Table 1.

Summary of in vitro and in vivo studies done on various cell types and animal models, respectively, using low level laser irradiation (LILI).

Species/cell type	Study design	Outcomes	Author reference
In vitro studies
Diabetic wounded human skin fibroblasts	Cells were irradiated at 660 nm with 5 J/cm2 and incubated for 48 or 72 h. Control cells received no laser irradiation.	Irradiation resulted in increased cellular migration, viability, proliferation, and collagen production.	Ayuk et al. [45]
Diabetic wounded and hypoxic human skin fibroblast cells (WS1)	Cells were irradiated at 636 nm with 5 J/cm2 and incubated for 1 or 24 h. Control cells received no laser irradiation.	Irradiated diabetic wounded cells showed increased cellular migration, viability, and proliferation and a decrease in apoptosis (caspase 3/7) and proinflammatory cytokine interleukin (IL)-1β. Nuclear factor kappa B (NF-κB) also translocated into the nucleus. Irradiated hypoxic cells regained their normal morphology and showed an increase in cellular viability, proliferation, and IL-6 and decreased apoptosis (caspase 3/7) and proinflammatory cytokine tumor necrosis factor (TNF)-α. NF-κB also translocated into the nucleus.	Sekhejane et al. [46]
Human skin fibroblasts (HSFs)	Cells were cultured in physiologic glucose (5.5 mM/L) or high glucose concentration (11.1 and 15 mM/L) and irradiated at 632.8 nm with 0.5, 1, and 2 J/cm2 on 3 consecutive days.	Densities of 0.5 and 1 J/cm2 had stimulatory effects on the viability and proliferation rate of HSFs cultured in physiologic glucose. Densities of 0.5, 1, and 2 J/cm2 had stimulatory effects on the proliferation rate of HSFs cultured in high glucose concentrations.	Esmaeelinejad et al. [44]
Diabetic wounded skin fibroblast cells (WS1)	Cells were irradiated at 632.8 nm with 5 or 16 J/cm2. Control cells received no laser irradiation.	Cells irradiated at 5 J/cm2 showed increased cellular migration and proliferation, while cells irradiated at 16 J/cm2 showed decreased cellular migration and proliferation.	Houreld and Abrahamse [53]
NIH 3T3 fibroblast cells	For proliferation studies, cells were grown in 2.5% foetal bovine serum (FBS) and irradiated at 904 nm. Cells received two applications (6 h interval) of 2 J/cm2 each (4 J/cm2 total); 1 J/cm2 and then 2 J/cm2 (3 J/cm2 total); 2 J/cm2 and then 3 J/cm2 (5 J/cm2 total). Control cells received no laser irradiation. Cells were incubated for 2, 4, 5, and 6 days. For procollagen studies, cells were grown in 2.5% FBS and irradiated at 904 nm, 3 J/cm2 and incubated for 4 days.	Cells irradiated with 3 and 4 J/cm2 showed increased cellular proliferation. No significant increase in procollagen was seen in any of the irradiated cells.	Pereira et al. [54]
Murine fibroblast 3T3 cells and primary human keloid fibroblast cell cultures	Cells were irradiated at 660 nm for 3 consecutive days (24, 48, and 72 h) with 3 or 21 J. For the MTT assay (proliferation), a power density of 0.26 W/cm2 was used, while 0.63 W/cm2 was used for viability assays (Trypan blue).	A dose of 3 J stimulated proliferation, while 21 J inhibited proliferation of human keloid fibroblast cells. Laser irradiation is affected by the physiological state of the cells; high-metabolic rate and short-cell-cycle 3T3 cells were not responsive to LILI. A dose of 3 J reduced cell death but did not stimulate cell cycle. A dose of 21 J had negative effects on the cells, as it increased cell death and inhibited cell proliferation.	Frigo et al. [55]
HIG-82 rabbit synovial fibroblasts	Cells were synchronized at G1 by serum starvation (0.2% FBS for 24 h) and irradiated at 660 nm with 1.2, 4.8, or 7.2 J/cm2 and cultured for another 24 h. Control cells received no laser irradiation.	Cellular proliferation was significantly stimulated at 4.8 and 7.2 J/cm2, while no effect was observed at 1.2 J/cm2. The proportion of cells at S phase in the laser irradiation group (4.8 J/cm2) was significantly higher; thus LILI enhances cell cycle progression and as it promotes synovial fibroblast proliferation.	Taniguchi et al. [56]
Porcine primary aortic smooth muscle cells (SMCs)	Cells were irradiated at 780 nm with 1 or 2 J/cm2. Cells were incubated for different time periods depending on the assay.	LILI stimulated porcine aortic SMC proliferation, increased collagen synthesis, modulated activity and expression of matrix metalloproteinase (MMP)-2, gene expression of MMP-1, and tissue inhibitor of metalloproteinases (TIMP)-2, and inhibited gene expression of proinflammatory cytokine IL-1β.	Gavish et al. [49]
