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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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