Low-level laser (light) therapy (LLLT) has been known since 1967 but still remains controversial due to incomplete understanding of the basic mechanisms and the selection of inappropriate dosimetric parameters that led to negative studies. The biphasic dose-response or Arndt-Schulz curve in LLLT has been shown both in vitro studies and in animal experiments. This review will provide an update to our previous (Huang et al. 2009) coverage of this topic. In vitro mediators of LLLT such as adenosine triphosphate (ATP) and mitochondrial membrane potential show biphasic patterns, while others such as mitochondrial reactive oxygen species show a triphasic dose-response with two distinct peaks. The Janus nature of reactive oxygen species (ROS) that may act as a beneficial signaling molecule at low concentrations and a harmful cytotoxic agent at high concentrations, may partly explain the observed responses in vivo. Transcranial LLLT for traumatic brain injury (TBI) in mice shows a distinct biphasic pattern with peaks in beneficial neurological effects observed when the number of treatments is varied, and when the energy density of an individual treatment is varied. Further understanding of the extent to which biphasic dose responses apply in LLLT will be necessary to optimize clinical treatments.
Keywords: low level laser therapy, photobiomodulation, biphasic dose response, reactive oxygen species, nitric oxide, traumatic brain injury
Low level laser (light) therapy (LLLT) employs visible (generally red) or near-infrared light generated from a laser or light emitting diode (LED) system to treat diverse injuries or pathologies in humans or animals. The light is typically of narrow spectral width between 600nm – 1000nm. The fluence (energy density) used is generally between 1 and 20 J/cm2 while the irradiance (power density) can vary widely depending on the actual light source and spot size; values from 5 to 50 mW/cm2 are common for stimulation and healing, while much higher irradiances (up to W/cm2) can be used for nerve inhibition and pain relief. LLLT is typically used to promote tissue regeneration, reduce swelling and inflammation and relieve pain and is often applied to the injury for 30 seconds to a few minutes or so, a few times a week for several weeks. Unlike other medical laser procedures, LLLT is not an ablative or thermal mechanism, but rather a photochemical effect comparable to photosynthesis in plants whereby the light is absorbed and exerts a chemical change.
Within a decade of the introduction of LLLT in the 1970s it was realized that more does not necessarily mean better. The demonstration of the biphasic dose response curve in LLLT has been hampered by disagreement about exactly what constitutes a “dose”. Many practitioners concentrate on fluence as the principle metric of dose, while others prefer irradiance or illumination time. The use of very small spot sizes by some practitioners has led to the assertion that they delivered hundreds of mW/cm2 from a 50 mW laser. While this statement is mathematically correct it can give the impression that much higher doses of light were given than actually were delivered.
Two years ago we reviewed (Huang et al. 2009) the biphasic dose response in LLLT and found many reports in the literature concerning biphasic dose responses observed in cell cultures, some in animal experiments but no clinical reports. We now believe that the time is right to revisit this interesting topic for two reasons. Firstly because we have found more instances in our laboratory both in vitro with cultured cortical neurons, and in vivo with LLLT of traumatic brain injuries in mouse models. Secondly because advances have been made in mechanistic understanding of how LLLT works at a cellular level that may explain why a little light may be beneficial and at the same time a lot of light might be harmful.
According to the First Law of Photochemistry, the photons of light must be absorbed by some molecular photoacceptors or chromophores for photochemistry to occur (Sutherland 2002).The mechanism of LLLT at the cellular level has been attributed to the absorption of monochromatic visible and near infrared (NIR) radiation by components of the cellular respiratory chain (Karu 1989). Phototherapy is characterized by its ability to induce photobiological processes in cells. The effective tissue penetration of light and the specific wavelength of light absorbed by photoacceptors are two of the major parameters to be considered in light therapy. In tissue there is an “optical window” that runs approximately from 650 nm to 1200 nm where the effective tissue penetration of light is maximized. Therefore the use of LLLT in animals and patients almost exclusively involves red and near-infrared light (600–1100-nm) (Karu and Afanas’eva 1995). The action spectrum (a plot of biological effect against wavelength) shows which specific wavelengths of light are most effectively used for biological endpoints as well as for further investigations into cellular mechanisms of phototherapy (Karu and Kolyakov 2005). Fluence (J/cm2) is often referred to as “dose”, though many authors and practitioners of LLLT also refer to energy (Joules) as dose. Not only is this confusing to the novice student of LLLT but it also assumes that the product of power and time (and more importantly power density and time) is the goal rather than the right combination of individual values. This lack of reciprocity has been shown many times before and since our first paper on biphasic dose response and several more authors have reported finding these effects since. Examples of recently published “dose-rate” effects are also reviewed later in this article.
Mitochondria play an important role in energy generation and metabolism and are involved in current research about the mechanism of LLLT effects. The absorption of monochromatic visible and NIR radiation by components of the cellular respiratory chain has been considered as the primary mechanism of LLLT at the cellular level (Karu 1989). Cytochrome c oxidase (Cco) is proposed to be the primary photoacceptor for the red-NIR light range in mammalian cells. Absorption spectra obtained for biological responses to light were found to be very similar to the absorption spectra of Cco in different oxidation states (Karu and Kolyakov 2005).LLLT on isolated mitochondria increased proton electrochemical potential, ATP synthesis (Passarella et al. 1984), increased RNA and protein synthesis (Greco et al. 1989) and increases in oxygen consumption, mitochondrial membrane potential, and enhanced synthesis of NADH and ATP.
Mitochondria are an important source of reactive oxygen species (ROS) within most mammalian cells. Mitochondrial ROS may act as a modulatable redox signal, reversibly affecting the activity of a range of functions in the mitochondria, cytosol and nucleus. ROS are very small molecules that include oxygen ions such as superoxide, free radicals such as hydroxyl radical, hydrogen peroxide, and organic peroxides. ROS are highly reactive with biological molecules such as proteins, nucleic acids and unsaturated lipids. ROS are also involved in the signaling pathways from mitochondria to nuclei. It is thought that cells have ROS or redox sensors whose function is to detect potentially harmful levels of ROS that may cause cell damage, and then induce expression of anti-oxidant defenses such as superoxide dismutase and catalase.
LLLT was reported to produce a shift in overall cell redox potential in the direction of greater oxidation (Karu 1999) and increased ROS generation and cell redox activity have been demonstrated (Lubart et al. 2005). These cytosolic responses may in turn induce transcriptional changes. Several transcription factors are regulated by changes in cellular redox state, but the most important one is nuclear factor κB (NF-κB). Figure 1 graphically illustrates some of the intracellular signaling pathways that are proposed to occur after LLLT.
Schematic depiction of the cellular signaling pathways triggered by LLLT. After photons are absorbed by chromophores in the mitochondria, respiration and ATP is increased but in addition signaling molecules such as reactive oxygen species (ROS) and nitric oxide (NO) are also produced.
There have been reports of the production and/or release of NO from cells after in vitro LLLT. It is possible that the delivery of low fluences of red/NIR light produces a small amount of NO from mitochondria by dissociation from intracellular stores (Shiva and Gladwin 2009), such as nitrosothiols (Borutaite et al. 2000), NO bound to hemoglobin or myoglobin (Lohr et al. 2009; Zhang et al. 2009) or by dissociation of NO from Cco (Lane 2006) as depicted in Figure 2. A second mechanism for NO production is by light-mediated increase of the nitrite reductase activity of cytochrome c oxidase (Lane 2006). A third possibility is that light can cause increase of the activity of an isoform of nitric oxide synthase (Poyton and Ball 2011), possibly by increasing intracellular calcium levels. This low concentration of NO produced by illumination is proposed to be beneficial through cell-signaling pathways (Ball et al. 2011).
One possible theory that can explain the simultaneous increase in respiration an production of nitric oxide is the photodissociation of bound NO that is inhibiting cytochrome c oxidase by displacing oxygen.
Many reports of biphasic dose responses in LLLT were reviewed in our previous contribution and for convenience we have assembled these reports into Tables. Table 1 lists reports on cultured cells in vitro, Table 2 lists those reports in animal models in vivo, while Table 3 contains the only report of biphasic dose response in clinical studies.