Primary human gingival fibroblasts (GF)	Cells were irradiated at 780 nm with different settings used in dentistry: power: 2 W, pulse interval: 1 ms, pulse length: 1 ms, 20 s/cm2, 20 J/cm2 (infected pocket setting); power: 1.5 W, pulse interval: 20 ms, pulse length: 20 ms, 20 s/cm2, 15 J/cm2 (Perio pocket setting); power: 0.3 W in continuous wave, 20 s/cm2, 6 J/cm2 (biostimulation setting).	No significant difference in proliferation was observed in the different laser applications when compared to the control group. Significantly increased insulin-like growth factor (IGF) and vascular endothelial growth factor (VEGF) mRNA was observed in all irradiated groups. A significant increase in collagen type I mRNA expression was noted in only the biostimulation setting.	Hakki and Bozkurt [57]
Human foreskin fibroblast HS68 cells	Cells were grown in 1% FBS for 24 h and then irradiated with a light emitting diode (LED) array (630 nm) with 1 or 2 J. Cells were incubated for 1 or 3 days. Control cells received no laser irradiation.	A dose of 1 J induced a significant increase in viability. Irradiation increased the mRNA expression level of type I collagen and also affected basic fibroblast growth factor (bFGF) secretion levels.	Huang et al. [58]
Human dermal fibroblasts	LED array populated with 590 and 870 nm LEDs. The ratios of visible to infrared (IR) light were decreased (in the case of visible) and increased (in the case of IR) in series of 25% increments from no IR to fully IR. Cells were incubated for 24 h.	Photomodulation with a 590/870 nm LED array in different ratios has an effect on gene expression profiles and is effective for altering gene expression, collagen synthesis, and reduction of MMP-1 expression.	McDaniel et al. [59]
Human gingival fibroblasts, FMM1 cells	Cells were irradiated at 904 nm with 3 J/cm2 and incubated for 3 days. Control cells received no laser irradiation.	Irradiation produced no difference in the amount of procollagen between groups, and the amount of type I collagen as well as the total protein content was significantly smaller in control cultures. There were also ultrastructural changes in cytoplasmic organelles, especially the mitochondria and rough endoplasmic reticulum.	Marques et al. [66]
Diabetic and ischemic skin fibroblast cells (WS1)	Whole cells or isolated mitochondria were irradiated at 660 nm with 5 or 15 J/cm2. Control cells received no laser irradiation.	Irradiation of mitochondria with 15 J/cm2 resulted in increased adenosine triphosphate (ATP) production, a higher accumulation of activated mitochondria in diabetic cells, an increase in complex IV activity, and a decrease in complex III activity. There was an increase in complex IV activity in mitochondria and a higher accumulation of activated mitochondria in diabetic cells irradiated with 5 J/cm2. Irradiated ischemic cells showed no significant differences compared to their nonirradiated control.	Houreld et al. [31]
Diabetic wounded skin fibroblast cells (WS1)	Cells were irradiated at 830 nm with 5 J/cm2. Control cells received no laser irradiation.Cells were incubated for 15 min, 1, 24, or 48 h.	Irradiation resulted in increased cellular proliferation (24 and 48 h), nitric oxide (15 min), and reactive oxygen species (15 min) and decreased apoptosis (24 h), TNF-α (1 and 24 h), and IL-1β (24 h).	Houreld et al. [60]
Primary human gingival fibroblasts (hGF)	Cells irradiated at 685 nm with 2 J/cm2 and incubated for 24 h. Two study groups, namely, cells which were irradiated once (single-dose group) and cells which were irradiated twice with 24 h interval (double dose). Control cells received no laser irradiation.	Cells in the single-dose group showed a significant increase in proliferation and growth factors bFGF and IGF-1, with no change in IGF-binding protein (IGFBP)3. Cells in the double dose group showed a significant increase in proliferation and growth factors bFGF, IGF-1, and IGFBP3.	Saygun et al. [61]
Human gingival fibroblast cell line (FGH)	Cells were grown in 1% FBS for 24 h and then irradiated in media containing 10% FBS. Cells irradiated twice at 660 or 780 nm with 3 or 5 J/cm2 with 6 h between irradiations.	There was no significant difference in the expression of keratinocyte growth factor (KGF), while bFGF was significantly increased in cells irradiated at 660 nm (no difference at 780 nm).	Damante et al. [62]
Wounded, diabetic wounded, and ischemic skin fibroblast cells (WS1)	Cells were irradiated at 660 nm with 5 J/cm2. Control cells received no laser irradiation. Cells were incubated for 30 min.	Irradiation upregulated the expression of mitochondrial genes COX6B2 (complex IV), COX6C (complex IV), and PPA1 (complex V) in diabetic wounded cells and ATP4B (complex V) and ATP5G2 (complex V) in ischemic cells. COX6C (complex IV), ATP5F1 (complex V), NDUFA11 (complex I), and NDUFS7 (complex I) were upregulated in wounded cells.	Masha et al. [32]
