Irradiation at 830 nm stimulates nitric oxide production and inhibits pro-inflammatory cytokines in diabetic wounded fibroblast cells.

Abstract

Wound healing in diabetic patients remains a chief problem in the clinical setting and there is a strong need for the development of new, safe, reliable therapies. This study aimed to establish the effect of irradiating diabetic wounded fibroblast cells (WS1) in vitro on pro-inflammatory cytokines and the production of nitric oxide (NO).
Normal, wounded and diabetic wounded WS1 cells were exposed to an 830 nm laser with 5 J/cm(2) and incubated for a pre-determined amount of time. Changes in cellular viability, proliferation and apoptosis were evaluated by the Trypan blue assay, VisionBlue fluorescence assay and caspase 3/7 activity respectively. Changes in cytokines (interleukin--IL-6, IL-1 beta and tumour necrosis factor-alpha, TNF-alpha) were determined by ELISA. NO was determined spectrophotometrically and reactive oxygen species (ROS) was evaluated by immunofluorescent staining.
Diabetic wounded WS1 cells showed no significant change in viability, a significant increase in proliferation at 24 and 48 hours (P<0.001 and P<0.01 respectively) and a decrease in apoptosis 24 hours post-irradiation (P<0.01). TNF-alpha levels were significantly decreased at both 1 and 24 hours (P<0.05), while IL-1 beta was only decreased at 24 hours (P<0.05). There was no significant change in IL-6. There was an increase in ROS and NO (P<0.01) 15 minutes post-irradiation.
Results show that irradiation of diabetic wounded fibroblast cells at 830 nm with 5 J/cm(2) has a positive effect on wound healing in vitro. There was a decrease in pro-inflammatory cytokines (IL-1 beta and TNF-alpha) and irradiation stimulated the release of ROS and NO due to what appears to be direct photochemical processes.

Full text

Page 1

Lasers in Surgery and Medicine 42:494–502 (2010)
Irradiation at 830nm Stimulates Nitric Oxide Production
and Inhibits Pro-Inflammatory Cytokines in Diabetic
Wounded Fibroblast Cells
Nicolette N. Houreld, D.Tech, Palesa R. Sekhejane, M.Tech, and Heidi Abrahamse, PhD*
Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, P.O. Box 17011,
Doornfontein 2028, South Africa
Background and Objective: Wound healing in diabetic
patients remains a chief problem in the clinical setting and
there is a strong need for the development of new, safe,
reliable therapies. This study aimed to establish the effect
of irradiating diabetic wounded fibroblast cells (WS1)
in vitro on pro-inflammatory cytokines and the production
of nitric oxide (NO).
Materials and Methods: Normal, wounded and diabetic
wounded WS1 cells were exposed to an 830nm laser with
5J/cm2andincubatedforapre-determinedamountoftime.
Changes in cellular viability, proliferation and apoptosis
were evaluated by the Trypan blue assay, VisionBlueTM
fluorescence assay and caspase 3/7 activity respectively.
Changesincytokines(interleukin—IL-6,IL-1bandtumour
necrosis factor-alpha, TNF-a) were determined by ELISA.
NO was determined spectrophotometrically and reactive
oxygenspecies(ROS)wasevaluatedbyimmunofluorescent
staining.
Results: Diabetic wounded WS1 cells showed no signi-
ficant change in viability, a significant increase in prolif-
erationat 24 and 48hours
respectively) and a decrease in apoptosis 24hours post-
irradiation (P<0.01). TNF-a levels were significantly
decreased at both 1 and 24hours (P<0.05), while IL-1b
was only decreased at 24hours (P<0.05). There was no
significant change in IL-6. There was an increase in ROS
and NO (P<0.01) 15minutes post-irradiation.
Conclusion: Results show that irradiation of diabetic
wounded fibroblast cells at 830nm with 5J/cm2has a
positive effect on wound healing in vitro. There was a
decrease in pro-inflammatory cytokines (IL-1b and TNF-a)
and irradiation stimulated the release of ROS and NO due
to what appears to be direct photochemical processes.
Lasers Surg. Med. 42:494–502, 2010.
? 2010 Wiley-Liss, Inc.
(P<0.001and P<0.01
Key words: IL-1b; IL-6; lasers; NO; ROS; TNF-a
INTRODUCTION
The process of wound healing is a highly co-ordinated
process that involves a series of overlapping events
controlled by a variety of cells, growth factors, cytokines
and metabolic enzymes released at the wound site.
Dysregulation of this co-ordinated event leads to impaired
wound healing; an abnormality which is frequently seen in
conditions such as diabetes. There are many causes of
chronic wounds, with diabetes, pressure ulcers and venous
stasis as the three most common causes [1]. Impaired
wound healing is an incapacitating complication of dia-
betes often necessitating amputation and poses a serious
challenge in clinical practice.
Growth factors and cytokines such as interleukin-1-beta
(IL-1b), IL-6 and tumour necrosis factor-alpha (TNF-a)
have diverse modes of action and are released during
wound repair [2]. IL-1b and TNF-a are both well-known
pro-inflammatory cytokines and have similar functions or
effects; however, they do not share chemical or structural
resemblance and their effects are interceded by specific
receptors. Together with IL-1, TNF-a is the first cytokine
knownto beupregulated during theinflammatory phaseof
wound healing and contributes to the oxidative stress
within the wound by generating reactive oxygen species
(ROS)[3].IL-6isinducedduring acutephasereactionsand
usually expressed in response to or together with IL-1 and
TNF-a [4]. However, contradictory effects have been
reported [5]; it suppresses TNF-a, IL-1 and IL-12. Its
vital role in wound healing is its ability to cause cell
differentiation and proliferation. TNF-a is the most critical
accelerator of diabetes [6].
ROS and reactive nitrogen species (RNS) act as mole-
cular messengers during cell signalling; however, they
have a biphasic effect, being both beneficial and detrimen-
tal depending on their concentration. ROS and RNS are
generated during wound healing and are important
mediators in this carefully controlled process, however in
chronicwoundsthereisanuncontrolledproductionofthese
molecules. Nitric oxide (NO) is significantly reduced in
chronic ulcers and impaired healing of diabetic wounds is
Contract grant sponsor: University of Johannesburg (UJ);
Contract grant sponsor: National Research Foundation (NRF) of
South Africa; Contract grant sponsor: Medical Research Council
(MRC) of South Africa.
*Correspondence to: Heidi Abrahamse, PhD, Laser Research
Centre, Faculty of Health Sciences, University of Johannesburg,
P.O. Box 17011, Doornfontein 2028, South Africa.
E-mail: habrahamse@uj.ac.za
Accepted 5 August 2009
Published online 15 July 2010 in Wiley InterScience
(www.interscience.wiley.com).
DOI 10.1002/lsm.20812
? 2010 Wiley-Liss, Inc.

