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Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation

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Abstract

As Light Emitting Diode (LED) devices are commercially introduced as an alternative for Low Level Laser (LLL) Therapy, the ability of LED in influencing wound healing processes at cellular level was examined. Cultured fibroblasts were treated in a controlled, randomized manner, during three consecutive days, either with an infrared LLL or with a LED light source emitting several wavelengths (950 nm, 660 nm and 570 nm) and respective power outputs. Treatment duration varied in relation to varying surface energy densities (radiant exposures). Statistical analysis revealed a higher rate of proliferation (p < 0.001) in all irradiated cultures in comparison with the controls. Green light yielded a significantly higher number of cells, than red (p < 0.001) and infrared LED light (p < 0.001) and than the cultures irradiated with the LLL (p < 0.001); the red probe provided a higher increase (p < 0.001) than the infrared LED probe and than the LLL source. LED and LLL irradiation resulted in an increased fibroblast proliferation in vitro. This study therefore postulates possible stimulatory effects on wound healing in vivo at the applied dosimetric parameters.
ORIGINAL ARTICLE
Elke M. Vinck Æ Barbara J. Cagnie
Maria J. Cornelissen Æ Heidi A. Declercq
Dirk C. Cambier
Increased fibroblast proliferation induced by light emitting diode
and low power laser irradiation
Received: 15 October 2002 / Accepted: 7 May 2003
Ó Springer-Verlag London Limited 2003
Abstract Background and Objective: As Light Emitting
Diode (LED) devices are commercially introduced as an
alternative for Low Level Laser (LLL) Therapy, the
ability of LED in influencing wound healing processes at
cellular level was examined. Study Design/Materials and
Methods: Cultured fibroblasts were treated in a con-
trolled, randomized manner, during three consecutive
days, either with an infrared LLL or with a LED light
source emitting several wavelengths (950 nm, 660 nm
and 570 nm) and respective power outputs. Treatment
duration varied in relation to varying surface energy
densities (radiant exposures). Results: Statistical analysis
revealed a higher rate of proliferation (p < 0.001) in all
irradiated cultures in comparison with the controls.
Green light yielded a significantly higher number of
cells, than red (p < 0.001) and infrared LED light
(p < 0.001) and than the cultures irradiated with the
LLL (p < 0.001); the red probe provided a higher in-
crease (p < 0.001) than the infrared LED probe and
than the LLL source. Conclusion: LED and LLL irra-
diation resulted in an increased fibroblast proliferation
in vitro. This study therefore postulates possible stimu-
latory effects on wound healing in vivo at the applied
dosimetric parameters.
Keywords Biostimulation Æ Fibroblast proliferation Æ
Light Emitting Diodes Æ Low Level Laser Æ
Tetrazolium salt
Introduction
Since the introduction of photobiostimulation into
medicine, the effectiveness and applicability of a variety
of light sources, in the treatment of a wide range of
medical conditions [1–5] has thoroughly been investi-
gated, in vitro as well as in vivo. The results of several
investigations are remark ably contradictory. This is at
least in part a consequence of the wide range of indi-
cations, as well as the wide range of suitable parameters
for irradiation and even the inability to measure the
possible effects after irradiation with the necessary
objectivity [4,6,7]. A lack of theoretical understanding
can also be responsible for the existing controversies. In
fact, theoretical understanding of the mechanisms is not
necessary to establish effects, though it is necessary to
simplify the evaluation and interpretation of the ob-
tained results. As a consequence, the widespread
acceptance of especially Low Level Laser (LLL) ther-
apy, in the early seventies is faded nowadays and bio-
stimulation by light is often viewed with scepticism [8].
According to Baxter [4,9], contemporary research and
consumption in physiotherapy is in particular focused
on the stimulation of wound healing. Tissue repair and
healing of injured skin are complex processes that in-
volve a dynamic series of events including coagulation,
inflammation, granulation tissue formation, wound
contraction and tissue remodelling [10]. This complexity
aggravates research within this cardinal indication.
Research in this domain mostly covers LLL studies,
but the current commercial availability of other light
sources, appeals research to investigate as well the effects
of those alternative light sour ces, e.g. Light Emitting
Diode (LED) apparatus.
The scarcity of literature on LED is responsible for
consultation of literature originating from LLL studies
Lasers Med Sci (2003) 18: 95–99
DOI 10.1007/s10103-003-0262-x
E.M. Vinck Æ B.J. Cagnie Æ D.C. Cambier
Department of Rehabilitation Sciences and Physiotherapy,
Ghent University, 9000 Ghent, Belgium
M.J. Cornelissen Æ H.A. Declercq
Department of Human Anatomy, Embryology,
Histology and Medical Physics, Ghent University,
9000 Ghent, Belgium
E.M. Vinck (&)
Ghent University, Faculty of Medicine and Health Sciences,
Department of Rehabilitation Sciences and Physiotherapy
(REVAKI), University Hospital De Pintelaan 185 (6K3),
9000 Ghent, Belgium
Tel.: +32 (0)9/240 52 65
Fax: +32 (0)9/240 38 11
E-mail: elke.vinck@ugent.be
[11] but it may be wondered if this literature is rep-
resentative for that purpose. As in the early days of
LLL therapy, the stimulating effects upon biological
objects were expl ained by its coherence [12,13], while
the beam emitted by LED’s on the contrary produces
incoherent light. Though the findings of some scien-
tists [9,14,15,16,17] pose nowadays that the coherence
of the light beam is not responsible for the effects of
LLL therapy. Given that the cardinal difference be-
tween LED and LLL therapy, coherence, is not of
remarkable importance in providing biological re-
sponse in cellular monolayers [5], one may consult
literature from LLL studies to refer to in this LED
studies.
