at
72 hours.

17. • Cytotoxicity was generally reduced under LED irradiation, and
a significant difference compared to the non-irradiated Ctrl
was noted in 5 J/cm2 LED-irradiated specimens at 24 (P <0.05)
and 72 (P <0.01) hours.
Cytotoxicity by LDH assay. Data are
presented as the cytotoxicity relative
to the specimens without LED light
irradiation (set as 0) at the same time
point.

18. • However, wound closure was significantly promoted with 5
J/cm2 LED irradiation at 12 hours.
Cell migration by the distance cells
traveled from the edges of the wound
relative to the baseline. Compared to
non-LED-irradiated specimens at the
same time point

19. Descriptive Histology
• At day 4, the underlying alveolar bone was exposed and
partially covered by a necrotic tissue mass, aggregated
erythrocytes, and inflammatory cells in all groups.
• Epithelium approximation was noted in all groups at day 7,
and the wound mostly consisted of inflammatory cells and
primitive fiber matrix, and sequestrum was noted in most
specimens.

20. • The wound was nearly completely covered by the epithelium
without prominent rete pegs, and hypertrophy of the CT
matrix was noted in most specimens at day 14.

21. • Inflammatory cells and the degraded collagen matrix were
surrounded by a sequestrum.
• The sequestrum was engaged with downgrown epithelium in
four of six specimens in the Ctrl and HD groups, and the
sequestrum was apparently smaller in the HD group.

22. • The sequestrum was clear in all LED-irradiated specimens at
day 28, whereas a sequestrum still presented in four of six
specimens in the Ctrl group.

23. • Inflammation subsided in the lamina propria of most
specimens, and the extension of rete pegs was noted in the
Ctrl and LD groups.

24. Collagen Matrix Deposition and TNF-a
Infiltration
• The collagen fibril matrix deposition was initiated after 7 days
of wound creation in all groups and was gradually deposited
and aligned within the cervical part of lamina propria of the
Ctrl and HD groups at day 14.

25. • In the LD group, the lamina propria was nearly occupied with
aligned collagen fibril matrix at day 14, and the fibril bundles
were apparently more densely packed at day 28.

26. • TNF-a was widely distributed on the edge of the sequestrum
and the necrotic tissue mass in the Ctrl group at day 7,
whereas the TNF-a-positive area was relatively small in the LD
and HD groups.

27. • TNF-a was restricted to the edge of the sequestrum and
epithelial approximation in the Ctrl and HD groups at day 14.

28. • Without the presence of a sequestrum, TNF-a was scattered
and lightly distributed in the collagen matrix in the LD group
from day 14 and in the HD group from day 28.
• However, TNF-a infiltration was still evident on the edge of
the sequestrum in the Ctrl group at day 28.

29. Quantitative Histologic Assessments
• The wound closure and reepithelialization were apparently
accelerated in both the HD and LD groups relative to the Ctrl
group at day 7, and the difference was less among groups at
the later stages.

30. • The SEI was increased in the LED light irradiated groups at day
7, and a significant difference compared to the Ctrl group was
noted in the HD group (P <0.05).

31. • The lamina propria was hypertrophic at day 14, but it
recessed at day 28, without an obvious difference among
groups.
• Inflammatory cell infiltration was gradually reduced in the Ctrl
group from days 7 to 28, and the level was consistently
significantly lower in the LD group.

32. • Collagen deposition was increased dramatically at day 14 in all
groups. The deposition was consistently higher in the LD and
HD groups at days 14 and 28, and the LD group was
significantly different compared to the Ctrl group at both time
points (P <0.01).

33. • The TNF-a-positive area was gradually reduced in all groups
from days 7 to 28 and consistently smaller in the LD and HD
groups, and a significantly smaller TNF-a-positive area relative
to the Ctrl group was noted in the LD group at days 7 and 14
(P <0.05).

34. Gene Expression Profiles
• The expression profiles of VEGF, periostin, fibronectin, and
type I collagen were generally similar.
• At day 4, there was slightly but insignificantly increased
expression of these genes compared to the Ctrl group in the
HD group.
• However, at day 7, significant upregulation of these genes was
noted in the LD and HD groups (P <0.001), whereas the levels
of periostin and Type I collagen were significantly higher in
the HD group compared to those of the LD group (P <0.001).
On days 14 and 28, there were no significant differences in
gene expression among the groups.

35. DISCUSSION
• It is generally accepted that LLLT facilitates wound healing by
altering the cellular behavior of fibroblasts and keratinocytes
and enhancing collagen synthesis, angiogenesis, and growth
factor release and the effect of LLLT irradiation is dose
dependent.
Walsh LJ 2003, Medrado AP et al 2008, Aleksic V et al 2010
• As an alternative source of LLLT, the results from the present
study show that the proliferation of the GFs was significantly
promoted, accompanied by significantly inhibited cytotoxicity
and significantly accelerated in vitro wound closure with 5
J/cm2 LED light irradiation.