Biphasic dose response studies of LLLT in vitro.
|1978||Lymphocytes in vitro||“threshold phenomenon”||Mester et al. 1978|
|1990||Macrophage cell lines (U-937)||820nm Laser; 120mW/cm2; 2.4J/ cm2 to 9.6J/cm2||Cell proliferation: Maximum at 7.2J/cm2 least at 9.6J/cm2||Bolton et al. 1990|
|1991||Macrophage cell lines (U-937)||820nm Laser; 2.4J/cm2 or 7.2J/cm2; 400mW/ cm2 or 800mW/ cm2||cell proliferation increased at 400mW/ cm2; Cell viability reduced at 800mW/cm2||Bolton et al. 1991|
|1994||Human oral mucosal fibroblast cells||812nm laser; 4.5mW/cm2;||Cell proliferation peak at 0.45 J/cm2; less at 1.422J/cm2||Loevschall and Arenholt-Bindslev 1994|
|2001||Chinese hamster ovary and human fibroblast cells||He-Ne laser;1.25 mW/cm2; 0.06 to 0.6J/cm2||Cell proliferation peak at 0.18 J/cm2; less at 0.6J/cm2.||al-Watban and Andres 2001|
|2003||human fibroblast cells||628nm LED; 11.46 mW/cm2; 0, 0.44, 0.88, 2.00, 4.40, and 8.68 J /cm2||Cell proliferation maximum at 0.88 J/cm2; reduced at 8.68 J/cm2||Zhang et al. 2003|
|2005||Human HEP-2 and murine L-929 cell lines||670 nm LED; 5 J/cm2 per treatment; Total 50J/cm2/day; 1 to 4 treatments/day||Cell proliferation bigger at 2 treatments/day||Brondon et al. 2005|
|2005||Hela cells||wavelength range of 580–860 nm||DNA synthesis rate maximum at 0.1 J/cm2 with 0.8 mW/cm2||Karu and Kolyakov 2005|
|2005||Wounded fibroblasts||632.8nm laser; 2mW/cm2; 0.5, 2.5, 5.0 or 10.0 J/cm2||Cell proliferation maximum at a single dose of 2.5J/cm2; Cellular damage at 10J/cm2||Hawkins and Abrahamse 2005|
|2006||Wounded fibroblasts||632.8nm laser; 5.0 J/ cm2 or 16J/ cm2||Cell proliferation and cell viability increased at 5 J/cm2; decreased at 10 and 16 J/cm2||Hawkins and Abrahamse 2006a|
|2006||Wounded fibroblasts||632.8nm laser; 5.0 J/cm2 or 16J/cm2||Cell migration and proliferation increased at a single dose of 5.0 J/cm2 and two or three doses of 2.5 J/cm2; inhibited at 16 J/cm2||Hawkins and Abrahamse 2006b|
|2007||Human Neural Progenitor Cells (NHNPCs)||810nm; 0.2J/ cm2; 50mW/cm2 and 100mW/ cm2||Neurite outgrowth greater at 50mW/cm2; less at 100mW/cm2||Anders et al. 2007|
|2009||Rheumatoid arthritis synoviocytes||810nm laser_1, 3, 5, 10, 20 and 50 J/cm2||Cell proliferation increased at 5 J/cm2 (16.7 mW/cm2); Lower at 50 J/cm2||Yamaura et al. 2009|
|2009||Mouse embryonic fibroblasts||810nm laser; 0.003,0.03,0.3,3 or 30J/cm2||NF-κB activation maximum at 0.3 J/cm2; decreased at 3 J/cm2 and 30 J/cm2||Chen et al. 2009|
Biphasic dose response studies of LLLT in vivo (animal models).
|1979||wound closure||He-Ne laser4 J/cm2||Wound healing best at 45 mW/cm2; least at 12.4 mW/cm2||Ginsbach 1979|
|2001||Induced heart attacks in rats||810 nm laser; 2.5 to 20mW/cm2 ;||Reductions of infarct size maximum at 5mW/cm2
Lower effects both at 2.5mW/cm2 and 20mW/cm2
|Oron et al. 2001|
|2005||Mouse pleurisy induced by Carrageenan||650nm laser; 2.5 mW in 0.08 cm2; 3 J/cm2, 7.5 J/cm2, and 15 J/cm2||Inflammatory cell migration reduction most at 7.5 J/cm2; Less at 3 and 15 J/cm2||Lopes-Martins et al. 2005|
|2007||Healing of pressure ulcers in mice||670nm LED; 5 J/cm2 at 0.7, 2, 8 or 40mW/cm2||Healing significant improved only at 8mW/cm2;Less at 0.7, 2, and 40 mW/cm2||Lanzafame et al. 2007|
|2007||Full thickness dorsal excisional wound in BALB/c mice||a single exposure from 635, 670, 720 or 820nm filtered lamp; 1, 2, 10 and 50 J/cm2; 100 mW/cm2 10, 20, 100 and 500 seconds||Healing effect best at 2 J/cm2 for 635nm light; worse at 50 J/cm2 for most wavelengths compared to no treatment||820nm was the best wavelength||Demidova-Rice et al. 2007|
|2007||Inflammatory arthritis induced by zymosan in rats||810-nm laser; 3 and 30 J/cm2; 5 mW/cm2 and 50 mW/cm2||30 J/cm2 was better than 3 J/cm2 at 50mW/cm2||3 J/cm2 has effective at 5mW/cm2 but not 50mW/cm2||Castano et al. 2007|
Biphasic dose response studies of LLLT in clinical studies.
|1997||Patients with post herpetic neuralgia of the facial type||830nm lasers; 60mW laser and 150mW laser; irradiance point at 4mm in diameter||Pain reduction greater at 150mW laser; less at 60mW laser when exposure to the same time.||Hashimoto et al. 1997|
Figure 3 shows a 3D depiction of the Arndt Schulz model to illustrate a possible dose “sweet spot” at the target tissue. This graph suggests that insufficient power density or too short a time will have no effect on the pathology, that too much power density and / or time may have inhibitory effects and that there may be an optimal balance between power density and time that produces a maximal beneficial effect. There even may be a (low) power density for which infinite irradiation time would only have positive effects and no inhibitory effect. We believe that the absolute figures will be different at different wavelengths, tissue types, redox states, and may be affected further by different pulse parameters.
Three-dimensional model of the Arndt-Schulz curve illustrating how either irradiance or illumination time (fluence) can have biphasic dose response effects in LLLT.
In this section we cover the new reports of biphasic dose responses in LLLT that have been published in the last two years since our previous review.
In an oral mucositis hamster model Lopes and coworkers (Lopes et al. 2009) delivered 660-nm laser at two different irradiances (55 mW/cm2 for 16 seconds per point or 155 mW/cm2 for 6 seconds per point). Both regimens delivered 0.9 J/cm2 per point. On day 7, 11 and 15 the authors reported reduced severity of clinical mucositis and lower levels of COX-2 staining in the 55 mW/cm2 group and that the 155 mW/cm2 had no significant differences when compared with controls. This data is summarized in Figure 4.
Mean grading of oral mucositis (OM) in a hamster cheek pouch model treated with 0.9 J/cm2 of 660-nm laser at two different irradiances (55 mW/cm2 for 16 seconds per point or 155 mW/cm2 for 6 seconds per point). Graph redrawn from data contained in (Lopes, Plapler et al. 2009).
Gal et al (Gal et al. 2009) compared the effects of delivering 5 J/cm2 of 670-nm laser at different power densities on wound tensile strength in a rat model. They found (Figure 5) that 670 nm laser achieved a significant effect using 4mW/cm2 applied for 1,250 seconds (20 mins 50 seconds) but that this effect was lost if the same 5J/cm2 fluence was delivered at 15 mW/cm2 for 333 seconds (5 mins 33 seconds).
Mean wound tensile strength obtained after delivering 5 J/cm2 of 670-nm laser at different power densities (4mW/cm2 applied for 1,250 seconds or 15 mW/cm2 for 333 seconds). Graph redrawn from data contained in (Gal, Mokry et al. 2009).
(Skopin and Molitor 2009) studied the effects of different influences of 980 nm laser on a human fibroblast in vitro model of wound healing. A small pipette was used to induce a wound in fibroblast cell cultures, which were exposed to a range of laser doses (1.5–66 J/cm2). Exposure to low- and medium-dose laser light accelerated cell growth, whereas high-intensity light negated the beneficial effects of laser exposure as shown in Figure 6.
Mean percentage of healing induced in a scratch wounded culture of human fibroblasts using different fluences (constant time, increasing irradiance) of 980-nm laser. Graph redrawn from data contained in (Gal, Mokry et al. 2009).