The purpose of this preliminary study is to examine
the hypothesis that LED irradiation at specific output
parameters can influence fibroblast proliferation.
Therefore, irradiated fibroblasts cultures were compared
with controls. The article reports the findings of this
study in an attempt to promote further discussion and
establish the use of LED.
Materials and methods
Cell isolation and culture procedures
Fibroblasts were obtained from 8-days old chicken embryos.
Isolation and disaggregation of the cells was performed with
warm trypsin according the protocol described by Ian Freshney
(1994) [18]. The primary explants were cultivated at 37 ° Cin
Hanks’ culture Medium supplemented with 10% Fetal Calf
Serum, 1% Fungizone, 1% L-Glutamine and 0.5% Penicillin-
Streptomycin. When cell growth from the explants reached
confluence, cells were detached with trypsine and subcultured
during 24 hours in 80-cm
2
culture flasks (Nunc
TM
)in12mlof
primary culture medium. After 72 hours the cells were removed
from the culture flasks by trypsinization and counted by Bu
¨
rker
hemocytometry. For the experiment, cells from the third passage
were plated in 96-well plates (Nunc
TM
) with a corresponding area
of 0.33 cm
2
, they were subcultured at a density of 70.000 cell/
cm
2
. Cultures were maintained in a humid atmosphere at 37 °C
during 24 hours.
All supplies for cell culture were delivered by N.V. Life
Technologies, Belgium, except for Fetal Calf Serum (Invitrogen
Corporation, UK)
Irradiation sources
In this study two light sources, a Light Emitting Diode (LED)
device and a Low Level Laser (LLL) device, were used in com-
parison to control cultures.
The used LLL was an infrared, GaAlAs Laser (Unilaser 301P,
MDB-Laser, Belgium) with an area of 0.196 cm
2
, a wavelength of
830 nm, a power output ranging from 1–400 mW and a frequency
range from 0–1500 Hz.
The Light Emitting Diode device (BIO-DIO preprototype,
MDB-Laser, Belgium), consisted of three wavelengths emitted by
separate probes. A first probe, emitting green light, had a wave-
length of 570 nm (power-range, 10–0.2 mW), the probe in the red
spectrum, had a wavelength of 660 nm (power-range, 80–15 mW)
and the third probe had a wavelength of 950 nm (power-range,
160–80 mW) and emitted infrared light. The area of all three
probes was 18 cm
2
and their frequency was variable within the
range of 0–1500 Hz.
Exposure regime
Prior to irradiation, the 96-well plates were microscopically veri-
fied, to guarantee that the cells were adherent, and to assure that
there was no confluence, nor contamination. Following aspiration
of 75% Hanks’ culture Medium irradiation started. The remaining
25% (50 ll) medium avoided dehydration of the fibroblasts
throughout irradiation.
The 96-well plates were randomly assigned in the treated (LLL
or green, red or infrared LED’s) or the control group.
For the treatments in this study, the continuous mode was
applied as well for the LLL as for the three LED-probes. The
distance from light source to fibroblasts was 0.6 cm. LLL therapy
consisted of 5 seconds irradiation at a power output of 40 mW
resulting in a radiant exposure of 1 J/cm
2
. The infrared and the red
beam delivered radiant exposures of 0.53 J/cm
2
and the green beam
emitted 0.1 J/cm
2
, corresponding to exposure-times of respectively
1 minute, 2 minutes or 3 minutes and a respective power output of
160 mW, 80 mW or 10 mW.
After these handlings, the remaining medium was removed and
new Hanks’culture medium was added, followed by 24 hours of
incubation.
One irradiation (LLL or LED) was performed daily, during three
consecutive days according to the aforementioned procedure. Con-
trol cultures underwent the same handling, but were sham-irradiated.
Determination of cell proliferation
The number of cells within the 96-well plates, as a measure for
repair [19], was quantified by a sensitive and reproducible colori-
metric proliferation assay [20, 21]. The colorimetric assay was
performed at two different points of time to determine the duration
of the effect of the used light sources.
This assay exists of a replacement of Hanks’culture medium by
fresh medium containing tetrazolium salt, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) 24 or 72 hours after
the third irradiation, for MTT analysis as described by Mosmann
(1983) [22]. Following a 4 hour incubation at 37 °C, the MTT
solution was substituted by lysing buffer, isopropyl alcohol. The
plates were temporarily shaken to allow dissolution of the pro-
duced formazan crystals. After 30 minutes of exposure to the lysing
buffer, absorbance was measured. The absorbance at 400 to
750 nm, which was proportional to fibroblast proliferation, was
determined using an ELx800 counter (Universal Microplate
Reader, Bio-Tek Instruments INC).