Page 2

thoughttoberelatedtothisdecrease[7,8].Burrowetal.[9]
demonstrated that normal skin fibroblasts produce more
NO than diabetic human skin fibroblasts. Various studies
show that phototherapy modulates NO both in vitro and in
vivo [10–14].
Hyperglycemia is the key metabolic abnormality in
diabetesmellitusthatisbelievedtoplaythemostprominent
role in the development of diabetic complications [15]. A
number of earlier studies showed that exposure of cells to
hyperglycaemic conditions (20–40mM), and thus mimick-
inguncontrolleddiabetes,resultedinarestrictionofcellular
proliferation [16–19]. This restraint is more pronounced
for higher glucose concentrations [16] and is expressed
especially after protracted exposure to high glucose levels
[20].Previousstudieshaveshownthatcontinuousexposure
of fibroblast cells to a glucose concentration of 22.6mMol/L
(17mMol/L glucose added to media with a basal concen-
tration of 5.6mMol/L) slowed cellular migration and there
was an increase in both cellular and DNA damage and
apoptosis [21].
In cell culture, normal cells show contact inhibition of
growth and population density stabilises at low levels; it is
these properties that provide a suitable environment to
study the cellular responses of cells as they react to an
insult or injury [22]. The central scratch method is an
in vitro wound model whereby the monolayer of cells are
scratcheddownthecentreofthecultureflask.Thismethod
hasbeenusedwithavarietyofcellsand,asthemonolayers
heal the wound in a characteristic manner, they have been
used to study cell polarisation, matrix remodelling, cell
migration and numerous other processes [23,24]. The
injury model simulates in vivo mechanical trauma and
the processes reflect the behaviour of individual cells as
well as the properties of the cell sheet as a surrogate tissue
[22]. The wounds heal in a stereotypical fashion with cells
polarising toward the central scratch, initiate protrusion,
migrate and close the wound. Progression of these events
can be monitored by manually imaging samples at
fixed time points [25–27].
The objective of this study was to determine if a
wavelength of 830nm at a dose of 5J/cm2speeds up wound
healinginwoundeddiabeticinducedhumanskinfibroblast
cells by increasing IL-6, ROS and NO and decreasing pro-
inflammatory cytokines TNF-a and IL-1 b.
MATERIALS AND METHODS
Cell Culture
Human skin fibroblast cells were purchased from
the American Type Culture Collection (WS1; ATCC CRL
1502; Adcock Ingram, Midrand, South Africa) and grown
in complete Dulbecco’s Modified Eagle’s Medium as
previously described by Hawkins and Abrahamse [28].
Normal, normal wounded and diabetic wounded cells were
used in this study. An in vitro diabetic wound model was
basedonRigauetal.[26],Hamuroetal.[29]andVincketal.
[15]. Briefly, diabetic cells were continuously cultured
in complete media containing an additional 17mMol/L
D-glucose. To determine the effects of the lasers, cells were
detached by trypsinisation (1ml/25cm2, 0.25% trypsin–
0.03%ethylenediaminetetraaceticacid),and6?105cellsin
3ml complete culture media were seeded into 3.3-cm-
diameter culture plates as determined by the Trypan blue
exclusion test [28]. Plates were incubated overnight to
allow cells to attach. A wound was induced 30minutes
before laser irradiation by scratching the cellular mono-
layer with a sterile 1ml pipette [26].
Laser Set-Up and Irradiation
Cell cultures were chosen at random and WS1 cells were
irradiated in the dark from the top with an 830nm diode
laser(poweroutput40mW;spotsize9.1cm2;powerdensity
4.4mW/cm2). Cells were irradiated once with a fluence
of 5J/cm2, which was calculated at 18minutes and
56seconds. Unirradiated cells were treated in the same
manner as irradiated cells, barring irradiation. Prior to
irradiation, culture media was discarded and cells were
rinsed with warm Hanks Balanced Salt Solution (HBSS),
and replaced with 1ml fresh media. Post-irradiation,
cultures were incubated for a pre-determined amount
of time (Table 1). Post-incubation cells were detached by
trypsinisation and re-suspended in 500ml culture media.
All tests were performed on different populations (n¼6) of
cellsforeachsamplegroupandeachbiochemicalassaywas
performed in duplicate.
Changes following laser irradiation were determined
by measuring cellular viability (Trypan blue exclusion
test), apoptosis (caspase 3/7 activity) and proliferation
(VisionBlueTMfluorescentassay).Cytokineexpressionwas
TABLE 1. Study Design (n¼6)
ProliferationApoptosis ViabilityNOROS
Cytokines (TNF-a;
IL-1b; IL-6)
Incubation time24 or 48hours1 or 24hours 15minutes, 1,
24 or 48hours
Trypan blue
exclusion test
Light microscopy
15minutes or
1hour
IF staining
15minutes 1 or 24hours
MethodFluorescence Caspase 3/7
activity
Luminescence
Griess reagent
system
Fluorescence
microscopy
ELISA
Data collectionFluorescent
spectroscopy
SpectroscopySpectroscopy
NO,nitricoxide;ROS,reactiveoxygenspecies;TNF-a,tumournecrosisfactor-alpha;IL-1b,interleukin1-beta;IL-6,interleukin-
6; IF, immunofluorescent; ELISA, enzyme linked immunosorbent assay.
DIABETIC WOUNDED FIBROBLAST CELLS 495

