Light-Emitting Diode IrradiationPromotes Donor Site Wound

Healingof the Free Gingival Graft

Chen-Ying Wang et al

JOP 2015.

Shilpa ShivanandIII MDS

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

• 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

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

• 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 2014WHY 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

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.

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.

• 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.

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.

• 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.

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.

• 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.

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).

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.

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.

• 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.

• 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 thesame time point

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.

• 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.

• 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.

• 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.

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

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.

• 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.

• 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.

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

• 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.

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.

• 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).

• 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.

• 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).

• 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).

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.

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.

• 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

• 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

• 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.

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.

CROSS REFERENCE

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.

• 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)).

• 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.

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.

• 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.

• 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.

• 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.