The complete procedure from isolation to MTT assay was
executed six times (Trial A, B, C, D, E and F) while it was
impossible to irradiate all the investigated number of wells with the
same LED apparatus on one day. All the trials included as much
control as irradiated wells, but the number of control and irradi-
ated wells in each trial varied, depending on the number of avail-
able cells after the second subculturing. A further consequence of
the available number of cells is the number of probes examined per
trial. Varying from 4 probes in trial A and F to 1 probe in trial B,
C, D and E.
Incubation period before proliferation analyses numbered 24
hours. To investigate if the stimulatory effect tends to occur
immediately after irradiation or after a longer period of time,
incubation in trial F lasted 72 hours.
An overview of the followed procedures regarding incubation
time before proliferation analysis, number of analysed wells for
each trial and the number of probes examined per trial is given in
Table 1. As a consequence of the differences in procedures followed
and because each trial started from a new cell line, the results of the
five trials must be discussed separately.
Statistical analysis
Depending on the amount of groups to be compared within each
trial and depending on the p-value of the Kolmogorov-Smirnov
96
test of normality, a T-test or one-way ANOVA was used for
parametrical analyses and a Kruskal-Wallis or Mann-Whitney-U
test was used for nonparametrical comparisons. Statistical signifi-
cance for all tests was accepted at the 0.05 level. For this analysis
Statistical Package for Social Sciences 10.0 (SPSS 10.0) was used.
Results
The results, presented in Table 1, show that cell counts
by means of MTT assay revealed a significant
(p < 0.001) increase in the number of cells in compar-
ison to their respective sham-irradiated controls, for all
the irradiated cultures of trial A, B, C, D, and E, except
the irradiated groups in trial F.
Moreover, the results of trial A showed that the effect
of the green and red LED probe was significantly
(p < 0.001) higher than the effect of the LLL probe.
With regard to the amount of proliferation the green
probe yielded a significantly higher number of cells , than
the red (p < 0.001) and the infrared probe (p < 0.001).
Furthermore, the red probe provided a higher increase
in cells (p < 0.001) than the infrared probe.
The infrared LED source and the LLL provided a
significant (p < 0.001) higher number of cells than the
control cultures but no statistical significant difference
was recorded between both light sources.
The trials A, B, C, D, and E, regardless of the number
of probes used in each trial, were analysed after 24 hours
of incubation after the last irradiation. The incubation
period of trial F lasted 72 hours.
The means of trial F illustrated that the effect was
opposite after such a long incubation. The control cul-
tures had significantly (p < 0.001) more fibroblasts than
the irradiated cultures, with the exception of the LED-
infrared group that showed a not significant increase of
cells. Further analysis, revealed that the green probe
yielded a significantly lower numb er of cells, than the red
(p < 0.001) and the infrared probe (p < 0.001) and
that the red probe provided a higher decrease
(p < 0.001) than the infrared probe. Laser irradiation
induced a significant decrease of fibroblasts in compar-
ison to the infrared irradiated cultures (p < 0.001) and
the control cultures (p ¼ 0.001). LED irradiation with
the green and the red probe revealed no statistical sig-
nificant differences.
Discussion
Despite the failure of some studies [2,23] to demonstrate
beneficial effects of laser and photodiode irradiation at
relatively low power levels (< 500 mW) on fibroblast
proliferation, this study provides experimental support
for a significant increased cell proliferation. Therefore
these results confirm previous studies that yielded
beneficial stimulating effect [1,15,24,25]. Remarkably
though is the higher increase, noted after irradiation at
lower wavelengths (570 nm). Van Breughel et al. [26]
observed a general decrease in absorption at longer
wavelengths and concluded that several molecules in fi-
broblasts serve as photoacceptors, resulting in a range of
absorption peaks (420, 445, 470, 560, 630, 690 and
730 nm). The wavelength of the used ‘green’ LED probe
is the closest to one of these peaks.
Karu [5] also emphasises that the use of the appro-
priate wavelength, namely within the bandwidth of the
absorption spectra of photoacc eptor molecules, is an
important factor to consider.
In this particular context, penetration depth can
almost be ignored as virtuall y all wavelengths in the
visible and infrared spectrum will pass through a
monolayer cell culture [12]. The irradiance (W/cm
2
)on
the contrary, could have had an important influence on
the outcome of this study. The higher increased prolif-
eration by the lower wavelengths is possibly a result of
the lower irradiance of these wavelengths. Lower irra-
diances are confir med by other experiments to be more
effective than higher irradiances [11,16,26].