(Prabhu et al. 2010) performed a dose response study by applying a 7 mW HeNe (632.8-nm) laser with a power density of 4 mW/cm2 to 15×15 mm excisional wounds on Swiss albino mice for a range of irradiation times from 249 seconds (4.15 mins) up to 2,290 seconds (41.46 mins). As Figure 7 shows, there was a clear biphasic response (including a possible inhibitory effect) with changes in irradiation time and therefore fluence.
Mean area under the curve of wound area over time in a mouse excisional wound healing model treated with a 7 mW (power density of 4 mW/cm2) HeNe (632.8-nm) laser for times ranging from 249 to 2,290 seconds. Graph redrawn from data contained in (Prabhu, Rao et al. 2010).
In order to elucidate the mechanism responsible for the beneficial effect reported by LLLT for brain related disorders, we carried out studies to look into effects of 810 nm laser on different cellular signaling molecules in primary cortical neurons. The primary cortical neurons were isolated from brains taken from embryonic mice. We irradiated the neurons with different fluences of 0.03, 0.3, 3, 10 or 30 J/cm2 delivered at a constant irradiance of 25 mW/cm2, and subsequently the intracellular levels of ROS, mitochondrial membrane potential (MMP) and ATP was measured. The changes in mitochondrial function were studied in terms of ATP and MMP. Low-level light was found to induce a significant increase in ATP and MMP at lower fluences and a decrease at higher fluence. ROS was induced significantly by light at all light doses but there was a distinctive pattern of a double peak with the first peak coinciding with the other peaks of ATP and MMP at 3 J/cm2 (Figure 8). However in contrast to ATP and MMP there was a second larger rise in ROS at 30 J/cm2 that coincided with the reduction in MMP below baseline. The results of the this study suggested that LLLT at lower fluences is capable of inducing mediators of cell signaling process which in turn may be responsible for the biomodulatory effects of the low level laser. Conversely at higher fluences beneficial mediators are reduced but potentially harmful mediators are increased. Thus this study offered an explanation for the biphasic dose response induced by LLLT.
Mean expression levels of reactive oxygen species (ROS, measured by MitoSox red fluorescence), mitochondrial membrane potential (MMP, measured by red/green fluorescence ration of JC1 dye) and ATP (measured by firefly luciferase assay) in primary mouse cortical neurons treated with various fluences of 810-laser delivered at 25 mW/cm2 over times varying from 1.2 to 1200 seconds.
We have been studying the effect of transcranial laser (810-nm) on mouse models of traumatic brain injury. The model involves a controlled cortical impact using a pneumatic piston device through a craniotomy followed by closure of the head. This injury can be adjusted in severity to produce a neurological severity score (NSS based on a panel of standardized behavioral tests) of 7–8 on a scale of 0 (normal mice) to 10 (severe brain injury that causes death). The basic finding was that delivering a single dose of 36 J/cm2 810-nm laser delivered at 50 mW/cm2 (12 minutes illumination time) in a spot of 1-cm diameter centered on the top of the mouse head at a time point of 4 hours post-TBI was highly effective in ameliorating the neurological symptoms suffered by the mice (Figure 9A). When we delivered 10 times as much 810-nm laser (360 J/cm2 at 500 mW/cm2) also taking 12 minutes the beneficial effect totally disappeared, and at early time points (1–6 days) the high fluence appeared to be worse than no treatment (Figure 9B).
Transcranial laser therapy (36 J/cm2 of 810-nm laser delivered at 50 mW/cm2 (12 minutes illumination time) in a spot of 1-cm diameter centered on the top of the mouse head) was used to treat mice with controlled cortical impact TBI four hours after injury. (A) Significant improvement in neurological severity score continuing for 4 weeks after a single treatment. (B) Delivering ten times more light by increasing irradiance tenfold (500 mW/cm2) loses all therapeutic benefit, and produces worse performance soon after laser. (C) Repeating beneficial laser treatment daily for 14 days loses benefit in performance after 5 days.
When we repeated the effective laser treatments 14 times (36 J/cm2 delivered at 50-mW/cm2 once a day for 14 days starting 4 hours post-TB) we found a very interesting result (Figure 9C). For the first 4 days the improvement in NSS in the repeated laser group was marginally better than the single treatment. However on day 5 the gradual improvement ceased and as the laser was repeated the NSS got closer to that of untreated TBI mice until at day 14 it actually crossed over. Although the differences were not statistically significant it appeared that from day 16 until day 28 the mice that received 14 laser treatments did worse than those that received no treatment at all.
The triphasic dose response we have observed for ROS production in cultured cortical neurons (see Fig 7) suggests an explanation for the biphasic dose response. The hypothesis is that there are two kinds of ROS. Good ROS are produced at fairly low fluences of light. The reason for the production of good ROS is likely to be connected with stimulation of mitochondrial electron transport as shown by increases in MMP and increases in ATP production. These good ROS can initiate beneficial cell signaling pathwas leading to activation of redox sensitive transcription factors such as NF-κB (Chandel et al. 2000; Groeger et al. 2009). NF-κB activation induces expression of a large number of gene products related to cell proliferation and survival (Karin and Lin 2002; Brea-Calvo et al. 2009). As the fluence of light is increased the beneficial ROS production in the mitochondria decreases in tandem with reductions in MMP and a drop-off in ATP production. Then when even more light is delivered there is a second peak in ROS production, which we will call bad ROS. Bad ROS can damage the mitochondria leading to a drop in MMP below baseline levels and presumably can lead to initiation of apoptosis by the mitochondrial pathway including cytochrome c release. It remains to be seen whether the good and bad ROS are identical species and just differ in amount, or whether they are chemically different species. For instance it may be hypothesized that the good ROS consists mainly of superoxide while the bad ROS consists of more damaging ROS such as hydroxyl radicals and peroxynitrite. In Figure 7 we used just one type of fluorescent ROS indicator (mitoSOX red), which is commonly supposed to be specific for superoxide but will likely also be activated by hydroxyl radicals and peroxynitrite.
There have been several studies showing that relatively high doses of light can induce apoptosis in various cell types via ROS-mediated signaling pathways (Huang et al. 2011). Meanwhile, there is an important proapoptotic signaling pathway has been identified which involv
The medical use of low level laser (LLL) irradiation has been occurring for decades, primarily in the area of tissue
healing and inflammatory conditions. Despite little mechanistic knowledge, the concept of a non-invasive, nonthermal
intervention that has the potential to modulate regenerative processes is worthy of attention when searching
for novel methods of augmenting stem cell-based therapies. Here we discuss the use of LLL irradiation as a
“photoceutical” for enhancing production of stem cell growth/chemoattractant factors, stimulation of angiogenesis,
and directly augmenting proliferation of stem cells. The combination of LLL together with allogeneic and autologous
stem cells, as well as post-mobilization directing of stem cells will be discussed.
Introduction (Personal Perspective)
We came upon the field of low level laser (LLL) therapy
by accident. One of our advisors read a press release
about a company using this novel technology of specific
light wavelengths to treat stroke. Given the possible role
of stem cells in post-stroke regeneration, we decided to
cautiously investigate. As a background, it should be
said that our scientific team has been focusing on the
area of cord blood banking and manufacturing of disposables
for processing of adipose stem cells for the past 3
years. Our board has been interested in strategically
refocusing the company from services-oriented into a
more research-focused model. An unbiased exploration
into the various degenerative conditions that may be
addressed by our existing know-how led us to explore
the condition of chronic obstructive pulmonary disease
(COPD), an umbrella term covering chronic bronchitis
and emphysema, which is the 4th largest cause of death
in the United States. As a means of increasing our probability
of success in treatment of this condition, the
decision was made to develop an adjuvant therapy that
would augment stem cell activity. The field of LLL therapy
attracted us because it appeared to be relatively
unexplored scientific territory for which large amounts
of clinical experience exist. Unfortunately, it was difficult
to obtain the cohesive “state-of-the-art” description of
the molecular/cellular mechanisms of this therapy in
reviews that we have searched. Therefore we sought in
this mini-review to discuss what we believe to be relevant
to investigators attracted by the concept of “regenerative
photoceuticals”. Before presenting our synthesis
of the field, we will begin by describing our rationale for
approaching COPD with the autologous stem cell based
approaches we are developing.