Page 3

determined by ELISA, while ROS was determined by IF
staining and NO by the Griess Reagent System.
Cellular Viability
The Trypan blue exclusion test was used to determine
cellular viability in cells which had been incubated
for 15minutes, 1, 24 or 48hours post-laser irradiation.
An equal volume of 0.4% Trypan blue (Sigma-Aldrich,
Johannesburg, South Africa, T8154) in HBSS was added
to re-suspended cells and allowed to incubate at room
temperature for 5–15minutes. The number of viable
(unstained) and non-viable (blue) cells were counted and
the percentage viability calculated (number of viable cells
divided by the number of total cells, multiplied by 100).
Apoptosis
The Caspase-GloTM3/7 assay (Whitehead Scientific,
Johannesburg, South Africa, Promega, TB323) was used
to measure the activity of caspase-3 and -7. The addition of
reagent results in cellular lysis followed by substrate
cleavage by caspase, and as a result, a luminescent signal
is generated by luciferase. Negative controls consisted of
reagent and culture media without cells. A positive control
was included by inducing apoptosis in 1?106cells/ml using
0.5mg/ml Actinomycin D (Sigma-Aldrich, A5156-1VL). An
equal volume of cells and reagent was added (25ml),
contents mixed and incubated at room temperature for
3hours.LuminescencewasreadusingtheVictor-3(Perkin-
Elmer, Separation Scientific, Johannesburg, South Africa)
and reported in reading light units (RLU).
Cellular Proliferation
Cellular proliferation of cells was determined using the
VisionBlueTMFluorescence Cell Viability Assay Kit (Bio-
comBiotech, Pretoria, South Africa, BioVision, K303-500),
which provides a sensitive and easy means for quantifying
cell proliferation. One hundred microlitres of cells was
addedtoa96-wellmicrotitreplateandincubatedat378Cin
5% CO2for 2hours to allow the cells to settle and attach.
Following incubation, 10ml (10% medium volume) Vision-
BlueTMreagent was added and plates incubated (378C in
5% CO2) for 2hours. Fluorescence was then measured
using the Victor-3 (Perkin-Elmer, Separation Scientific) at
Ex/Em 560/595.
Cytokine Expression
The optEIATMsandwich type enzyme-linked immuno-
sorbent assay (ELISA) sets for human cytokine from BD
Biosciences(ScientificGroup,Johannesburg,SouthAfrica)
was used to determine IL-1b (BD 557953), TNF-a (BD
555212)andIL-6(BD555220).ELISA’swererunaccording
to the manufacturers’ protocol. Briefly, each microwell
plate was coated overnight at 48C with specific capture
antibody (1:250 in coating buffer). Plates were washed
three times, blocked with assay diluent and incubated for
1hour at room temperature. Plates were washed as before.
Serial dilutions of standards were performed from the
stock standard to generate a 9-point standard curve. One
hundred microlitres of sample or standard was pipetted
into their respective wells and incubated for 2hours at
room temperature. Plates were washed as before and
incubated for 1hour with working detector (biotinylated
anti-human monoclonal detection antibody) conjugated to
streptavidin-horseradish peroxidase. Plates were washed
as before. Tetramethylbenzidine (TMB) substrate was
added and plates were incubated for 30minutes at room
temperature in the dark. Stop solution was added and
absorbance was determined at A450nm (Perkin-Elmer
Victor 3).
Nitric Oxide
NO was determined spectrophotometrically using the
Griess Reagent system (Whitehead Scientific, Promega,
G2930). One means to investigate NO formation is to
measure nitrite (NO2), which is one of two primary, stable
and non-volatile breakdown products of NO. This assay
relies on a diazotisation reaction that was originally
described by Griess in 1879 [30]. The Griess Reagent
System uses sulphanilamide and N-1-napthylethylenedi-
amine dihydrochloride (NED) under acidic (phosphoric
acid) conditions. A serial dilution of 100mM nitrite was
made in complete media (100–0mM) and added to the
respectivewellsofa96-wellplate.Equalamountsofculture
media and sulphanilamide solution was added to the wells
and incubated at room temperature, protected from light,
for 10minutes. Fifty microlitres of NED was added and
cells incubated as before. Absorbance was measured at
A540nm (Perkin-Elmer, Victor-3).
Reactive Oxygen Species
ROS was determined in irradiated (5J/cm2) and control
cells (0J/cm2) by immunofluorescent (IF) staining using
the Image-iTTMLIVE Green Reactive Oxygen Species
(ROS) Detection Kit (Scientific Group; Invitrogen, Mole-
cular Probes, 136007). The assay is based on 5-(and-6)-
carboxy-20,70-dichlorodihydrofluoresceindiacetate(carboxy-
H2DCFDA), a reliable fluorogenic marker for ROS in live
cells. In addition to carboxy-H2DCFDA, the kit provides
the common inducer of ROS production tert-butyl hydro-
peroxide (TBHP), as a positive control, and the blue-
fluorescent cell-permeant nucleic acid stain Hoechst
33342. H2DCFDA detects ROS such as hydrogen peroxide,
singlet oxygen and hydroxyl radicals in living cells, but not
superoxide anions or NOs [31]. Briefly, 6?105cells were
grown on heat sterilised coverslips in 3ml complete
culture media in a 3.3-cm-diameter culture plates. Post-
laser irradiation, cells were washed with warm HBSS/Ca/
Mg and labelled with 25mM Carboxy-H2DCFDA and
incubated for 30minutes at 378C, protected from light.
During the last 5minutes of incubation, 1.0mM Hoechst
33342 was added. Cells were washed and mounted using
0.1Mpropylgallate(Sigma-Aldrich,P3130)inglycerol/PBS
(9:1). For the positive control, 100mM TBHP was added
tocellsadheringtothecoverslipsandincubated(378Cin5%
CO2) for 60minutes. Fluorescence was viewed and images
were taken with the Zeiss Live-Cell Imager.
496HOURELD ET AL.