The used radiant exposures reached the tissue inter-
action threshold of 0.01 J/cm
2
as described by Po
¨
ntinen
[17], but in the scope of these results it also needs to be
noticed that there is a substantial difference in radiant
exposure between the LLL (1 J/cm
2
), the green LED
probe (0.1 J/cm
2
) and the remaining LED probes
(0.53 J/cm
2
). Consequently, the results of especially trial
Table 1 Fibroblast proliferation after LED and LLL irradiation
Groups Mean number of
fibroblasts
a
Trial A
n = 64 Control 0.595 ± 0.056
TP = 24 h Irradiated (LLL) 0.675 ± 0.050*
Irradiated (LED-infrared) 0.676 ± 0.049*
Irradiated (LED-red) 0.741 ± 0.059*
Irradiated (LED-green) 0.775 ± 0.043*
Trial B
n = 368 Control 0.810 ± 0.173
TP = 24 h Irradiated (LLL) 0.881 ± 0.176*
Trial C
n = 368 Control 0.810 ± 0.173
TP = 24 h Irradiated (LED-infrared) 0.870 ± 0.178*
Trial D
n = 192 Control 0.886 ± 0.084
TP = 24 h Irradiated (LED-red) 0.917 ± 0.066*
Trial E
n = 192 Control 0.818 ± 0.075
TP = 24 h Irradiated (LED-green) 0.891 ± 0.068*
Trial F
n = 64 Control 0.482 ± 0.049
TP = 72 h Irradiated (LLL) 0.454 ± 0.065*
Irradiated (LED-infrared) 0.487 ± 0.044
Irradiated (LED-red) 0.446 ± 0.044*
Irradiated (LED-green) 0.442 ± 0.035*
a
Mean number of fibroblasts as determined by MTT analy-
sis ± SD and significances (*p < 0.001) in comparison to the
control group
n = number of analysed wells for each group within a trial
TP = Time Pre-analysis, incubation time before proliferation
analysis was performed
97
A and F must be interpreted with the necessary caution.
It is possible that the determined distinction between the
used light sources and the used probes is a result from
the various radiant exposures applied during the treat-
ments of the cultures.
Notwithstanding the increased proliferation revealed
with MTT analysis 24 hours after the last irradiation,
this study was unable to demonstrate a stimulating ef-
fect when analysis was performed 72 hours after the last
irradiation. Moreover, this longer incubation period
even yielded an adverse effect. Although a weakening of
the photostimulating influence over time is acceptable,
it can not explain a complete inversion. Especially in the
knowledge that a considerable amount of authors still
ascertain an effect after a longer incubation period
[24,27]. In an attempt to illuminate this finding, one can
suppose that the circadian response of the cells triggered
by the LED and the LLL [12,28] forfeited after a pro-
longed period (72 hours) in the dark. The most obvious
explanation is even though a decreased vitality and
untimely cell death in the irradiated cell cultures as a
result of reaching confluence at an earlier point of time
than the control cultures. The cells of a confluent
monolayer have the tendency to inhibit growth and fi-
nally die when they are not subcultured in time. No
other reasonable explanations could be found for this
discrepancy.
Photo-modulated stimulation of wound healing is
often viewed with scepticism. The real benefits of Lig ht
Emitting Diodes, if any, can only be established by
histological and clinical investigations performed under
well controlled protocols. Despite these remarks, this
study suggests beneficial effects of LED and LLL
irradiation at the cellular lev el, assuming potential
beneficial clinical results. LED application on cutane-
ous wounds of human skin may be assumed useful at
the appl ied dosimetric parameters, but future investi-
gation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in
the use of the most effective parameters for LED
treatment. Subsequently res olving the lack of scientific
evidence and nullifying the controversial acknowledge-
ments of the effect of LED can bring about a wide-
spread acceptance for the use of LED in clini cal
settings.
Persons in good health rarely require treatment for
wound healing, as posed by Reddy et al. [1,3] light has a
possible optimal effect under conditions of impaired
healing. Postponed wound healing is a time-consuming
and often expensive complication. Thus, future pros-
pects must remind to examine the therapeutic efficacy of
LED on healing-resistant wounds.
Acknowledgements The authors are grateful to Prof. Deridder for
supplying the laboratory as well as the material necessary for this
investigation, and to Ms. Franc¸ ois, laboratory worker, for pro-
viding the culture medium and for the technical support.
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... Vinck et al. reported that different wavelengths of the light-emitting diode (950 nm, 660 nm, and 570 nm) and low-power laser irradiation could increase fibroblast proliferation. Among them, the wavelength of 570 nm exhibited higher efficacy [38]. Thus, we assume that DPL, which encompasses the wavelength of the above reports, is beneficial for acne treatment and may accelerate the onset of the effect of oral isotretinoin, even at a low dose. ...
... In our study, LLLT by DPL at low energy had multivalent effects on the modulation of the skin barrier and PIH prevention. A significant reduction in TEWL and EI by DPL was observed, which might be attributed to LLLT-mediated fibroblast proliferation and inflammation reduction [38,40]. Additionally, MI was reduced. ...
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... Accordingly, this wavelength can also inhibit the plasminogen activator [28]. The results of the present study show that 808-nm diode laser radiation has a significant stimulatory effect on human gingival fibroblast proliferation rate, which is consistent with the results of the Kreisler et al.'s study [19]. Notably, the laser investigated in the present study was radiated at the dose of 5.2.J/cm 2 /a day (a total of 15.2J/cm 2 ) for three consecutive days. ...