COPD as an Indication for Stem Cell Therapy
COPD possesses several features making it ideal for
stem cell based interventions: a) the quality of life and
lack of progress demands the ethical exploration of
novel approaches. For example, bone marrow stem cells
have been used in over a thousand cardiac patients with
some indication of efficacy [1,2]. Adipose-based stem
cell therapies have been successfully used in thousands
of race-horses and companion animals without adverse
effects , as well as numerous clinical trials are
ongoing and published human data reports no adverse
effects (reviewed in ref ). Unfortunately, evaluation of
stem cell therapy in COPD has lagged behind other
areas of regenerative investigation; b) the underlying
cause of COPD appears to be inflammatory and/or
immunologically mediated. The destruction of alveolar
tissue is associated with T cell reactivity [5,6], pathological
pulmonary macrophage activation , and auto-antibody
production . Mesenchymal stem cells have been
demonstrated to potently suppress autoreactive T cells
[9,10], inhibit macrophage activation , and autoantibody
responses . Additionally, mesenchymal stem
cells can be purified in high concentrations from adipose
stromal vascular tissue together with high concentrations of T regulatory cells , which in animal
models are approximately 100 more potent than peripheral
T cells at secreting cytokines therapeutic for COPD
such as IL-10 [13,14]. Additionally, use of adipose
derived cells has yielded promising clinical results in
autoimmune conditions such as multiple sclerosis ;
and c) Pulmonary stem cells capable of regenerating
damaged parenchymal tissue have been reported .
Administration of mesenchymal stem cells into neonatal
oxygen-damaged lungs, which results in COPD-like
alveoli dysplasia, has been demonstrated to yield
improvements in two recent publications [16,17].
Based on the above rationale for stem cell-based
COPD treatments, we began our exploration into this
area by performing several preliminary experiments and
filing patents covering combination uses of stem cells
with various pharmacologically available antiinflammatories,
as well as methods of immune modulation. These
have served as the basis for two of our pipeline candidates,
ENT-111, and ENT-894. As a commerciallyoriented
organization, we needed to develop a therapeutic
candidate that not only has a great potential for efficacy,
but also can be easily implemented as part of the
standard of care. Our search led us to the area of low
level laser (LLL) therapy. From our initial perception as
neophytes to this field, the area of LLL therapy has been
somewhat of a medical mystery. A pubmed search for
“low level laser therapy” yields more than 1700 results,
yet before stumbling across this concept, none of us, or
our advisors, have ever heard of this area of medicine.
On face value, this field appeared to be somewhat of a
panacea: clinical trials claiming efficacy for conditions
ranging from alcoholism , to sinusitis , to
ischemic heart disease . Further confusing was that
many of the studies used different types of LLL-generating
devices, with different parameters, in different model
systems, making comparison of data almost impossible.
Despite this initial impression, the possibility that a simple,
non-invasive methodology could exist that augments
regenerative potential in a tissue-focused manner
became very enticing to us. Specific uses envisioned, for
which intellectual property was filed included using light
to concentrate stem cells to an area of need, to modulate
effects of stem cells once they are in that specific
area, or even to use light together with other agents to
modulate endogenous stem cells.
The purpose of the current manuscript is to overview
some of the previous work performed in this area that was
of great interest to our ongoing work in regenerative medicine.
We believe that greater integration of the area of
LLL with current advancements in molecular and cellular
biology will accelerate medical progress. Unfortunately, in
our impression to date, this has been a very slow process.
What is Low Level Laser Irradiation?
Lasers (Light amplification by stimulated emission of
radiation) are devices that typically generate electromagnetic
radiation which is relatively uniform in wavelength,
phase, and polarization, originally described by Theodore
Maiman in 1960 in the form of a ruby laser . These
properties have allowed for numerous medical applications
including uses in surgery, activation of photodynamic
agents, and various ablative therapies in cosmetics that are
based on heat/tissue destruction generated by the laser
beam [22-24]. These applications of lasers are considered
“high energy” because of their intensity, which ranges
from about 10-100 Watts. The subject of the current
paper will be another type of laser approach called low
level lasers (LLL) that elicits effects through non-thermal
means. This area of investigation started with the work of
Mester et al who in 1967 reported non-thermal effects of
lasers on mouse hair growth . In a subsequent study
, the same group reported acceleration of wound healing
and improvement in regenerative ability of muscle
fibers post wounding using a 1 J/cm2 ruby laser. Since
those early days, numerous in vitro and in vivo studies
have been reported demonstrating a wide variety of therapeutic
effects involving LLL, a selected sample of which
will be discussed below. In order to narrow our focus of
discussion, it is important to first begin by establishing the
current definition of LLL therapy. According to Posten et
al , there are several parameters of importance: a)
Power output of laser being 10-3 to 10-1 Watts; b) Wavelength
in the range of 300-10,600 nm; c) Pulse rate from 0,
meaning continuous to 5000 Hertz (cycles per second); d)
intensity of 10-2-10 W/cm(2) and dose of 0.01 to 100 J/
cm2. Most common methods of administering LLL radiation
include lasers such as ruby (694 nm), Ar (488 and 514
nm), He-Ne (632.8 nm), Krypton (521, 530, 568, and 647
nm), Ga-Al-As (805 or 650 nm), and Ga-As (904 nm).
Perhaps one of the most distinguishing features of LLL
therapy as compared to other photoceutical modalities is
that effects are mediated not through induction of thermal
effects but rather through a process that is still not clearly
defined called “photobiostimulation”. It appears that this
effect of LLL is not depend on coherence, and therefore
allows for use of non-laser light generating devices such as
inexpensive Light Emitting Diode (LED) technology .
To date several mechanisms of biological action have
been proposed, although none are clearly established.
These include augmentation of cellular ATP levels ,
manipulation of inducible nitric oxide synthase (iNOS)
activity [30,31], suppression of inflammatory cytokines
such as TNF-alpha, IL-1beta, IL-6 and IL-8 [32-36],
upregulation of growth factor production such as PDGF,
IGF-1, NGF and FGF-2 [36-39], alteration of mitochondrial
membrane potential [29,40-42] due to chromophores found in the mitochondrial respiratory
chain [43,44] as reviewed in , stimulation of protein
kinase C (PKC) activation , manipulation of NF-!B
activation , direct bacteriotoxic effect mediated by
induction of reactive oxygen species (ROS) , modification
of extracellular matrix components , inhibition
of apoptosis , stimulation of mast cell
degranulation , and upregulation of heat shock proteins
. Unfortunately these effects have been demonstrated
using a variety of LLL devices in noncomparable
models. To add to confusion, dose-dependency
seems to be confined to such a narrow range or
does not seem to exist in that numerous systems therapeutic
effects disappear with increased dose.
In vitro studies of LLL
In areas of potential phenomenology, it is important to
begin by assessing in vitro studies reported in the literature
in which reproducibility can be attained with some
degree of confidence, and mechanistic dissection is simpler
as compared with in vivo systems. In 1983, one of
the first studies to demonstrate in vitro effects of LLL
was published. The investigators used a helium neon
(He-Ne) laser to generate a visible red light at 632.8 nm
for treatment of porcine granulosa cells. The paper
described upregulation of metabolic and hormone-producing
activity of the cells when exposed for 60 seconds
to pulsating low power (2.8 mW) irradiation . The
possibility of modulating biologically-relevant signaling
proteins by LLL was further assessed in a study using an
energy dose of 1.5 J/cm2 in cultured keratinocytes.
Administration of He-Ne laser emitted light resulted in
upregulated gene expression of IL-1 and IL-8 . Production
of various growth factors in vitro suggests the
possibility of enhanced cellular mitogenesis and mobility
as a result of LLL treatment. Using a diode-based
method to generate a similar wavelength to the He-Ne
laser (363 nm), Mvula et al reported in two papers that
irradiation at 5 J/cm2 of adipose derived mesenchymal
stem cells resulted in enhanced proliferation, viability
and expression of the adhesion molecule beta-1 integrin
as compared to control [54,55]. In agreement with possible
regenerative activity based on activation of stem
cells, other studies have used an in vitro injury model to
examine possible therapeutic effects. Migration of fibroblasts
was demonstrated to be enhanced in a “wound
assay” in which cell monolayers are scraped with a pipette
tip and amount of time needed to restore the
monolayer is used as an indicator of “healing”. The cells
exposed to 5 J/cm2 generated by an He-Ne laser
migrated rapidly across the wound margin indicating a
stimulatory or positive influence of phototherapy.
