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.
Article: Impaired wound healing[show abstract] [hide abstract]
ABSTRACT: Nonhealing wounds represent a significant cause of morbidity and mortality for a large portion of the population. One of the underlying mechanisms responsible for the failure of chronic wounds to heal is an out-of-control inflammatory response that is self-sustaining. Underappreciation of the inherent complexity of the healing wound has led to the failure of monotherapies, with no significant reduction in wound healing times. A model of the inflammatory profile of a nonhealing wound is one in which the equilibrium between synthesis and degradation has been shifted toward degradation. This review summarizes the current information regarding acute wound healing responses as contrasted to the delayed response characteristic of chronic wounds. In addition, some initial complexity theoretical models are proposed to define and explain the underlying pathophysiology.Clinics in Dermatology 01/2007; 25:19-25. · 2.33 Impact Factor
Article: Major enzymatic pathways in dermal wound healing: current understanding and future therapeutic targets.[show abstract] [hide abstract]
ABSTRACT: Skin is an essential protective organ for vertebrate animals. During skin injury, a plethora of cells and mediators occupy the wound site and, through a collective effort, perform repair of the tissue. This complex pathophysiological process is referred to as wound healing. The efficiency of wound repair is governed by the sequential influx of a variety of cell types to the wound site, upregulation/downregulation of many signaling molecules, and the interaction of various enzymatic pathways. Any dysregulation in this highly complex, but orderly, pathophysiological process results in impaired wound repair. A variety of metabolic enzymes are induced upon injury and are responsible for driving the key physiological processes within the wound milieu during the inflammatory and resolution phases of wound repair. This review will focus on the contribution of major enzymatic biosystems to the inflammatory, remodeling and resolution phases of normal wound healing, including the arachidonic acid metabolic pathway, L-arginine metabolism and the endogenous oxidant-antioxidant redox systems of the body. The major therapeutic targets within these processes will also be highlighted.Current opinion in investigational drugs (London, England: 2000) 06/2006; 7(5):418-22. · 3.31 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Neutrophils gather at the wound site shortly after trauma and release bactericidal reactive oxygen species (ROS) and H2O2 to kill bacteria and prevent infection. Macrophages arrive at the wound in response to environmental stimuli, phagocytose foreign particles, and release vascular endothelial growth factor (VEGF), an angiogenic factor crucial for wound healing. Because oxidants are released early in inflammation and have been found to regulate transcription factors, we investigated a possible role of H2O2 in VEGF stimulation. Human U937 macrophages exposed to H2O2 and allowed to recover in H2O2-free medium rapidly showed an increase in VEGF mRNA. The H2O2-mediated mRNA increase was dose dependent, blocked by catalase, and associated with elevated VEGF in conditioned media. The increase in VEGF was also found in primary rat peritoneal macrophages and the RAW 264.7 murine macrophage cell line. Transcriptional inhibition with actinomycin D revealed no significant difference in mRNA half-life. Transient transfections with a 1.6-kb VEGF promoter-luciferase construct (Shima DT, Kuroki M, Deutsch U, Ng YS, Adamis AP, and D'Amore PA. J Biol Chem 271: 3877-3883, 1996) showed a ninefold stimulation of VEGF gene promoter activity. We concluded that H2O2 increases macrophage VEGF through an oxidant induction of VEGF promoter. This oxidant stimulation can be mediated by activated neutrophils.AJP Heart and Circulatory Physiology 06/2001; 280(5):H2357-63. · 3.71 Impact Factor
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
Changes in cellular viability, proliferation and apoptosis
were evaluated by the Trypan blue assay, VisionBlueTM
fluorescence assay and caspase 3/7 activity respectively.
necrosis factor-alpha, TNF-a) were determined by ELISA.
NO was determined spectrophotometrically and reactive
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.
Key words: IL-1b; IL-6; lasers; NO; ROS; TNF-a
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 . 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 . 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
usually expressed in response to or together with IL-1 and
TNF-a . However, contradictory effects have been
reported ; 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 .
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
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.
Accepted 5 August 2009
Published online 15 July 2010 in Wiley InterScience
? 2010 Wiley-Liss, Inc.