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Statement of the problem: In recent years, regeneration of periodontal soft tissues in the reconstruction of periodontal defects and the finding of suitable membranes and graft materials for the placement of autogenous grafts have been of great interest in various studies. In this regard, the proliferation and adhesion of regenerative cells are two linchpins of the complete regenerative process. Purpose: This study aimed to evaluate the effects of low-level laser beams on the attachment and the proliferation of human gingival fibroblasts in the presence of acellular dermal matrix (ADM). Materials and method: All the experiments were conducted compared to tissue culture plate in four groups as follows: (1) Fibroblast+ADM+laser, (2) Fibroblast+ADM+ no laser, (3) Fibroblast + laser radiation, and (4) Fibroblast+ no laser. In this experimental study, the primary attachment was evaluated by passing 8h from seeding of 5×105 gingival fibroblasts with or without a single dose (15.6 J/cm2) of laser radiation. Cell proliferation rate was also examined at 24, 48, and 72 hours after cell culture, following exposure to 5.2 J/cm2 of laser at each day of examination. Thereafter, fibroblasts were incubated under the normal culture condition (at 37°C, 5% CO2) in high glucose Dulbecco's Modified Eagle's medium (DMEM) medium supplemented with 10% fetal bovine serum, 1% glutamax, and 1% penicillin/streptomycin. Subsequently, the cellular viability was assessed on each time point using MTS calorimetric assay. The obtained data were statistically analyzed by applying ANOVA and Tukey tests. Results: There was a significant difference among the means of these four groups in terms of the proliferation of fibroblasts at 24, 48 and 72 hours (p< 0.001). Moreover, there was no significant difference among the means of two groups in terms of fibroblastic attachment in 8 hours (p< 0.2). The fibroblast group has shown the highest proliferation rate among all groups after laser radiation. Conclusion: It was indicated that the laser radiation increases the fibroblast cell proliferation. Accordingly, although this increase was higher in the fibroblast group alone compared to the fibroblasts cultured on acellular dermal matrix, the laser radiation did not significantly increase the attachment of fibroblast cells to acellular dermal matrix.
... Histologically, several studies have revealed that LLLT increases collagen fiber deposition, promotes fibroblast proliferation, and enhances microcirculation in local tissues, which results in virtuous physiological cycles [32][33][34]. Our histopathological outcomes based on epithelial thickness revealed similar results, showing that LLLT can significantly reduce tissue fibrosis based on epithelial thicknesses. ...
Article
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Little is known about alternative treatment options for rhinosinusitis (RS). We aimed to evaluate the efficacy of low-level laser therapy (LLLT) for RS in experimentally induced rabbit models of RS. A total of 18 rabbits were divided into four groups: a negative control group (n = 3), an RS group without treatment (n = 5, positive control group), an RS group with natural recovery (n = 5, natural recovery group), and an RS group with laser irradiation (n = 5, laser-treated group). Computed tomography and histopathological staining were performed for each group. mRNA and protein expression levels of local cytokines (IFN-γ, IL-17, and IL-5) were also measured. Tissue inflammation revealed a significant improvement in the laser-treated group compared with the RS and natural recovery groups (p < 0.01). In addition, sinus opacification in the CT scans and cytokine expression was reduced in the laser-treated group, though without statistical significance. LLLT could be an effective option for the management of RS concerning radiological, histological, and molecular parameters.
... The results indicate that both wavelength and cell type affected cell growth response to laser therapy [4]. In vitro, laser low power therapy with wavelengths of different induces a greater rate of breed in fibroblasts, suggesting that at particular dosimetric parameters, this treatment may have a stimulating influence on incision healing in vivo [5]. The wound-healing effect of visible and infrared irradiation occurs resulting from the local light impact, but many details of their processes are unknown [6]. ...
Conference Paper
Objective: This research aimed at evaluating whether broad-spectrum light can support and accelerate cell proliferation in conjunction with laser therapy. Background Data: Broad-spectrum light can reduce the effect of laser light to some extent or completely. There are a few experiments that examine the gain or downside of integrating broad- spectrum light with laser irradiation. Methods: Fibroblasts were irradiated at various doses (J/cm²) using 635 and 808nm laser in the dark and light. Using MTT (3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assays, changes in cell proliferation have been testing. Results: The findings show that low-level laser therapy (LLLT) has a beneficial effect on this cell type. The results revealed that dosages of 3.7, 5, 7.4, and 11 J/cm² were adequate for fibroblast cells to undergo observable changes. The MTT assay shows that various dosages using 808 nanometers in the lit medium were more active than 635 nm in enhancing cell-reproduction and no as effective as 635 nm in the dark. Conclusion: Our findings show that cells respond optimally in the dark medium to wavelengths of 635 nm and in the light to wavelengths of 808 nm, preserving cell viability.