Page 4

Statistical Analysis
Experiments were repeated six times (n¼6). All assays
were performed in duplicate and the mean was used. The
results are represented as percentage change between
irradiated cells (5J/cm2) and non-irradiated control
cells (0J/cm2). Results were graphically presented and
statistically analysed using Sigma Plot Version 8.0. A
student t test and one-way ANOVA was performed to
detect differences between the control and experiments,
and as well as between experimental groups. Bonferroni
correction was taken into account and all results remain-
ed significant (P¼0.017). Results were considered to
be statistically significant when P<0.05. Statistical sig-
nificance, compared to their respective control (0J/cm2)
is shown in graphs as P<0.05 (*), P<0.01 (**) or P<0.001
(***).
RESULTS
Cellular Viability
IrradiationofWS1cellsatawavelengthof830nmwitha
fluence of 5J/cm2did not have any significant effect on the
viability of cells. Percentage viability was above 95% in all
cell types.
Apoptosis
Post-irradiation, normal, normal wounded and diabetic
wounded human skin fibroblast cells were incubated
for 1 or 24hours and caspase 3/7 activity was determined
(Fig. 1a). There were no significant changes 1hour post-
irradiation,however,after24hours,therewasasignificant
decrease in apoptosis of 82% and 31% in normal wounded
(P<0.001) and diabetic wounded (P<0.01) cells respec-
tively. Unirradiated normal cells showed a significant
decrease in apoptosis at both 1 and 24hours compared
to unirradiated stressed cells (P<0.001), as did irradiated
normal cells compared to irradiated normal wounded and
diabetic wounded cells (P<0.001 at 1hour and P<0.05 at
24hours). Both unirradiated and irradiated diabetic
wounded cells showed a significant increase in caspase
3/7activityascomparedtonormalwoundedcells(P<0.001)
at both 1 and 24hours. Caspase 3/7 activity significantly
decreased in all irradiated cell types 24hours post-
incubation compared to 1hour (P<0.01).
Cellular Proliferation
Cellularproliferation wasdetermined in normal,normal
wounded and diabetic wounded WS1 cells 24 or 48hours
post-irradiationat830nmwith5J/cm2(Fig.1b).Therewas
an increase in proliferation of 51% and 19% in normal
wounded cells irradiated for 24 or 48hours respectively
(P<0.01).Diabeticcellsshowedanincreaseof53%and28%
respectively (P<0.01). Comparison of unirradiated cells
showedanincreaseinproliferationinnormalwoundedand
diabetic wounded cells 48hours post-irradiation compared
to normal cells (P<0.05). Comparison of irradiated cells
showed an increase in normal wounded and diabetic
wounded cells at both 1 and 24hours (P<0.01) compared
to normal cells. At 24hours irradiated diabetic wounded
cells showed an increase compared to irradiated normal
wounded cells (P<0.05). All cells incubated at 378C for
48hours showed a significant increase in proliferation as
compared to the same cells incubated for 24hours
(P<0.001).
Cytokine Expression
The optEIATMsandwich type ELISA sets was used to
determine TNF-a, IL-1b and IL-6 in cells incubated for 1 or
24hours post-irradiation. Normal, normal wounded and
diabetic wounded cells incubated for 1hour all showed a
significant decrease in TNF-a by 18%, 20% and 13%
respectively (P<0.01, P<0.01 and P<0.05 respectively)
compared to non-irradiated controls (Table 2). At 24hours,
TNF-a levels returned to their natural levels in normal
cells, however, levels were still significantly decreased in
normal wounded and diabetic wounded cells (P<0.05) by
23% and 17% respectively. There was no significant
difference between unirradiated cells or irradiated cells,
except for the increase seen in unirradiated diabetic
wounded cells at 24hours (P<0.05) compared to unirra-
diated normal cells. The only difference seen between the
twoincubationtimeswasinnormalcells,withadecreasein
TNF-a seen at 24hours (P<0.01).
When IL-1b
wasdetermined
wounded and diabetic wounded cells, normal cells
irradiated at 830nm with 5J/cm2and incubated for 1hour
showed a significant decrease of 30% (P<0.05) compared
to unirradiated control cells (Table 2), while diabetic
wounded cells showed a significant decrease of 39%
innormal, normal
Fig. 1. Apoptosis(a)(luminescence,measuredinrelativelight
units—RLU) and proliferation (b) (measured in fluorescence,
Ex/Em 560/595) was determined in normal (N), normal
wounded (NW) and diabetic wounded (DW) cells irradiated at
5J/cm2with 830nm. Results are shown as percentage change
between irradiated (5J/cm2) and non-irradiated control cells
(0J/cm2) with the actual value and standard error written in.
Significant differences are shown as P<0.01 (**) and P<0.001
(***). There was a significant decrease in caspase 3/7 activity
24hours post-incubation in NW and DW cells. Note the
significant increase in proliferation in NW and DW cells
24 and 48hours post-incubation.
DIABETIC WOUNDED FIBROBLAST CELLS 497