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
Hyperglycemia is the key metabolic abnormality in
role in the development of diabetic complications . A
number of earlier studies showed that exposure of cells to
hyperglycaemic conditions (20–40mM), and thus mimick-
proliferation [16–19]. This restraint is more pronounced
for higher glucose concentrations  and is expressed
especially after protracted exposure to high glucose levels
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
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 . The central scratch method is an
in vitro wound model whereby the monolayer of cells are
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
. 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
cells by increasing IL-6, ROS and NO and decreasing pro-
inflammatory cytokines TNF-a and IL-1 b.
MATERIALS AND METHODS
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 .
Normal, normal wounded and diabetic wounded cells were
used in this study. An in vitro diabetic wound model was
. 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–
3ml complete culture media were seeded into 3.3-cm-
diameter culture plates as determined by the Trypan blue
exclusion test . 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 .
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
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
performed in duplicate.
Changes following laser irradiation were determined
by measuring cellular viability (Trypan blue exclusion
test), apoptosis (caspase 3/7 activity) and proliferation
TABLE 1. Study Design (n¼6)
Incubation time24 or 48hours1 or 24hours 15minutes, 1,
24 or 48hours
15minutes 1 or 24hours
MethodFluorescence Caspase 3/7
6; IF, immunofluorescent; ELISA, enzyme linked immunosorbent assay.
DIABETIC WOUNDED FIBROBLAST CELLS 495
determined by ELISA, while ROS was determined by IF
staining and NO by the Griess Reagent System.
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).
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
Elmer, Separation Scientific, Johannesburg, South Africa)
and reported in reading light units (RLU).
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
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
The optEIATMsandwich type enzyme-linked immuno-
sorbent assay (ELISA) sets for human cytokine from BD
was used to determine IL-1b (BD 557953), TNF-a (BD
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
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 . 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
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)-
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 . 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
(9:1). For the positive control, 100mM TBHP was added
CO2) for 60minutes. Fluorescence was viewed and images
were taken with the Zeiss Live-Cell Imager.
496HOURELD ET AL.
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
fluence of 5J/cm2did not have any significant effect on the
viability of cells. Percentage viability was above 95% in all
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-
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
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).
Cellularproliferation wasdetermined in normal,normal
wounded and diabetic wounded WS1 cells 24 or 48hours
an increase in proliferation of 51% and 19% in normal
wounded cells irradiated for 24 or 48hours respectively
respectively (P<0.01). Comparison of unirradiated cells
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
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
TNF-a seen at 24hours (P<0.01).
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%
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
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
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-
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).
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
% ChangeAbsolute value% Change Absolute value
0.9992 ? 0.071
1.1365 ? 0.073
1.5147 ? 0.141
1.2365 ? 0.042
1.3834 ? 0.087
1.3052 ? 0.057
0.0433 ? 0.005
0.0451 ? 0.002
0.0438 ? 0.001
0.0489 ? 0.002
0.0476 ? 0.002
0.0498 ? 0.002
0.0356 ? 0.004
0.0453 ? 0.003
0.0486 ? 0.003
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
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
are shown as P<0.01 (**).
498 HOURELD ET AL.
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
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.
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 . 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
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,
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.
be beneficial in hastening the healing process in diabetic
a dose of 10J/cm2beneficial. Al-Watban  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 . In addition, TNF-a is auto-
in a persisting cycle of inflammation . 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  and
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.  found that irradiation of rats with an
reduced TNF-a concentration in bronchoalveolar lavage
fluid. Mafra de Lima et al.  and Boschi et al.  also
found a reduction in TNF-a post-irradiation, while Safavi
et al.  found a significant decrease in the gene
expression of IL-1b and no significant difference in TNF-a
irradiations caused the inhibition of IL-1b and Boschi et al.
 found a decrease in NO and IL-6.
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
diabetic cells, this corresponds with previous studies 
and work conducted by Susztak et al. .
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
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.  also found an
increase in proliferation in porcine aortic smooth muscle
cells irradiated at 780nm with 2J/cm2. However, they
Hawkins and Abrahamse  found an increase in
proliferation in wounded fibroblast cells (WS1) irradiated
with a He–Ne laser with 5 J/cm2, while Houreld and
Abrahamse  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 . 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
as a result of ROS which then participate in various redox
reactions. Eichler et al.  found that both red and
infrared light stimulated the production of ROS in rat
cardiocytes. Lindga ˚ rd et al.  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.  irradiated
normal human fibroblasts with a He–Ne laser (0.5–16J/
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
500 HOURELD ET AL.
of activating NF-kB which then translocates from the
cytosol to the nucleus where it initiates the production of
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-
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.
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