... Previous studies showed beneficial effects of green light for treating a wide range of dermatologic conditions such as chronic lichen sclerosus, solar keratoses, port wine stains, basal cell carcinoma, and actinic keratosis [22][23][24][25][26]. Fushimi reported the superior effects of green LEDs over red and blue LEDs for reducing wound size in mice and inducing HaCaT cell migration by enhancing migratory and proliferative mediators including leptin, interleukin-8, and VEGF [27]. Similar outcomes were reported by a different study in which green light exhibited a higher fibroblast proliferation rate than red light and infrared light [28]. Interestingly, green light could be a new powerful therapeutic method beyond the red light. ...
Article
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Light penetration depth in the scalp is a key limitation of low-level light therapy for the treatment of androgenetic alopecia (AGA). A novel light emitting diode (LED) microneedle patch was designed to achieve greater efficacy by enhancing the percutaneous light delivery. The study aimed to investigate the efficacy and safety of this device on hair growth in mice. Thirty-five male C57BL/6 mice which their dorsal skin was split into upper and lower parts to receive either LED irradiation alone or LED irradiation with a microneedle patch. Red (629 nm), green (513 nm), and blue light (465 nm) at an energy dose of 0.2 J/cm2 were applied once daily for 28 days. Outcomes were evaluated weekly using digital photographs. Histopathological findings were assessed using a 6 mm punch biopsy. A significant increase in hair growth was observed in the green light, moderate in the red light, and the lowest in the blue light group. The addition of the microneedle patch to LED irradiation enhanced greater and faster anagen entry in all the groups. Histopathology showed an apparent increase in the number of hair follicles, collagen bundles in the dermis, angiogenesis, and mononuclear cell infiltration after treatment with the green-light LED microneedle patches. No serious adverse effects were observed during the experiment. Our study provides evidence that the newly developed green-light LED microneedle patch caused the optimal telogen-to-anagen transition and could lead to new approaches for AGA. Microneedle stimulation may aid percutaneous light delivery to the target hair follicle stem cells.
... In previous studies , energy densities higher than the 10 J/cm 2 used in this study, such as 160 or 320 J/cm 2 , were applied. Another study showed that fibroblasts proliferated after LED irradiation with a low energy level of 1 J/cm 2 or less, which supports this reasoning [22]. ...
Article
Purpose: This study aimed to investigate the proper wavelengths for safe levels of light-emitting diode (LED) irradiation with bactericidal and photobiomodulation effects in vitro. Methods: Cell viability tests of fibroblasts and osteoblasts after LED irradiation at 470, 525, 590, 630, and 850 nm were performed using the thiazolyl blue tetrazolium bromide assay. The bactericidal effect of 470-nm LED irradiation was analyzed with Streptococcus gordonii, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, and Tannerella forsythia. Levels of nitric oxide, a proinflammatory mediator, were measured to identify the anti-inflammatory effect of LED irradiation on lipopolysaccharide-stimulated inflammation in RAW 264.7 macrophages. Results: LED irradiation at wavelengths of 470, 525, 590, 630, and 850 nm showed no cytotoxic effect on fibroblasts and osteoblasts. LED irradiation at 630 and 850 nm led to fibroblast proliferation compared to no LED irradiation. LED irradiation at 470 nm resulted in bactericidal effects on S. gordonii, A. actinomycetemcomitans, F. nucleatum, P. gingivalis, and T. forsythia. Lipopolysaccharide (LPS)-induced RAW 264.7 inflammation was reduced by irradiation with 525-nm LED before LPS treatment and irradiation with 630-nm LED after LPS treatment; however, the effects were limited. Conclusions: LED irradiation at 470 nm showed bactericidal effects, while LED irradiation at 525 and 630 nm showed preventive and treatment effects on LPS-induced RAW 264.7 inflammation. The application of LED irradiation has potential as an adjuvant in periodontal therapy, although further investigations should be performed in vivo.
... The use of FIR radiation on wound healing of rat thick skin has demonstrated an improvement in the formation of anti-inflammatory cytokine (TGF-1), the growth factor and which activates fibroblasts for enhanced wound healing. FIR radiation has also been utilised to directly promote fibroblast proliferation, which boosted fibroblast propagation in vitro [43]. ...
Article
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Far infrared (FIR) radiation (3-100 µm) is an electromagnetic spectrum commonly studied for biological effects. This article aims to discuss using Far infrared radiation with sub-division (4-24 µm) of this waveband to stimulate tissues and cells and is considered an effective therapeutic modality for specific medical disorders. The IR application as a medical therapy has advanced rapidly in recent years. For example, IR therapy like IR-emitting apparel and materials that can be run solely by body heat (does not need an external power supply) have been developed. New methods for providing FIR radiation to the human body have emerged due to technological advancements. Specialty saunas and lamps that emit pure FIR radiation have become effective, safe, and widely used therapeutic sources. Fibers infused with thermide, FIR emitting ceramic nanomaterials, and knitted into fabrics are used as clothes and apparel to produce FIR radiation and benefit from its effects. A deeper understanding of FIR's significant innovations and biological implications could aid in improving therapeutic efficacy or developing new methods that use FIR wavelengths.