Higher doses (10 and 16 J/cm2) caused a decrease in
cell viability and proliferation with a significant amount
of damage to the cell membrane and DNA . In
order to examine whether LLL may positively affect
healing under non-optimal conditions that mimic clinical
situations treatment of fibroblasts from diabetic animals
was performed. It was demonstrated that with the
He-Ne laser dosage of 5 J/cm2 fibroblasts exhibited an
enhanced migration activity, however at 16 J/cm2 activity
was negated and cellular damage observed . Thus
from these studies it appears that energy doses from 1.5
J/cm2 to 5 J/cm2 are capable of eliciting “biostimulatory
effects” in vitro in the He-Ne-based laser for adherent
cells that may be useful in regeneration such as fibroblasts
and mesenchymal stem cells.
Studies have also been performed in vitro on immunological
cells. High intensity He-Ne irradiation at 28
and 112 J/cm2 of human peripheral blood mononuclear
cells, a heterogeneous population of T cells, B cells, NK
cells, and monocytes has been described to induce chromatin
relaxation and to augment proliferative response
to the T cell mitogen phytohemaglutin . In human
peripheral blood mononuclear cells (PBMC), another
group reported in two papers that interleukin-1 alpha
(IL-1 alpha), tumor necrosis factor-alpha (TNF-alpha),
interleukin-2 (IL-2), and interferon-gamma (IFNgamma)
at a protein and gene level in PBMC was
increased after He-Ne irradiation at 18.9 J/cm2 and
decreased with 37.8 J/cm2 [59,60]. Stimulation of human
PBMC proliferation and murine splenic lymphocytes
was also reported with He-Ne LLL [61,62]. In terms of
innate immune cells, enhanced phagocytic activity of
murine macrophages have been reported with energy
densities ranging from 100 to 600 J/cm2, with an optimal
dose of 200 J/cm2 . Furthermore, LLL has been
demonstrated to augment human monocyte killing
mycobacterial cells at similar densities, providing a functional
Thus from the selected in vitro studies discussed, it
appears that modulation of proliferation and soluble factor
production by LLL can be reliably reproduced. However
the data may be to some extent contradictory. For
example, the over-arching clinical rationale for use of
LLL in conditions such as sinusitis , arthritis [66,67],
or wound healing  is that treatment is associated
with anti-inflammatory effects. However the in vitro studies
described above suggested LLL stimulates proinflammatory
agents such as TNF-alpha or IL-1 [59,60].
This suggests the in vivo effects of LLL may be very
complex, which to some extent should not be surprising.
Factors affecting LLL in vivo actions would include
degree of energy penetration through the tissue, the various
absorption ability of cells in the various tissues, and
complex chemical changes that maybe occurring in
paracrine/autocrine manner. Perhaps an analogy to the
possible discrepancy between LLL effects in vitro versus in vivo may be made with the medical practice of extracorporeal
ozonation of blood. This practice is similar to
LLL therapy given that it is used in treatment of conditions
such as atherosclerosis, non-healing ulcers, and
various degenerative conditions, despite no clear
mechanistic understanding [69-71]. In vitro studies have
demonstrated that ozone is a potent oxidant and inducer
of cell apoptosis and inflammatory signaling [72-74].
In contrast, in vivo systemic changes subsequent to
administration of ozone or ozonized blood in animal
models and patients are quite the opposite. Numerous
investigators have published enhanced anti-oxidant
enzyme activity such as elevations in Mg-SOD and glutathione-
peroxidase levels, as well as diminishment of
inflammation-associated pathology [75-78]. Regardless
of the complexity of in vivo situations, the fact that
reproducible, in vitro experiments, demonstrate a biological
effect provided support for us that there is some
basis for LLL and it is not strictly an area of
Animal Studies with LLL
As early as 1983, Surinchak et al reported in a rat skin
incision healing model that wounds exposed He-Ne
radiation of fluency 2.2 J/cm2 for 3 min twice daily for
14 days demonstrated a 55% increase in breaking
strength over control rats. Interestingly, higher doses
yielded poorer healing . This application of laser
light was performed directly on shaved skin. In a contradictory
experiment, it was reported that rats irradiated
for 12 days with four levels of laser light (0.0, 0.47, 0.93,
and 1.73 J/cm2) a possible strengthening of wounds tension
was observed at the highest levels of irradiation
(1.73 J/cm2), however it did not reach significance when
analyzed by resampling statistics . In another
wound-healing study Ghamsari et al reported accelerated
healing in the cranial surface of teats in dairy cows
by administration of He-Ne irradiation at 3.64 J/cm2
dose of low-level laser, using a helium-neon system with
an output of 8.5 mW, continuous wave . Collagen
fibers in LLL groups were denser, thicker, better
arranged and more continuous with existing collagen
fibers than those in non-LLL groups. The mean tensile
strength was significantly greater in LLL groups than in
non-LLL groups . In the random skin flap model,
the use of He-Ne laser irradiation with 3 J/cm2 energy
density immediately after the surgery and for the four
subsequent days was evaluated in 4 experimental
groups: Group 1 (control) sham irradiation with He-Ne
laser; Group 2 irradiation by punctual contact technique
on the skin flap surface; Group 3 laser irradiation surrounding
the skin flap; and Group 4 laser irradiation
both on the skin flap surface and around it. The percentage
of necrotic area of the four groups was determined
on day 7-post injury. The control group had an average
necrotic area of 48.86%; the group irradiated on the skin
flap surface alone had 38.67%; the group irradiated
around the skin flap had 35.34%; and the group irradiated
one the skin flap surface and around it had
22.61%. All experimental groups reached statistically significant
values when compared to control . Quite
striking results were obtained in an alloxan-induced diabetes
wound healing model in which a circular 4 cm2
excisional wound was created on the dorsum of the diabetic
rats. Treatment with He-Ne irradiation at 4.8 J/
cm2 was performed 5 days a week until the wound
healed completely and compared to sham irradiated animals.
The laser-treated group healed on average by the
18th day whereas, the control group healed on average
by the 59th day .
In addition to mechanically-induced wounds, beneficial
effects of LLL have been obtained in burn-wounds
in which deep second-degree burn wounds were
induced in rats and the effects of daily He-Ne irradiation
at 1.2 and 2.4 J/cm2 were assessed in comparison to
0.2% nitrofurazone cream. The number of macrophages
at day 16, and the depth of new epidermis at day 30,
was significantly less in the laser treated groups in comparison
with control and nitrofurazone treated groups.
Additionally, infections with S. epidermidis and S. aureus
were significantly reduced .
While numerous studies have examined dermatological
applications of LLL, which may conceptually be
easier to perform due to ability to topically apply light,
extensive investigation has also been made in the area
of orthopedic applications. Healing acceleration has
been observed in regeneration of the rat mid-cortical
diaphysis of the tibiae, which is a model of post-injury
bone healing. A small hole was surgically made with a
dentistry burr in the tibia and the injured area and LLL
was administered over a 7 or 14 day course transcutaneously
starting 24 h from surgery. Incident energy density
dosages of 31.5 and 94.5 J/cm2 were applied during
the period of the tibia wound healing. Increased angiogenesis
was observed after 7 days irradiation at an
energy density of 94.5 J/cm2, but significantly decreased
the number of vessels in the 14-day irradiated tibiae,
independent of the dosage . In an osteoarthritis
model treatment with He-Ne resulted in augmentation
of heat shock proteins and pathohistological improvement
of arthritic cartilage . The possibility that a
type of preconditioning response is occurring, which
would involve induction of genes such as hemoxygenase-
1 , remains to be investigated. Effects of LLL
therapy on articular cartilage were confirmed by another
group. The experiment consisted of 42 young Wistar
rats whose hind limbs were operated on in order to
immobilize the knee joint. One week after operation they were assigned to three groups; irradiance 3.9 W/
cm2, 5.8 W/cm2, and sham treatment. After 6 times of
treatment for another 2 weeks significantpreservation of
articular cartilage stiffness with 3.9 and 5.8 W/cm2 therapy
was observed .
Muscle regeneration by LLL was demonstrated in a rat
model of disuse atrophy in which eight-week-old rats
were subjected to hindlimb suspension for 2 weeks,
after which they were released and recovered. During
the recovery period, rats underwent daily LLL irradiation
(Ga-Al-As laser; 830 nm; 60 mW; total, 180 s) to
the right gastrocnemius muscle through the skin. After
2-weeks the number of capillaries and fibroblast growth
factor levels exhibited significant elevation relative to
those of the LLL-untreated muscles. LLL treatment
induced proliferation in satellite cells as detected by
Other animal studies of LLL have demonstrated
effects in areas that appear unrelated such as suppression
of snake venom induced muscle death ,
decreasing histamine-induced vasospasms , inhibition
of post-injury restenosis , and immune stimulation
by thymic irradiation .