Page 5

24hours post-irradiation (P<0.05). The decreases seen in
the other cell types were insignificant. Unirradiated
diabetic wounded cells showed a significant increase in
IL-1b at 24hours compared to unirradiated normal and
unirradiated normal wounded cells (P<0.01 and P<0.05
respectively). Comparison of irradiated cell types showed a
significant increase at both 1 and 24hours in diabetic
wounded cells compared to normal cells (P<0.05 and
P<0.01 respectively). The only significant difference seen
between the two incubation times was in unirradiated
normal cells, with a decrease seen at 24hours (P<0.05).
When normal, normal wounded and diabetic wounded
WS1 cells were irradiated once at 830nm with 5J/cm2and
incubatedfor1or24hours,therewasnosignificantchange
in IL-6 levels in irradiated cells compared to unirradiated
controls (Table 2). Comparison of unirradiated cell types
showed a significant increase in IL-6 in diabetic wounded
cells at both 1 and 24hours (P<0.05 and P<0.01 respec-
tively).Unirradiatednormalwoundedcellsonlyshowedan
increaseat24hours(P<0.05).Irradiateddiabeticwounded
cells showed a significant increase at 1hour compared
to both irradiated normal and normal wounded cells
(P<0.01 and P<0.05 respectively). Cells which were
incubated for 24hours showed an increase in IL-6 com-
pared to cells incubated for 1hour, with significances seen
in unirradiated and irradiated normal cells (P<0.05) and
irradiated normal wounded cells (P<0.05).
Nitric Oxide
Normal, normal wounded and diabetic wounded human
fibroblast cells were irradiated at 830nm with 5J/cm2and
incubated for 15minutes or 1hour at 378C. NO was
determined at A540nm. Fifteen minutes post-irradiation,
all cells showed a significant increase in NO (P<0.01),
(Fig. 2). Normal and diabetic wounded cells showed an
increase of 49%, while normal wounded cells showed an
increase of 45%. This increase was no longer evident at
TABLE 2. Interleukin-6 (IL-6), Tumour Necrosis Factor-Alpha (TNF-a) and
Interleukin-1-Beta (IL-1b) Was Determined by ELISA in Cells Irradiated at 5J/cm2
With 830nm and Incubated for 1 or 24Hours
Cell
1hour 24hours
% ChangeAbsolute value% Change Absolute value
IL-6
Normal
Normal wounded
Diabetic wounded
TNF-a
Normal
Normal wounded
Diabetic wounded
IL-1b
Normal
Normal wounded
Diabetic wounded
2.36
14.78
19.6
0.9992 ? 0.071
1.1365 ? 0.073
1.5147 ? 0.141
4.54
?0.74
?4.0
1.2365 ? 0.042
1.3834 ? 0.087
1.3052 ? 0.057
?17.60**
?19.70**
?13.00*
0.0433 ? 0.005
0.0451 ? 0.002
0.0438 ? 0.001
?1.80
?22.90*
?16.70*
0.0489 ? 0.002
0.0476 ? 0.002
0.0498 ? 0.002
?30.30*
?5.70
?6.60
0.0356 ? 0.004
0.0453 ? 0.003
0.0486 ? 0.003
?23.60
?23.50
?38.80*
0.0275 ? 0.004
0.0361 ? 0.004
0.0472 ? 0.004
Results are shown as percentage change between irradiated (5J/cm2) and non-irradiated
control cells (0J/cm2), with the absolute value and standard error included. Significant
differencesareshownasP<0.05(*),P<0.01(**)andP<0.001(***).After1hourincubation,
there was a significant decrease in TNF-a in all cells, which remained significantly decreased
in NW and DW cells 24hours later. There was a significant decrease in IL-1b in N cells 1hour
post-irradiation and in DW cells 24hours post-irradiation.
? Standard error.
Fig. 2. Nitricoxide(NO)wasdeterminedbytheGriessreagent
system in normal (N), normal wounded (NW) and diabetic
wounded (DW) cells irradiated at 5J/cm2with 830nm and
incubated for 15minutes. There was a significant increase in
NOinallcellsincubatedfor15minutes.Significantdifferences
are shown as P<0.01 (**).
498 HOURELD ET AL.

Page 6

1hour. Unirradiated cells showed no significant difference
between the incubation times, while irradiated normal,
normal wounded and diabetic wounded cells showed a
significant decrease 1hour post-irradiation compared to
cells incubated for 15minutes (P<0.001, P<0.05 and
P<0.01 respectively).
Reactive Oxygen Species
ROS was determined 15minutes post-laser irradiation
in unirradiated and irradiated normal and diabetic cells
(Fig. 3) by fluorescent staining. Post-irradiation, both
normal and diabetic cells showed more green fluorescence
than unirradiated normal and diabetic cells respectively.
Irrespective of irradiation, diabetic cells showed more ROS
than normal cells.
DISCUSSION
The development of new therapies for wound healing
requires an understanding of the mechanisms involved,
including underlying disease conditions, and translating
these mechanisms into useful agents. Diabetes is known to
be associated with poor wound healing and is responsible
for 50–70% of all non-traumatic amputations and it is
estimated that 15% of all diabetic patients will develop
an ulcer on the feet or ankles at some time during the
disease course [32]. Diabetic wounds are predominantly
Fig. 3. Reactive oxygen species (ROS) was determined by fluorescent microscopy in non-
irradiated (0J/cm2) and irradiated (5J/cm2) normal and diabetic cells. A positive control
(100mM tert-butyl hydroperoxide (TBHP)) was included. ROS fluoresced green, while the
nuclei fluoresced blue. Little ROS is seen in non-irradiated cells, while irradiated cells
show and abundance of ROS. Diabetic cells show more green fluorescence than normal cells.
[Figure can be viewed in color online via www.interscience.wiley.com.]
DIABETIC WOUNDED FIBROBLAST CELLS 499