... The authors called for more randomized controlled trials to support the use of PBMT, particularly Laser and LED light sources.42 There are data showing that there is no difference in the interaction of a laser and a LED with the human tissue.[43][44][45] Despite the need for further evaluation, we must acknowledge the advantages of LEDs, which include no laser safety considerations, the ability to irradiate a large area of tissue at once, much lower cost per mW, and the possibility of wearable or take-home devices. ...
Article
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Objective: This study aimed to compare the historical incidence rate of severe oral mucositis (OM) in head and neck cancer patients undergoing definitive concurrent chemoradiation therapy (CRT) versus a prospective cohort of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) treated with prophylactic photobiomodulation therapy (PBMT). Methods: This US-based, institutional, single-arm, phase Ⅱ prospective clinical trial was initiated in 50 patients (age ≥ 18 years, Karnofsky Performance Scale Index > 60, with locally advanced HNSCC (excluding oral cavity) receiving definitive or adjuvant radiation therapy (RT) with concurrent platinum-based chemotherapy (CT). PBMT was delivered three times per week throughout RT utilizing both an intraoral as well extraoral delivery system. Primary outcome measure was incidence of severe OM utilizing both the National Cancer Institute Common Toxicity Criteria, version 4.0 (NCI-CTCAE) Grade ≥3 and the World Health Organization Mucositis Grading Scale (WHO) Grade ≥3 versus historical controls; secondary outcome measures included time to onset of severe OM following therapy initiation. Results: At baseline, all patients included in final analysis (N = 47) had OM Grade 0. Average RT and CT dose was (66.3 ± 5.1) Gy and (486.1 ± 106.8) mg/m2, respectively. Severe OM was observed in 11 of 47 patients (23%, confidence interval: 12, 38). OM toxicity grade trended upward during treatment, reaching a maximum at 7 weeks (WHO: 1.8 vs. NCI-CTCAE: 1.7). Subsequently, OM grade returned to baseline 3 months following completion of RT. The mean time to onset of severe OM was (35 ± 12) days. The mean time to resolution of severe OM was (37 ± 37) days. Conclusions: Compared to historical outcomes, PBMT aides in decreasing severe OM in patients with locally advanced HNSCC. PBMT represents a minimally invasive, prophylactic intervention to decrease OM as a major treatment-related side effect.
... Application of low-intensity light therapy has gained increasing attention for medical purpose in cancer treatment [2][3][4][5][6]. According to this method, the energy is transferred to living organisms inducing changes in biological processes [7][8][9][10][11][12][13]. e concept of low-intensity light therapy requires the use of photosensitive molecules, which respond to external light stimuli. ...
Article
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This study aimed to evaluate the therapeutic efficacy of low-intensity visible light responsive nanocolloids of a Pt-based drug using a 2D and three-dimensional (3D) in vitro cancer cell model. Biocompatible and biodegradable polymeric nanocolloids, obtained using the ultrasonication method coupled with Layer by Layer technology, were characterized in terms of size (100 ± 20 nm), physical stability, drug loading (78%), and photoactivation through spectroscopy studies. The in vitro biological effects were assessed in terms of efficacy, apoptosis induction, and DNA-Pt adducts formation. Biological experiments were performed both in dark and under visible light irradiation conditions, exploiting the complex photochemical properties. The light-stimuli responsive nanoformulation gave a significant enhancement in drug bioactivity. This allowed us to achieve satisfying results by using nanomolar drug concentration (50 nM), which was ineffective in darkness condition. Furthermore, our nanocolloids were validated in 3D in vitro spheroids using confocal microscopy and cytofluorimetric assay to compare their behavior on culture in 2D monolayers. The obtained results confirmed that these nanocolloids are promising tools for delivering Pt-based drugs.
Article
This work reports an optomechanical, electronic system consisting of LEDs emitting light in the near-infrared region. The 147 light-emitting diodes were distributed in a semi-spherical dome so that each focused light upon the center of the radius. The LEDs received electrical polarization so that the optical power of each provided 16 ± 0.36 mW for a total of 2.4 W. The developed system allows multifunctional applications in phototherapy, making it possible to perform this clinical technique for the treatment of skin lesions and contusions at the musculoskeletal level. The geometry of the developed system also allows the use of phototherapy in the treatment of neurological pathologies. In terms of instrumentation, the three-dimensional light intensity distribution and the thermographic images were obtained using three working distances from the system to the target. Three-dimensional light intensity distributions were obtained considering each working distance. These were inserted in the thermographic images as indications of the emitted temperature distributions. This work verified that the optomechanical and electronic system developed with LEDs may be used for multiple applications in phototherapy.
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The entomopathogenic fungus Neozygites parvispora (Entomophthorales: Zygomycetes) grows in vitro as irregularly rod-shaped hyphal bodies in a complex medium. In order to simplify the medium composition and determine growth-promoting compounds for the cultivation of this fungus, we were looking for a rapid and quantitative method to estimate the number of living cells in small volumes of liquid culture. A colorimetric method for the determination of cell densities using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] proved to be more accurate and timesaving than conventional hemocytometer counting.