Clinical Studies Using LLL
Growth factor secretion by LLL and its apparent regenerative
activities have stimulated studies in radiationinduced
mucositis. A 30 patient randomized trial of carcinoma
patients treated by radiotherapy alone (65 Gy at
a rate of 2 Gy/fraction, 5 fractions per week) without
prior surgery or concomitant chemotherapy suffering
from radiation-induced mucositis was performed using a
He-Ne 60 mW laser. Grade 3 mucositis occured with a
frequency of 35.2% in controls and at 7.6% of treated
patients. Furthermore, a decrease in “severe pain” (grade
3) was observed in that 23.8% in the control group
experienced this level of pain, as compared to 1.9% in
the treatment group . A subsequent study reported
similar effects .
Healing ability of lasers was also observed in a study
of patients with gingival flap incisions. Fifty-eight extraction
patients had one of two gingival flap incisions lased
with a 1.4 mW He-Ne (670 nm) at 0.34 J/cm2. Healing
rates were evaluated clinically and photographically.
Sixty-nine percent of the irradiated incisions healed faster
than the control incisions. No significant difference
in healing was noted when patients were compared by
age, gender, race, and anatomic location of the incision
. Another study evaluating healing effects of LLL in
dental practice examined 48 patients subjected to surgical
removal of their lower third molars. Treated patients
were administered Ga-Al-As diode generated 808 nm at
a dose of 12 J. The study demonstrated that extraoral
LLL is more effective than intraoral LLL, which was
more effective than control for the reduction of postoperative
trismus and swelling after extraction of the
lower third molar .
Given the predominance of data supporting fibroblast
proliferative ability and animal wound healing effects of
LLL therapy, a clinical trial was performed on healing of
ulcers. In a double-blinded fashion 23 diabetic leg ulcers
from 14 patients were divided into two groups. Phototherapy
was applied (<1.0 J/cm2) twice per week, using a
Dynatron Solaris 705(R) LED device that concurrently
emits 660 and 890 nm energies. At days 15, 30, 45, 60,
75, and 90 mean ulcer granulation and healing rates
were significantly higher for the treatment group as
compared to control. By day 90, 58.3% of the ulcers in
the LLL treated group were fully healed and 75%
achieved 90-100% healing. In the placebo group only
one ulcer healed fully .
As previously mentioned, LLL appears to have some
angiogenic activity. One of the major problems in coronary
artery disease is lack of collateralization. In a 39
patient study advanced CAD, two sessions of irradiation
of low-energy laser light on skin in the chest area from
helium-neon B1 lasers. The time of irradiation was 15
minutes while operations were performed 6 days a week
for one month. Reduction in Canadian Cardiology
Society (CCS) score, increased exercise capacity and
time, less frequent angina symptoms during the treadmill
test, longer distance of 6-minute walk test and a
trend towards less frequent 1 mm ST depression lasting
1 min during Holter recordings was noted after therapy
Perhaps one of the largest clinical trials with LLL was
the NEST trial performed by Photothera. In this double
blind trial 660 stroke patients were recruited and randomized:
331 received LLL and 327 received sham. No
prespecified test achieved significance, but a post hoc
analysis of patients with a baseline National Institutes of
Health Stroke Scale score of <16 showed a favorable
outcome at 90 days on the primary end point (P <
0.044) . Currently Photothera is in the process of
repeating this trial with modified parameters.
Relevance of LLL to COPD
A therapeutic intervention in COPD would require
addressing the issues of inflammation and regeneration.
Although approaches such as administration of bone marrow
stem cells, or fat derived cellular components have
both regenerative and anti-inflammatory activity in animal
models, the need to enhance their potency for clinical
applications can be seen in the recent Osiris’s COPD trial
interim data which reported no significant improvement
in pulmonary function . Accordingly, we sought to
develop a possible rationale for how LLL may be useful as
an adjunct to autologous stem cell therapy.
Table 1 Examples of LLL Properties Relevant to COPD
LLL Experiment LLL Details Ref
Inflammation In vivo. Decreased joint inflammation in zymosan-induced
Semiconductor laser (685 nm and 830 nm) at (2.5 J/cm2)
In vitro. Suppression of LPS-induced bronchial inflammation and
655 nm at of 2.6 J/cm2
In vivo. Carrageenan-induced pleurisy had decreased leukocyte
infiltration and cytokine (TNF-alpha, IL-6, and MCP)
660 nm at 2.1 J/cm2
In vitro. LPS stimulated Raw 264.7 monocytes had reduced gene
expression of MCP-1, IL-1 and IL-6
780 nm diode laser at 2.2 J/cm2)
In vivo. Suppression of LPS-stimulated neutrophil influx,
myeloperoxidase activity and IL-1beta in bronchoalveolar lavage
660 nm diode laser at 7.5 J/cm2
In vitro. Inhibition of TNF-alpha induced IL-1, IL-8 and TNF-alpha
mRNA in human synoviocytes
810 nm (5 J/cm2) suppressed IL-1 and TNF, (25 J/cm2) also
In vivo. Reduction of TNF-alpha in diaphragm muscle after
intravenous LPS injection.
4 sessions in 24 h with diode Ga-AsI-Al laser of 650 nm and
a total dose of 5.2 J/cm2
In vivo. Inhibition of LPS induced peritonitis and neutrophil influx 3 J/cm2 and 7.5 J/cm2
Growth Factor Production
In vivo. Upregulation of TGF-b and PDGF in rat gingiva after
He-Ne laser (632.8 nm) at a dose of 7.5 J/cm2
In vitro. Osteoblast-like cells were isolated from fetal rat calvariae
had increased IGF-1
Ga-Al-As laser (830 nm) at (3.75 J/cm2).
In vitro. Upregulated production of IGF-1 and FGF-2 in human
685 nm, for 140 s, 2 J/cm2
In vivo. Increased fiber to capillary ratio in rabbits with ligated
Gallium-aluminum-arsenide (Ga-Al-As) diode laser, 904 nm
and power of 10 mW
In vitro. Stimulation of HUVEC proliferation by conditioned media
from LLL-treated T cells
820 nm at 1.2 and 3.6 J/cm2.
In vitro. 7-fold increased production of VEGF by cardiomyocytes,
1.6-fold increase by smooth muscle cells (SMC) and fibroblasts.
Supernatant of SMC had increased HUVEC-stimulating potential.
He:Ne continuous wave laser (632 nm). 0.5 J/cm2 for SMC,
2.1 J/cm2 for fibroblasts and 1.05 J/cm2 for cardiomyocytes.
In vitro. Direct stimulation of HUVEC proliferation 670 nm diode device at 2 and 8 J/cm2
Direct Stem Cell Effects
In vivo. LLL precondition significantly enhanced early cell survival
rate by 2-fold, decreased the apoptotic percentage of implanted
BMSCs in infarcted myocardium and increased the number of
newly formed capillaries.
635 nm at 0.96 J/cm2
In vitro. LLL stimulated MSC proliferation, VEGF and NGF
production, and myogenic differentiation after 5-aza induction.
635 nm diode laser at 0.5 J/cm2 for MSC proliferation, 5 J/
cm2 for VEGF and NGF production and for augmentation of
induced myogenic differentiation
In vitro. Increased proliferation of rat MSC. Red light LED 630 nm at 2 and 4 J/cm(2)
In vitro. Augmented proliferation of bone marrow and cardiac
specific stem cells.
GA-Al-As 810 nm at 1 and 3 J/cm2
In vitro/In vivo. Administration of LLL-treated MSC resulted 53%
reduction in infarct size, 5- and 6.3-fold significant increase in cell
density that positively immunoreacted to BrdU and c-kit,
respectively, and 1.4- and 2-fold higher level of angiogenesis and
vascular endothelial growth factor, respectively, when compared
to non-laser-treated implanted cells
Ga-Al-As laser (810 nm wavelength), 1 J/cm2
In vitro. Enhanced proliferation of adipose derived MSC in
presence of EGF.