Page 7

characterised
deformity, altered immune function or increased suscept-
ibility to infection, decreased wound NO production, and
often hypoxia/ischemia [33,34]. Treatment of diabetic
wounds includes debridement, mechanical load relief,
topical antibiotics and dressings, while newer develop-
ments include the use of bioengineered skin equivalents,
growth-factortherapyandhyperbaricoxygentreatment.A
number of studies have shown that laser irradiation, using
appropriate parameters, is beneficial to a wide range of
conditions, including wound healing in diabetic patients.
Al-Watbanetal.[35]foundawavelengthof633nmwitha
doseof10J/cm2tobethemostbeneficialintreatingwounds
indiabeticmice.Maiyaetal.[36]alsofoundlasertherapyto
be beneficial in hastening the healing process in diabetic
rats(632.8nmwith4.8J/cm2),whileRabeloetal.[37]found
a dose of 10J/cm2beneficial. Al-Watban [38] suggests that
633nm laser therapy should be given three times per week
at 4.71J/cm2per dose for diabetic burns, and three times
per week at 2.35J/cm2per dose for diabetic wound healing
as actual doses for human clinical trials.
Inflammatory cytokines such as IL-1b and TNF-a have
also been shown to be increased in non-healing wounds, as
well as in diabetic patients [39]. In addition, TNF-a is auto-
stimulativeandcaninducethesecretionofIL-1b,resulting
in a persisting cycle of inflammation [40]. TNF-a is
responsible for apoptosis by binding to its receptor, TNFRI
which contains a death domain, and activating the caspase
cascade. IL-1b enhances TNF-a induced apoptosis [41] and
iscapableofinducingothercytokinessuchasTNF,IL-6,IL-
2 and apoptosis which can be mediated via NO production
by increasing secretion of inducible nitric oxide synthase
(iNOS). IL-6 exerts a variety of effects on cells and is
involved in immune activity, the acute-phase response to
injury and infection, inflammation, oncogenesis and hem-
atopoiesis, as well as exerting growth-inducing, growth-
inhibitory and differentiation-induction effects [42–45].
Aimbire et al. [46] found that irradiation of rats with an
immune-complexinducedlunginjuryat650nmwith0.11J
reduced TNF-a concentration in bronchoalveolar lavage
fluid. Mafra de Lima et al. [47] and Boschi et al. [48] also
found a reduction in TNF-a post-irradiation, while Safavi
et al. [49] found a significant decrease in the gene
expression of IL-1b and no significant difference in TNF-a
inirradiatedrats.Shimizuetal.[50]alsoshowedthatlaser
irradiations caused the inhibition of IL-1b and Boschi et al.
[48] found a decrease in NO and IL-6.
Therewasnosignificantdifferenceinviability,prolifera-
tion, IL-6, TNF-a, IL-1b and NO between unirradiated
normal wounded and unirradiated diabetic wounded
cells at both 1 and 24hours, thus any differences seen in
irradiateddiabeticwoundedcellswasadirectresultoflaser
irradiation.Therewasanincreaseincaspase3/7activityin
diabetic cells, this corresponds with previous studies [21]
and work conducted by Susztak et al. [51].
Irradiation at a wavelength of 830nm with a fluence of
5J/cm2stimulated cell survival. There was no negative
effect on cellular viability in irradiated cells, thus laser
irradiation did not induce additional damage on cells. In
by peripheralneuropathy, structural
factitprotectedcellsfromapoptosisasseenbythedecrease
in caspase 3/7 activity. This study showed a significant
increase in proliferation in both normal wounded and
diabetic wounded cells in vitro at 24 and 48hours. The
increase was more pronounced 24hours post-irradiation,
with increases of 50%. Gavish et al. [52] also found an
increase in proliferation in porcine aortic smooth muscle
cells irradiated at 780nm with 2J/cm2. However, they
foundagreaterincreaseat24hoursasopposedto48hours.
Hawkins and Abrahamse [28] found an increase in
proliferation in wounded fibroblast cells (WS1) irradiated
with a He–Ne laser with 5 J/cm2, while Houreld and
Abrahamse [53] found an increase in diabetic wounded
WS1 cells irradiated at 830nm with 5J/cm2.
Irradiation of normal wounded and diabetic wounded
cells at 830nm with 5J/cm2had an anti-inflammatory
effect on cells, with decreases in TNF-a seen in normal,
normal wounded and diabetic wounded cells 1hour post-
irradiation, and decreases innormal wounded and diabetic
wounded cells 24hours post-irradiation. A decrease in
IL-1b was seen in normal cells 1hour post-irradiation and
in diabetic wounded cells 24hours post-irradiation. These
decreases in pro-inflammatory cytokines corresponds with
other studies [46–50,54]. There was no TNF-a induced
apoptosis in cells as seen by the decrease in TNF-a and
caspase 3/7 activity and an increase in proliferation. Cells
were stimulated to enter the cell survival pathway.
Several papers on laser irradiation have shown signifi-
cant increases in IL-6 [42,55,56], this study showed an
insignificant increase at 1 and 24hours (P¼0.08 and
P¼0.514 respectively). IL-6 has been linked to the patho-
genesis of type 1 diabetes [43,57,58] and altered IL-6 levels
have also been associated with delayed wound healing in
diabetes [59]. This study showed that although there was
an initial insignificant increase in IL-6, levels decreased
and there was no negative effect on wound healing in vitro.
At a molecular level, the effects of laser therapy remain
illusive.Manystudieshavesuggestedthattheseeffectsare
as a result of ROS which then participate in various redox
reactions. Eichler et al. [11] found that both red and
infrared light stimulated the production of ROS in rat
cardiocytes. Lindga ˚ rd et al. [13] demonstrated that irradi-
ation at 634nm (35.7W/cm2) could stimulate the release of
NO in human monocytes within 20minutes and that the
release was not coupled to the activation of iNOS or
endothelial NOS (eNOS). They also demonstrated the
intracellular release of ROS. Pal et al. [31] irradiated
normal human fibroblasts with a He–Ne laser (0.5–16J/
cm2;0.64–1.16mW/cm2)andfoundthatthekineticsofROS
generation was strongly dependent on laser fluence rather
than laser intensity.
In this study, all cell types showed an increase in NO
15minutes post-irradiation. There were no significant
changes at 1hour, and the decrease at 1hour was
significant compared to 15minutes. In this study ROS
production was determined by immunofluorescence stain-
ing. WS1 cells irradiated with 5J/cm2showed more
fluorescence than unirradiated cells. As expected, diabetic
cellsshowedmoreROSthannormalcells.TNF-aiscapable
500 HOURELD ET AL.