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This work is supported and managed through the NASA Marshall Space Flight Center-SBIR Program. Studies on cells exposed to microgravity and hypergravity indicate that human cells need gravity to stimulate cell growth. As the gravitational force increases or decreases, the cell function responds in a linear fashion. This poses significant health risks for astronauts in long termspace flight. LED-technology developed for NASA plant growth experiments in space shows promise for delivering light deep into tissues of the body to promote wound healing and human tissue growth. This LED-technology is also biologically optimal for photodynamic therapy of cancer. .
Chapter
The first publications about low-power laser therapy (then called laser biostimulation) appeared more than 30 years ago. Since then, approximately 2000 studies have been published on this still controversial topic. In the 1960s and 1970s, doctors in Eastern Europe, and especially in the Soviet Union and Hungary, actively developed laser biostimulation. However, scientists around the world harbored an open skepticism about the credibility of studies stating that low-intensity visible-laser radiation acts directly on an organism at the molecular level. The coherence of laser radiation for achieving stimulative effects on biological objects was more than suspect. Supporters in Western countries, such as Italy, France, and Spain, as well as in Japan and China also adopted and developed this method, but the method was — and still remains — outside mainstream medicine. The controversial points of laser biostimulation, which were topics of great interest at that time, were analyzed in reviews that appeared in the late 1980s.
Article
Laboratory studies of biostimulation are predominantly of Eastern Block origin and for the most part are confined to measurements of DNA, RNA and protein synthesis in E. coli, yeasts and transformed mammalian cells. Few of these studies have compared the effects induced by coherent versus incoherent sources and fewer provide sufficient details of the exposure regime to enable the work to be repeated. We, like many other laboratories, found the published claims unconvincing. Given our scepticism we have undertaken a series of pilot experiments in an attempt to highlight possible sources of error in previous studies. This paper reports our results and is an attempt to promote further discussion.
Article
The surgical, ophthalmological, and dermatological applications of high power lasers are well known and easily understood. What is neither as well known nor as easily understood is that lasers at powers that are orders of magnitude smaller have also been used in the laboratory and clinic for nearly 30 years to modulate cell function, lessen pain, and accelerate healing of soft tissue injuries. This article analyzes the rationale of this approach, examines the utility of laser therapy in its most common clinical applications, reviews and synthesizes the findings, and concludes that although laboratory findings seem authentic, clinical utility remains unestablished. © 1995 Wiley-Liss, Inc.
Article
Low energy laser photostimulation at certain wavelengths can enhance tissue repair by releasing growth factors from fibroblasts and stimulate the healing process. This study was designed to evaluate the influence of laser photostimulation on collagen production in experimentally tenotomized and repaired rabbit Achilles tendons. A total of 24 male New Zealand rabbits, ages 10-12 weeks, were used. Following tenotomy and repair, the surgical hind limbs of the rabbits were immobilized in customized polyurethane casts. The experimental animals were treated with a 632.8 nm He:Ne laser daily at 1.0 J cm(-2) for 14 days. Control animals were sham treated with the laser head. On the fifth day after repair, the casts were removed to allow the animals to bear weight on the lower extremity. The animals were euthanized on the 15th postoperative day, then, the Achilles tendons were excised, processed and analyzed. Biochemical analyses of the tendons revealed a 26% increase in collagen concentration with laser photostimulation indicating a more rapid healing process in treated tendons compared to controls. Sequential extractions of collagen from regenerating tissues revealed that the laser photostimulated tendons had 32% and 33% greater concentrations of neutral salt soluble collagen and insoluble collagen, respectively, than control tendons suggesting an accelerated production of collagen with laser photostimulation. A significant decrease (9%) in pepsin soluble collagen was observed in laser-treated tendons compared to controls. There were no statistically significant differences recorded in the concentrations of hydroxypyridinium crosslinks and acid soluble collagen between treated and control tendons. This study of laser photostimulation on tendon healing in rabbits suggests that such therapy facilitates collagen production in a manner that enhances tendon healing.
Article
A two-stage postal survey was carried out among Chartered physiotherapists in Northern Ireland to assess current clinical practice in low level laser therapy (LLLT), and thus indicate appropriate areas for future research. Results from the analysis of 116 returned completed questionnaires (response rate=63.1%) are presented. These show that LLLT has become a relatively popular treatment modality within the province. Based upon the subjective ratings of respondents, LLLT seems indicated for a range of conditions, including rheumatoid arthritis, shingles, and various types of ulcer — but especially for burns, for which LLLT was rated ‘very effective’. However, 94% of respondents complained about the lack of information/instruction available to them on LLLT, especially concerning the selection of optimal treatment parameters. This may represent one reason for the variable results reported by physiotherapists in the treatment of a number of conditions which are recommended for LLLT by manufacturers (eg osteoarthritis and muscle spasms). Given the popularity of LLLT, its apparent efficacy, and the dissatisfaction expressed by physiotherapists, future research should aim to establish laser's mechanisms of action, the optimal treatment parameters for therapeutic effects, and (based upon this) its efficacy in the treatment of selected conditions by means of properly organised and controlled clinical trials.