Isolated mouse embryonic fibroblasts	Cells irradiated at 810 nm with 0.003, 0.03, 0.3, 3, or 30 J/cm2. Control cells received no laser irradiation.	A dose of 0.3, 3, and 30 J/cm2 produced an increase in reactive oxygen species (ROS). No increase in ATP was seen with 0.003 J/cm2, a small increase was seen at 0.03 J/cm2 and a large increase was seen with fluencies of 0.3, 3, and 30 J/cm2. A dose of 0.3 J/cm2 increased NF-κB 1 h after irradiation. Activation of NF-κB is mediated via ROS generation.	Chen et al. [63]
In vivo studies
Rat, Sprague-Dawley, diabetic (streptozotocin induced), and nondiabetic	Full-thickness wound (102.5 ± 9 mm2) or a burn 1 (48 ± 12.5 mm2) was made on each rat. Rats were irradiated with various wavelengths (532, 633, 810, 980, and 10,600 nm) and polychromatic LED clusters (510–543, 594–599, 626–639, 640–670, and 842–872 nm) with a dose of 5, 10, 20, or 30 J/cm2 three times per week.	The best effects on wound and burn healing were exhibited with a laser with a wavelength of 633 nm. Based on the results, phototherapy at 633 nm, 4.71 J/cm2, 3 times/week is recommended for diabetic burn wounds, and phototherapy at 633 nm, 2.35 J/cm2, 3 times/week for diabetic wounds is recommended for human clinical trials.	Al-Watban [74]
Mice, diabetic (BKS.Cg-m+/+Lepr  db/J), male and female	A full-thickness circular wound was made on the left flank in each mouse using a sterile 5 mm diameter skin punch, and the wound extended down to the fascial layer over the abdominal musculature. Wounds were irradiated at 660 nm, with 0, 0.8, 1.6, or 3.2 J/day. Mice were euthanized on day 14.	Irradiation of splintered wounds at 660 nm with 1.6 J/day (3.7–5.0 J/cm2/day) for 7 days was shown to cause the maximal stimulation of healing on day 14. Wounds healed mainly by reepithelization and granulation tissue formation.	Chung et al. [75]
Rats, Wistar, diabetic (streptozotocin induced), and male	A 2 × 8 cm cutaneous flap was raised on the dorsum of each animal. A plastic sheet was introduced between the flap and the bed to impair blood supply, and the flap was then sutured. Rats were treated transcutaneously every other day with 680 or 790 nm, on 16 contact points at the wound margin (2.5 J/cm2/point; total of 40 J/cm2). Rats were euthanized on day 8.	The results suggest that the best responses of the flaps were observed on irradiated subjects, in particular those treated with 790 nm. There was increased angiogenesis, reduced tissue necrosis and inflammation, and increased fibroblastic proliferation.	Santos et al. [76]
Rats, Wistar, diabetic (streptozotocin induced), and male	Rats were divided into 4 groups: control (untreated, nondiabetic); laser (laser treated, nondiabetic); diabetic (diabetic rats, nonlaser treated); and diabetic + laser (diabetic rats laser treated). Scars were irradiated once at 660 nm with 4 J/cm2, and rats were euthanized 24 h after irradiation.	In untreated diabetic rats there was increased MMP-2 and MMP-9 expression compared to untreated nondiabetic rats. Irradiation of diabetic rats significantly reduced MMP-2 and MMP-9 expression compared to untreated diabetic rats, and there was also increased production of collagen.	Aparecida et al. [77]
Rats, Wistar, diabetic (streptozotocin induced), male	Full-thickness wounds were made in the hard palates using a 3 mm biopsy punch. Rats were divided into 2 groups: control group (nonirradiated) and experimental group (irradiated). Wounds were irradiated at 940 nm with 10 J/cm2 after surgery and on days 2, 4, and 6 after surgery. Rats were euthanized on days 7, 14, and 21. Irradiation resulted in decreased numbers of inflammatory cells and increased mitotic activity of fibroblasts, collagen synthesis, and vascularization. Oxidative status was also significantly decreased on day 21.	Decreased inflammatory cells, and oxidative stress and increased collagen and vascularization	Firat et al. [78]
Rat, Sprague-Dawley, normal or diabetic (streptozotocin induced), male	Left and right maxillary first molars were extracted, and extraction sockets on the left were not irradiated, while the right ones were irradiated at 980 nm with 13.95 J/cm2. Rats were euthanized 3, 5, 7, or 14 days after extraction.	Irradiation promoted new bone formation. In normal rats, osteoblasts and osteoid tissue were observed at day 5, which was earlier than in the control group, and new bone reached the top of the extraction socket at day 14. In diabetic irradiated rats, less infiltration of inflammatory cells and blood clots were observed at day 3, and more new bone formed at days 7 and 14 than in the nonirradiated diabetic group. Laser irradiation stimulated the differentiation of osteoblasts and increased the expression of collagen type I and osteocalcin mRNA.	Park and Kang [79]
Rats, Wistar, diabetic (streptozotocin induced) male	Rats were divided into 7 groups: control (normoglycemic, no injury), diabetic (no injury), sham (Normoglycemic, sham irradiated), diabetic sham, nondiabetic cryoinjured submitted to LLLT, diabetic cryoinjured submitted to LLLT, and diabetic cryoinjured nontreated. Cryoinjury was carried out on the left posterior leg: the muscle fascia was carefully removed, and the tibialis anterior muscle was surgically exposed and cryoinjured for 10 s with a cooled (in liquid nitrogen) round 3 mm metal probe. After the frozen muscle had thawed, the procedure was repeated on the same area for another 10 s. Surgical wounds were closed with sutures and rats were allowed recovering. Two hours after injury, the muscle was irradiated at 780 nm with 5 J/cm2 to 8 points within the area (energy per point was 0.2 J, totalizing 1.6 J per treatment). Irradiations were performed daily (24 h interval). Rats that were euthanized on day 7 received 6 treatments, while rats euthanized on day 14 received 13 treatments.	Diabetic animals that received LLLT exhibited morphological aspects of skeletal muscle healing similar to those found in the normoglycemic animals having received LLLT, with the organization of immature fibers in the collagen meshwork. The diabetic sham irradiated group exhibited fibrosis. Thus, LLLT can help avoid fibrosis and reduce muscle atrophy	França et al. [80]
Double-blind, randomized, placebo-controlled study. Twenty patients with 32 chronic lower extremity venous ulcers	Inclusion criteria included the following: ulcer in the lower extremity, (2) ulcers larger than 1.0 cm2, (3) ulcer duration >6 wk, (4) presence of classical signs of venous insufficiency such as edema, varicosities, lipodermatosclerosis, eczema, and elephantiasis nostra, (1) and (5) controlled systemic arterial hypertension (diastolic arterial pressure <95 mm Hg). Each group of ulcers was treated 2x/week. Ulcers were covered with 1% silver sulfadiazine (SDZ) cream, dressed, and then bandaged. Group 1 received placebo phototherapy; group 2 were irradiated at 660 and 890 nm (LEDs) 30 s per point until the entire ulcer surface was treated with the probe; and the control group 3 received standard care without phototherapy. Ulcers were treated for a maximum of 90 days.	Laser therapy increased wound healing. At all time points, light treated ulcers healed faster than the control group treated with SDZ cream dressing alone, as well as the placebo treatment group.	Caetano et al. [81]
Double-blind, randomized placebo-controlled, experimental design, 14 patients with 23 chronic diabetic leg ulcers	Inclusion criteria: (1) diagnosis of type II diabetes independent of glycemic control with neuropathic or mixed (venous and arterial) ulcers, (2) ulcer located on the lower extremity, (3) ulcer present for a minimum of 4 weeks during which it has been either stable or worsening, (4) willingness to participate in the study and commitment to the follow-up protocol, and (5) signed written consent. Ulcers were cleaned with 0.9% physiological saline and dried before phototherapy was applied twice per week for a maximum of 90 days. Ulcers were dressed with 1% silver sulfadiazine cream covered with gauze and bandaged. Ulcers in the irradiated group were treated with 660 and 890 nm probes (LEDs) 30 s per point until the entire ulcer surface was treated.	Laser irradiation using a combination of 660 and 890 nm promoted tissue granulation and rapid healing of diabetic ulcers that failed to respond to other forms of treatment.	Minatel et al. [82]
Double-blind, randomized controlled clinical trial, 23 patients with chronic diabetic ulcers	Patients having a diabetic foot ulcer for a minimum of 12 weeks with ulcer stages I and II who were capable of giving informed consent, understanding instructions, and cooperating with study protocol were enrolled. Patients were divided into laser treated and conventional therapy or conventional therapy alone (placebo group). Ulcers were treated 6x/week for two successive weeks and then every other day up to complete healing. Ulcers were treated with a 685 nm laser at a dose of 10 J/cm2. Patients in the placebo treatment group received sham irradiation.	Laser irradiation increased wound healing. Four weeks after beginning treatment, the size of ulcers was significantly decreased and by 20 weeks a greater number of patients in the irradiated group showed complete healing than in the placebo group, and the mean time of healing was lower.	Kaviani et al. [83]