Page 8

of activating NF-kB which then translocates from the
cytosol to the nucleus where it initiates the production of
ROS.InthisstudyitappearsasthoughtheincreaseinROS
and NO is directly released due to a photochemical process
since the increases were seen 15minutes post-irradiation.
It appears plausible that TNF-a could not have stimulated
ROS production (via NF-kB) since TNF-a levels were
decreased 1hour post-irradiation.
This study shows that laser therapy might prove
beneficial for wound healing, including healing of diabetic
wounds. Irradiation of wounded diabetic cells in vitro at a
wavelength of 830nm using 5J/cm2did not induce addi-
tional damage, significantly increased proliferation, ROS
and NO production and significantly decreased pro-inflam-
matorycytokinesTNF-aandIL-1bandcaspase3/7activity.
Irradiation of normal and diabetic induced WS1 cells
stimulated the release of intracellular ROS and NO due
to what appears to be direct photochemical processes.
ACKNOWLEDGMENTS
Alllasersweresuppliedandset-upbytheNationalLaser
Centre (NLC) of South Africa.
REFERENCES
1. Menke NB, Ward KR, Witten TM, Bonchev DG, Diegelmann
RF. Impaired wound healing. Clin Dermatol 2007;25:19–25.
2. Kapoor M, Kojima F, Appleton I, Kawai S, Crofford LJ. Major
enzymatic pathways in dermal wound healing: Current
understanding and future therapeutic targets. Curr Opin
Invest Drugs 2006;7(5):418–422.
3. Cho M, Hunt TK,HussainMZ. Hydrogenperoxide stimulates
macrophage vascular endothelial growth factor release. Am
J Physiol Heart Circ Physiol 2001;280(5):H2357–H2363.
4. Xing Z, Gauldie J, Cox G, Baumann H, Jordana M, Lei X,
Achong MK. IL-6 is an antiinflammatory cytokine required
for controllinglocal or
responses. J Clin Invest 1998;101(2):311–320.
5. Deon D, Ahmed S, Tai K, Scaletta N, Herrero C, Lee IH,
Krause A, Ivashkiv LB. Cross-talk between IL-1 and IL-6
signalling pathways in rheumatoid arthritis. J Immunol
2001;167(9):5395–5403.
6. Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose
expression of tumor necrosis factor-alpha: Direct role in
obesitylinkedinsulinresistance.Science1993;259(5091):87–
91.
7. Bulgrin JP, Shanbani M, Chakravarthy D, Smith DJ. Nitric
oxide synthesis is suppressed in steroid-impaired and
diabetic wounds. Wounds 1995;7:48.
8. Schwentker A, Vodovotz Y, Weller R, Billiar TR. Nitric oxide
and wound repair: Role of cytokines? Nitric Oxide 2002;7:1–
10.
9. Burrow JW, Koch JA, Chuang H, Zhong W, Dean DD, Sylvia
VL. Nitric oxide donors selectively reduce the expression of
matrix metalloproteinases-8 and -9 by human diabetic skin
fibroblasts. J Surg Res 2007;140(1):90–98.
10. Zhu Q, Yu W, Yang X, Hicks GL, Lanzafame RJ, Wang T.
Photo-irradiation improved functional preservation of iso-
lated rat heart. Lasers Surg Med 1997;20(3):332–339.
11. Eichler M, Lavi R, Friedmann H, Shainberg A, Lubart R. Red
light-induced redox reactions in cells observed with TEMPO.
Photomed Laser Surg 2007;25(3):170–174.
12. Lubart R, Eichler M, Lavi R, Friedman H, Shainberg A. Low-
energy laser irradiation promotes cellular redox activity.
Photomed Laser Surg 2005;23(1):3–9.
13. Lindga ˚ rd A, Hulte ´n LM, Svensson L, Soussi B. Irradiation at
634nm releases nitric oxide from human monocytes. Lasers
Med Sci 2007;22(1):30–36.
systemicacuteinflammatory
14. Gavish L, Perez LS, Reissman P, Gertz SD. Irradiation with
780nm diode laser attenuates inflammatory cytokines but
upregulates nitric oxide in lipopolysaccharide-stimulated
macrophages: Implications for the prevention of aneurysm
progression. Lasers Surg Med 2008;40(5):371–378.
15. Vinck EM, Cagnie BJ, Cornelissen MJ, Declercq HA,
Cambier DC. Green light emittingdiode irradiation enhances
fibroblast growth impaired by high glucose level. Photomed
Laser Surg 2005;23(2):167–171.
16. Kamal K, Du W, Mills I, Sumpio BE. Antiproliferative effect
of elevated glucose in human microvascular endothelial cells.
J Cell Biochem 1998;71(4):491–501.
17. LorenziM, Montisano DF,ToledoS,BarrieuxA. Highglucose
induces DNA damage in cultured human endothelial cells.
J Clin Invest 1986;77(1):322–325.
18. Lorenzi M, Cagliero E, Toledo S. Glucose toxicity for human
endothelial cells in culture. Delayed replication, disturbed
cell cycle, and accelerated death. Diabetes 1985;34(7):621–
627.
19. Stout RW. Glucose inhibits replication of cultured human
endothelial cells. Diabetologia 1982;23(5):436–439.
20. Lorenzi M, Nordberg JA, Toledo S. High glucose prolongs
cell-cycle traversal of cultured human endothelial cells.
Diabetes 1987;36(11):1261–1267.
21. Houreld NN, Abrahamse H. Effectiveness of helium-neon
laser irradiation on viability and cytotoxicity of diabetic-
wounded fibroblast cells. Photomed Laser Surg 2007;25(6):
474–481.
22. Zungu IL, Hawkins Evans D, Houreld N, Abrahamse H.
Biological responses of injured human skin fibroblasts to
assess the efficacy of in vitro models for cell stress studies. Afr
J Biochem Res 2007;1(4):060–071.
23. Cha D, O’Brien P, O’Toole EA, Woodley DT, Hudson L.
Enhanced modulation of keratinocyte motility by TGF
relative to EGF. J Invest Dermatol 1996;106:590–597.
24. Hawkins D, Abrahamse H. Biological effects of Helium-Neon
(632.8nm) laser irradiation on normal and wounded human
skin fibroblasts. Photomed Laser Surg 2005;23(3):251–
259.
25. Yarrow JC, Perlman ZE, Westwood NJ, Mitchison TJ. A high
throughput cell migration assay using scratch wound heal-
ing, a comparison of imaged based readout methods. BMC
Biotechnol 2004;9(4):21.
26. Rigau J, Sun C, Trelles MA, Berns M. Effects of the 633nm
laser on the behaviour and morphology of primary fibroblasts
in culture. In: Karu T, Young A, editors. Proceedings, effects
of low power light on biological systems. Barcelona, Spain:
Progress in Biomedical Optics; 1995. pp 38–42.
27. Wong MK, Gotlieb AI. The reorganization of microfilaments,
centrosomes, andmicrotubules
wound reendothelialization. J Cell Biol 1988;107:1777–
1783.
28. Hawkins D, Abrahamse H. The role of laser fluence in cell
viability, proliferation, and membrane integrity of wounded
human skin fibroblasts following helium-neon laser irradi-
ation. Lasers Surg Med 2006;38:74–83.
29. Hamuro M, Polan J, Natarajan M, Mohan S. High glucose
induced nuclear factor kappa B mediated inhibition of
endothelial cell migration. Atherosclerosis 2002;162(2):277–
287.
30. Promega. Griess reagent system instructions for use of
product G2930. 2005: Part# TB229.
31. Pal G, Dutta A, Mitra K, Grace MS, Amat A, Romanczyk TB,
Wu X, Chakrabarti K, Anders J, Gorman E, Waynant RW,
Tata DB. Effect of low intensity laser interaction with human
skin fibroblast cells using fiber-optic nano-probes. J Photo-
chem Photobiol B Biol 2007;86:252–261.
32. Bild DE, Selby JV, Sinnock P, Browner WS, Braveman P,
Showstack JA. Lower-extremity amputations in people with
diabetes. Epidemiology and prevention. Diabetes Care 1989;
12(1):24–31.
33. SchafferMR, TantryU, vanWesepRA, BarbulA. Nitric oxide
metabolism in wounds. J Surg Res 1997;71:25–31.
34. Reiber GE, Pecoraro RE, Keopsell TD. Risk factors for
amputation in patients with diabetes mellitus: A case control
study. Ann Intern Med 1992;117:97–105.
duringinvitro small
DIABETIC WOUNDED FIBROBLAST CELLS 501