36. • With the implementation of 10 or 20 J/cm2 LED light on the
palatal wound, wound closure and reepithelialization were
accelerated, and there was apparent, rapid recovery from scar
hypertrophy, reduced inflammatory cell infiltration and
sequestrum, and collagen deposition promotion.
• These findings are in agreement with previous studies and
support LED light as favorable in facilitating palatal wound
healing.
Sacono NT et al 2008, Chang PC et al 2013

37. • The reduction of sequestrum formation appeared to be
parallel to the reduction of inflammatory cell infiltration and
TNF-a infiltration.
• However, the expression of TNF-a was temporaril upregulated
in the HD group.
• It is noteworthy that LED light may also activate immune cell
activity such that the upregulation of inflammation-associated
genes in the wound might not be preventable.
Redlich K et al 2012

38. • It appeared that the biomodulation of LED light irradiation
was attenuate after 14 days, whereby no significant difference
between the LED and non-LED light irradiated groups was
noted in any histologic parameter or examined gene.
• Furthermore, the healing capability of animals might not be
relevant to humans, and the rationalization of the LED light
condition for human use is still controversial.
• Future studies to justify the indications and conditions of LED
light for human use are still needed.

39. CONCLUSIONS
• Notwithstanding the limitations of the study, it is concluded
that 660 nm LED light irradiation, specifically within 10 to 20
J/cm2 energy density, facilitates the healing of opened palatal
wounds, potentially via reducing ROS production and
angiogenesis and by promoting provisional matrix and tissue
reorganization in vivo.
• Additional justifications of the LED light and experimental
models, as well as the investigation of the inflammation
profiles are warranted.

40. CROSS REFERENCE

41. I. Laser and LED phototherapies on angiogenesis.
de Sousa AP, Paraguassu´ GM, Silveira NT, et al
Lasers Med Sci 2013;28:981-987.
ABSTRACT:
• Angiogenesis is a key process for wound healing.
• There are few reports of LED phototherapy on angiogenesis,
mainly in vivo.
• The aim of the present investigation was to evaluate histologically
the angiogenesis on dorsal cutaneous wounds treated with laser
(660 and 790 nm) or LEDs (700, 530, and 460 nm) in a rodent
model.

42. • 24 young adult male Wistar rats weighting between 200 and
250 g were used on the present study.
• Under general anesthesia, one excisional wound was created
on the dorsum of each animal that were then randomly
distributed into six groups with four animals each: G0control;
G1laser λ660 nm (60 mW, ϕ ∼2 mm, 10 J/cm(2)); G2laser
λ790 nm (50 mW, ϕ ∼2 mm, 10 J/cm(2)); G3LED λ700 ± 20 nm
(15 mW, ϕ ∼16 mm, 10 J/cm(2)); G4LED λ530 ± 20 nm (8 mW,
ϕ ∼16 mm, 10 J/cm(2)); G5LED λ460 ± 20 nm (22 mW, ϕ ∼16
mm, 10 J/cm(2)).

43. • Irradiation started immediately after surgery and was repeated
every other day for 7 days. Animal death occurred at the eighth
day after surgery.
• The specimens were removed, routinely processed to wax, cut
and stained with HE. Angiogenesis was scored by blood vessel
counting in the wounded area. Quantitative results showed that
green LED (λ530 ± 20 nm), red LED (λ700 ± 20 nm), λ790 nm laser
and λ660 nm laser caused significant increased angiogenesis
when compared to the control group. It is concluded that both
laser and LED light are capable of stimulating angiogenesis in vivo
on cutaneous wounds and that coherence was not decisive on the
outcome of the treatment.

44. II. Irradiation by light emitting diode light as an adjunct to
facilitate healing of experimental periodontitis in vivo.
Chang PC, Chien LY, Ye Y, Kao MJ .
J Periodontal Res 2013;48:135-143.
• AIM:
• This study evaluated the biostimulatory effect of 660 nm
lightemitting diode (LED) as an adjunct in the treatment of
experimental periodontitis.
• METHODOLOGY:
• 96 rats underwent experimental periodontitis by placement
of a silk ligature followed with or without additive P.gingivalis
lipopolysaccharide (PgLPS) injection.

45. • Irradiation with LED light was performed at varying energy
densities of 5, 10 and 15 J/cm2, 1 d after debridement and
detoxification.
• Rats were killed at 3, 7 and 14 d after irradiation with LED
light, and the effect of irradiation was evaluated by
descriptive histology and quantitative measurements of
periodontal bone loss, inflammatory infiltration and cellular
proliferation.

46. • RESULTS:
• Reduction of inflammation, accelerated collagen deposition
and realignment was noted following irradiation with LED
light at densities of 10 and 15 J/cm2, and temporary reduction
of periodontal bone loss, as well as bundle bone apposition,
was noted at day 3 in rats treated with 10 J/cm2 light.
• The biomodulatory effect was stronger in sites treated with
PgLPS injection. In sites without PgLPS injection, temporary
reduction of inflammation was noted in all LED lightirradiated
specimens at day 3. No significant change in cellular
proliferation was noted in any LED lighttreated group.

47. • CONCLUSION:
• LED light (660 nm) with an energy density of 10 J/cm2
appeared suitable as an adjunct modality for periodontitis by
temporarily reducing inflammation, facilitating collagen
realignment and bundle bone deposition.
• Future studies will aim to amplify the biostimulatory effect of
LED light by adding a supplementary medium or repeated
irradiation.