636 nm diode, 5 J/cm2
Lin et al. Journal of Translational Medicine 2010, 8:16
Table 1: Examples of LLL Properties Relevant to COPD (Continued)
In vitro. Enhanced proliferation and beta-1 integrin expression of
adipose derived MSC.
635 nm diode laser, at 5 J/cm2
Clinical. 660 stroke patients: 331 received LLL and 327 received
sham. No prespecified test achieved significance, but a post hoc
analysis of patients with a baseline National Institutes of Health
Stroke Scale score of <16 showed a favorable outcome at 90
days on the primary end point (P < 0.044).
808 nm. No density disclosed.
Table 1 depicts some of the properties of LLL that provide
a rationale for the combined use with stem cells. One
of the basic properties of LLL seems to be ability to inhibit
inflammation at the level of innate immune activation.
Representative studies showed that LLL was capable of
suppressing inflammatory genes and/or pathology after
administration of lipopolysaccharide (LPS) as a stimulator
of monocytes  and bronchial cells , in vitro, and
leukocyte infiltration in vivo [103,104]. Inflammation
induced by other stimulators such as zymosan, carrageenan,
and TNF-alpha was also inhibited by LLL
[32,105,106]. Growth factor stimulating activity of LLL
was demonstrated in both in vitro and in vivo experiments
in which augmentation of FGF-2, PDGF and IGF-1 was
observed [36,37,107]. Endogenous production of these
growth factors may be useful in regeneration based on
activation of endogenous pulmonary stem cells [108,109].
Another aspect of LLL activities of relevance is ability to
stimulate angiogenesis. In COPD, the constriction of
blood vessels as a result of poor oxygen uptake is results
in a feedback loop culminating in pulmonary hypertension.
Administration of angiogenic factors has been
demonstrated to be beneficial in several animal models of
pulmonary pathology [110,111]. The ability of LLL to
directly induce proliferation of HUVEC cells , as well
as to augment production of angiogenic factors such as
VEGF , supports the possibility of creation of an
environment hospitable to neoangiogenesis which is optimal
for stem cell growth. In fact, a study demonstrated in
vivo induction of neocapillary formation subsequent to
LLL administration in a hindlimb ischemia model .
The critical importance of angiogenesis in stem cell
mediated regeneration has previously been demonstrated
in the stroke model, where the major therapeutic activity
of exogenous stem cells has been attributed to angiogenic
as opposed to transdifferentiation effects .
Direct evidence of LLL stimulating stem cells has been
obtained using mesenchymal stem cells derived both
from the bone marrow and from the adipose tissue
[116,117]. Interestingly in vivo administration of LLL stimulated
MSC has resulted in 50% decrease in cardiac
infarct size . Clinical translation of LLL has been
performed in the area of stroke, in which a 660 patient
trial demonstrated statistically significant effects in post
trial subset analysis .
Despite clinical use of LLL for decades, the field is still
in its infancy. As is obvious from the wide variety of
LLL sources, frequencies, and intensities used, no standard
protocols exist. The ability of LLL to induce
growth factor production, inhibition of inflammation,
stimulation of angiogenesis, and direct effects on stem
cells suggests the urgent need for combining this modality
with regenerative medicine, giving birth to the new
field of “regenerative photoceuticals”. Development of a
regenerative treatment for COPD as well as for other
degenerative diseases would be of considerable benefit.
Regarding COPD, such treatment would be life-saving/
life extending for thousands of affected individuals.
Ceasing smoking or not starting to smoke would considerably
impact this disease.
The authors thank Victoria Dardov and Matthew Gandjian for critical
discussions and input.
1Entest BioMedical, San Diego, CA, USA. 2Georgetown Dermatology,
Washington DC, USA. 3Cromos Pharma Services, Longview, WA, USA. 4Center
for the Study of Natural Oncology, Del Mar, CA, USA. 5Department of
Hematology and Medical Oncology, St Francis Hospital and Medical Center,
Hartford, CT, USA. 6Moores Cancer Center, University of California San Diego,
CA, USA. 7Department of Cardiothoracic Surgery, University of Utah, Salt
Lake City, UT, USA.
FL, SFJ, DTA, FR, VB, VG, CAD, RDNC, ANP, EC, DRK contributed to literature
review, analysis and discussion, synthesis of concepts, writing of the
manuscript and proof-reading of the final draft.
David R Koos is a shareholder, as well as Chairman and CEO of Entest Bio.
Feng Lin is research director of Entest Bio. All other authors declare no
Received: 7 January 2010
Accepted: 16 February 2010 Published: 16 February 2010
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32. Yamaura M, Yao M, Yaroslavsky I, Cohen R, Smotrich M, Kochevar IE: Low
level light effects on inflammatory cytokine production by rheumatoid
arthritis synoviocytes. Lasers Surg Med 2009, 41:282-290.
33. Shiba H, Tsuda H, Kajiya M, Fujita T, Takeda K, Hino T, Kawaguchi H,
Kurihara H: Neodymium-doped yttrium-aluminium-garnet laser
irradiation abolishes the increase in interleukin-6 levels caused by
peptidoglycan through the p38 mitogen-activated protein kinase
pathway in human pulp cells. J Endod 2009, 35:373-376.
34. de Lima Mafra F, Costa MS, Albertini R, Silva JA Jr, Aimbire F: Low level
laser therapy (LLLT): attenuation of cholinergic hyperreactivity, beta(2)-
adrenergic hyporesponsiveness and TNF-alpha mRNA expression in rat
bronchi segments in E. coli lipopolysaccharide-induced airway
inflammation by a NF-kappaB dependent mechanism. Lasers Surg Med
35. Aimbire F, de Oliveira Ligeiro AP, Albertini R, Correa JC, de Campos
Ladeira CB, Lyon JP, Silva JA Jr, Costa MS: Low level laser therapy (LLLT)
decreases pulmonary microvascular leakage, neutrophil influx and IL-
1beta levels in airway and lung from rat subjected to LPS-induced
inflammation. Inflammation 2008, 31:189-197.
36. Safavi SM, Kazemi B, Esmaeili M, Fallah A, Modarresi A, Mir M: Effects of
low-level He-Ne laser irradiation on the gene expression of IL-1beta,
TNF-alpha, IFN-gamma, TGF-beta, bFGF, and PDGF in rat’s gingiva. Lasers
Med Sci 2008, 23:331-335.
37. Saygun I, Karacay S, Serdar M, Ural AU, Sencimen M, Kurtis B: Effects of
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38. Schwartz F, Brodie C, Appel E, Kazimirsky G, Shainberg A: Effect of helium/
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41. Wu S, Xing D, Gao X, Chen WR: High fluence low-power laser irradiation
induces mitochondrial permeability transition mediated by reactive
oxygen species. J Cell Physiol 2009, 218:603-611.
42. Lan CC, Wu CS, Chiou MH, Chiang TY, Yu HS: Low-energy helium-neon
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61. Gulsoy M, Ozer GH, Bozkulak O, Tabakoglu HO, Aktas E, Deniz G, Ertan C:
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62. Novoselova EG, Cherenkov DA, Glushkova OV, Novoselova TV,
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63. Dube A, Bansal H, Gupta PK: Modulation of macrophage structure and
function by low level He-Ne laser irradiation. Photochem Photobiol Sci
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66. Shen X, Zhao L, Ding G, Tan M, Gao J, Wang L, Lao L: Effect of combined
laser acupuncture on knee osteoarthritis: a pilot study. Lasers Med Sci
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68. Minatel DG, Frade MA, Franca SC, Enwemeka CS: Phototherapy promotes
healing of chronic diabetic leg ulcers that failed to respond to other
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69. Bocci V, Travagli V, Zanardi I: May oxygen-ozone therapy improves
cardiovascular disorders?. Cardiovasc Hematol Disord Drug Targets 2009,
70. Bocci V, Borrelli E, Travagli V, Zanardi I: The ozone paradox: ozone is a
strong oxidant as well as a medical drug. Med Res Rev 2009, 29:646-682.
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ozone in patients with diabetic foot. Eur J Pharmacol 2005, 523:151-161.
79. Surinchak JS, Alago ML, Bellamy RF, Stuck BE, Belkin M: Effects of low-level
energy lasers on the healing of full-thickness skin defects. Lasers Surg
Med 1983, 2:267-274.
80. Broadley C, Broadley KN, Disimone G, Riensch L, Davidson JM: Low-energy
helium-neon laser irradiation and the tensile strength of incisional
wounds in the rat. Wound Repair Regen 1995, 3:512-517.