Page 9

35. Al-Watban FAH, Zhang ZY, Andres BL. Low-level laser
therapy enhances wound healing in diabetic rats: A compar-
ison of different lasers. Photomed Laser Surg 2007;25(2):72–
77.
36. 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(2):187–190.
37. Rabelo SB, Balbin Villaverde A, Nicolau R, Salgado MC, Da
Silva Melo M, Pacheco MTT. Comparison between wound
healing in induced diabetic and nondiabetic rats after low-
level laser therapy. Photomed Laser Surg 2006;24(4):474–
479.
38. Al-Watban FA. Laser therapy converts diabetic wound
healing to normal healing. Photomed Laser Surg 2009;
27(1):127–135.
39. Brandner JM, Zacheja S, Houdek P, Moll I, Lobmann R.
Expression of matrix metalloproteinases, cytokines, and
connexins in diabetic and nondiabetic human keratinocytes
before and after transplantation into an ex vivo wound-
healing model. Diabetes Care 2008;31(1):114–120.
40. Mast BA, Schultz GS. Interactions of cytokines, growth
factors and proteases in acute and chronic wound. Wound
Repair Regen 1996;4:411–420.
41. Dinarello CA. Proinflammatorycytokines.Chest 2000;118(2):
503–508.
42. Hawkins D, Abrahamse H. The release of interleukin-6 after
low level laser therapy and the effect on migration and
proliferation of human skin fibroblasts. Med Technol S Afr
2004;18:11–15.
43. Kishimoto T. Interleukin-6 (IL-6). In: Thomson AW, Lotze
MT, editors. The Cytokine Handbook, Vol. 1, 4th edition.
London: Elsevier Science Ltd; 2003. pp 281–304.
44. Castell JV,Gomez-Lechon MJ, David M, Fabra R, Trullenque
R, Heinrich PC. Acute-phase response of human hepatocytes:
Regulation of acute-phase protein synthesis by interleukin-6.
Hepatology 1990;12:1179–1186.
45. Biffl WL, Moore EE, Moore FA, Petterson VM. Interleukin-6
in the injured patient. Marker of injury or mediator of
inflammation? Ann Surg 1996;224:647–664.
46. Aimbire F, Albertini R, Pacheco MTT, Castro-Faria-Neto HC,
Leonardo PSLM, Iversen VV, Lopes-Martins RAB, Bjordal
JM. Low-level laser therapy induces dose-dependent reduc-
tion of TNFa levels in acute inflammation. Photomed Laser
Surg 2006;24(1):33–37.
47. Mafra de Lima F, Costa MS, Albertini R, Silva JA, Jr.,
Aimbire F. Low level laser therapy (LLLT): Attenuation of
cholinergic hyperreactivity, b2-adrenergic hyporesponsive-
ness and TNF-a mRNA expression in rat bronchi segments in
E. coli lipopolysaccharide-induced airway inflammation by a
NF-kB dependent mechanism. Lasers Surg Med 2009;41(1):
68–74.
48. Boschi ES, Leite CE, Saciura VC, Caberlon E, Lunardelli A,
Bitencourt S, Melo DAS, Oliveira JR. Anti-Inflammatory
effects of low-level laser therapy (660nm) in the early phase
in carrageenan-induced pleurisy in rat. Lasers Surg Med
2008;40(7):68–74.
49. 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-1b, TNF-a, IFN-g, TGF-b, bFGF, and
PDGF in rat’s gingival. Lasers Med Sci 2008;23(3):331–
335.
50. Shimizu N, Yamaguchi M, Goseki T, Shibata Y, Takiguchi H,
Iwasawa T, Abiko Y. Inhibition of prostaglandin E2 and
interleukin 1-beta production by low-power laser irradiation
in stretched human periodontal ligament cells. J Dent Res
1995;74(7):1382–1388.
51. Susztak K, Raff AC, Schiffer M, Bottinger EP. Glucose-
induced reactive oxygen species cause apoptosis of podocytes
and podocyte depletion at the onset of diabetic nephropathy.
Diabetes 2006;55:225–233.
52. Gavish L, Perez L, Gertz SD. Low-level laser irradiation
modulates matrix metalloproteinase activity and gene
expression in porcine aortic smooth muscle cells. Lasers Surg
Med 2006;38:779–786.
53. Houreld NN, Abrahamse H. Laser light influences cellular
viability and proliferation in diabetic-wounded fibroblast
cells in a dose- and wavelength-dependent manner. Lasers
Med Sci 2008;23:11–18.
54. Yamaura M, Yao M, Yaroslavsky I, Cohen R, Smotrich M,
KochevarIE.Low levellighteffectson inflammatorycytokine
production by rheumatoid arthritis synoviocytes. 2009;41(4):
282–290.
55. Gavish L, Asher Y, Becker Y, Kleinman Y. Low level laser
irradiation stimulates mitochondrial membrane potential
and disperses subnuclear promyelocytic leukaemia protein.
Lasers Surg Med 2004;35:369–376.
56. Novoselova EG, Cherenkov DA, Glushkova OV, Novoselova
TV, Chudnovskii VM, Iusupov VI, Fesenko EE. Effect of
low-intensitylaser irradiation
cells isolated from mice [abstract]. Biofizika 2006;51:509–
518.
57. Morohoshi M, Fujisawa K, Uchimura I, Numano F. The
effects of glucose and advanced glycosylation end products on
IL-6 production by human monocytes. Ann N Y Acad Sci
1995;17:562–570.
58. CampbellIL, Kay TW, Oxbrow L, Harrison LC. Essential role
for interferon-gamma and interleukin-6 in autoimmune
insulin-dependent diabetes in NOD/ Wehi mice. J Clin Invest
1991;87:739–742.
59. Fahey TJ III, Sadaty A, Jones WG II, Barber A, Smoller B,
Shires GT. Diabetes impairs the late inflammatory response
to wound healing. J Surg Res 1991;50:308–313.
(632.8nm)on immune
502 HOURELD ET AL.