81. Ghamsari SM, Taguchi K, Abe N, Acorda JA, Yamada H: Histopathological
effect of low-level laser therapy on sutured wounds of the teat in dairy
cattle. Vet Q 1996, 18:17-21.
82. Ghamsari SM, Taguchi K, Abe N, Acorda JA, Sato M, Yamada H: Evaluation
of low level laser therapy on primary healing of experimentally induced
full thickness teat wounds in dairy cattle. Vet Surg 1997, 26:114-120.
83. Pinfildi CE, Liebano RE, Hochman BS, Ferreira LM: Helium-neon laser in
viability of random skin flap in rats. Lasers Surg Med 2005, 37:74-77.
84. Maiya GA, Kumar P, Rao L: Effect of low intensity helium-neon (He-Ne)
laser irradiation on diabetic wound healing dynamics. Photomed Laser
Surg 2005, 23:187-190.
85. Bayat M, Vasheghani MM, Razavi N, Taheri S, Rakhshan M: Effect of lowlevel
laser therapy on the healing of second-degree burns in rats: a
histological and microbiological study. J Photochem Photobiol B 2005,
86. Garavello I, Baranauskas V, da Cruz-Hofling MA: The effects of low laser
irradiation on angiogenesis in injured rat tibiae. Histol Histopathol 2004,
87. Lin YS, Huang MH, Chai CY, Yang RC: Effects of helium-neon laser on
levels of stress protein and arthritic histopathology in experimental
osteoarthritis. Am J Phys Med Rehabil 2004, 83:758-765.
88. Jamieson RW, Friend PJ: Organ reperfusion and preservation. Front Biosci
89. Akai M, Usuba M, Maeshima T, Shirasaki Y, Yasuoka S: Laser’s effect on
bone and cartilage change induced by joint immobilization: an
experiment with animal model. Lasers Surg Med 1997, 21:480-484.
Lin et al. Journal of Translational Medicine 2010, 8:16
90. Nakano J, Kataoka H, Sakamoto J, Origuchi T, Okita M, Yoshimura T: Lowlevel
laser irradiation promotes the recovery of atrophied gastrocnemius
skeletal muscle in rats. Exp Physiol 2009, 94:1005-1015.
91. Doin-Silva R, Baranauskas V, Rodrigues-Simioni L, da Cruz-Hofling MA: The
ability of low level laser therapy to prevent muscle tissue damage
induced by snake venom. Photochem Photobiol 2009, 85:63-69.
92. Gal D, Chokshi SK, Mosseri M, Clarke RH, Isner JM: Percutaneous delivery of
low-level laser energy reverses histamine-induced spasm in
atherosclerotic Yucatan microswine. Circulation 1992, 85:756-768.
93. Kipshidze N, Sahota H, Komorowski R, Nikolaychik V, Keelan MH Jr:
Photoremodeling of arterial wall reduces restenosis after balloon
angioplasty in an atherosclerotic rabbit model. J Am Coll Cardiol 1998,
94. Novoselova EG, Glushkova OV, Cherenkov DA, Chudnovsky VM, Fesenko EE:
Effects of low-power laser radiation on mice immunity. Photodermatol
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95. Bensadoun RJ, Franquin JC, Ciais G, Darcourt V, Schubert MM, Viot M,
Dejou J, Tardieu C, Benezery K, Nguyen TD, Laudoyer Y, Dassonville O,
Poissonnet G, Vallicioni J, Thyss A, Hamdi M, Chauvel P, Demard F: Lowenergy
He/Ne laser in the prevention of radiation-induced mucositis. A
multicenter phase III randomized study in patients with head and neck
cancer. Support Care Cancer 1999, 7:244-252.
96. Arun Maiya G, Sagar MS, Fernandes D: Effect of low level helium-neon
(He-Ne) laser therapy in the prevention & treatment of radiation
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97. Neiburger EJ: Rapid healing of gingival incisions by the helium-neon
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98. Aras MH, Gungormus M: Placebo-controlled randomized clinical trial of
the effect two different low-level laser therapies (LLLT)-intraoral and
extraoral-on trismus and facial swelling following surgical extraction of
the lower third molar. Lasers Med Sci 2009.
99. Zycinski P, Krzeminska-Pakula M, Peszynski-Drews C, Kierus A, Trzos E,
Rechcinski T, Figiel L, Kurpesa M, Plewka M, Chrzanowski L, Drozdz J: Laser
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preliminary observational study. Kardiol Pol 2007, 65:13-21, discussion 22-
100. Zivin JA, Albers GW, Bornstein N, Chippendale T, Dahlof B, Devlin T,
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Rymer M, Schellinger P, Schneider D, Schwab S, Veltkamp R, Walker M,
Streeter J, NeuroThera Effectiveness and Safety Trial-2 Investigators:
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101. Osiris Therapeutics Reports Interim Data for COPD Trial. http://www.
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prevention of aneurysm progression. Lasers Surg Med 2008, 40:371-378.
103. Correa F, Lopes Martins RA, Correa JC, Iversen VV, Joenson J, Bjordal JM:
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Translational Medicine 2010 8:16.
This tool is a searchable collection of technical publications, books, videos and other resources about the use of lasers and light for PhotoBioModulation (PBM). Enter a keyword above or see some of our favorite queries below.
Here are some of our favorite queries:
All the resources include links to the original source so we are not making any statement about the use of lasers for treating non-FDA cleared application, we are simple summarizing what others have said.
Where every possible, we have included a link to the orginal publication.
This tool uses a broad match query so:
The results of the search are sorted based on 3 quality factors on a scale of 1 to 10 with 10 being the best score. Originally all the resources were given a 5-5-5 until they could be individually evaluated. These scores are purely opinion and are only used to simplify the rank of the results from more valuable to least valuable. This should not be considered a critique of any work. This system was created to help researchers (including ourselves) find the most usable resources for any cold laser therapy research. The resources are assigned values based on the following 3 factors:
Over the past few years of working with research, we found that a majority of the published resources are lacking in one of these three ranking factors.
The original goal of this research tool was to tie published resources to the protocols in the laser-therapy.us library. This connection allows users to trace each protocol back to a list of resources so the protocol can be researched and improved.
When many of the first research papers were published, the most power laser available for therapy were less than 100mW and many systems had to be pulsed to keep the laser from burning out too quickly. Today, system are available that will deliver up to 60,000mW of continuous output. Because of these power limitation, many early studies were limited to extremely low dosages by today’s standards. It takes a 50mW system 17 minutes to deliver 50 joules at the surface of the skin. If this was spread over a large area of damage or was treating a deeper problem, the actual dosages were much less than 1J/cm2. Today, we know that these dosages typically produce very little or no results.
About 80% of the resources in this database are in the near infrared wavelength. There is also some interest in the red wavelength (600 to 660nm) . Other wavelengths like blue, purple, and green have very little scientific research behind them and have not gotten much traction in the core therapy market with the exception of some fringe consumer products.
This research tool is free to use but we make no claims about the accuracy of the information. It is an aggregation of existing published resources and it is up to the user to determine if the source of the resources has any value. The information provided through this web site should not be used for diagnosing or treating a health problem or disease. If you have or suspect you may have a health problem, you should consult your local health care provider.
Welcome to the BioPhotonica Education Center. There are over 5000 successful studies showing the efficacy of PBM, light therapy and sound therapy. This is a searchable collection of technical publications, books, videos and other resources about the best practices in the industry and about treating a wide variety of problems. All the resources include links to the original source (where available) so we are not making any claims about the use of our technology for treating "non-FDA cleared" applications, we are simply summarizing what the expert are saying about proper application of these technologies.
Enter a keyword above and click on one of the following links to see a set of publications about that subject. HINT: Shorter keywords work better.
Here are some of our favorite queries:
Research Info for other Applications
This tool uses a broad match query so:
Welcome to the Lighthouse Health Education Center. There are over 5000 successful studies showing the efficacy of PBM, light therapy and sound therapy. This is a searchable collection of technical publications, books, videos and other resources about the best practices in the industry and about treating a wide variety of problems. All the resources include links to the original source (where available) so we are not making any claims about the use of our technology for treating "non-FDA cleared" applications, we are simply summarizing what the expert are saying about proper application of these technologies.
Enter a keyword above and click on one of the following links to see a set of publications about that subject. HINT: Shorter keywords work better.
Here are some of our favorite queries:
Research Info for other Applications
This tool uses a broad match query so: