# Laser Irradiation Alters the Expression Profile of Genes Involved in the Extracellular MatrixIn Vitro

Authors: Sandra M. Ayuk; Nicolette N. Houreld; Heidi Abrahamse
Journal: International Journal of Photoenergy
 (2014)

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## Abstract

The extracellular matrix (ECM) forms the basis of every phase in wound healing. Healing may be impaired if some of these components are destroyed. Photobiostimulation has demonstrated a stimulatory response in biological processes. This study aimed to evaluate various genes involved in the ECM, in response to laser irradiation. Isolated human skin fibroblasts were used in three different cell models, namely, normal, normal wounded, and diabetic wounded. Cells were irradiated with 5 J/cm2 using a continuous wave diode laser emitting at a wavelength of 660 nm and incubated for 48 h. Nonirradiated (0 J/cm2) normal and diabetic wounded cells served as the control. Real-time reverse transcription (RT) quantitative polymerase chain reaction (qPCR) was used to determine the expression of 84 genes in a PCR array. There was a significant upregulation of 29 genes in the normal cells, 32 genes in the normal wounded cells, and 18 genes in the diabetic wounded cells as well as a downregulation of 19 genes (normal), 6 genes (normal wounded), and 31 genes (diabetic wounded). Low intensity laser irradiation (LILI) stimulates gene expression in various cell adhesion molecules (CAMs) and extracellular proteins at 660 nm in wounded fibroblasts in vitro.


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## Body

## 1. Introduction

Components of the extracellular matrix (ECM) have been shown to be useful in wound healing [1, 2]. They form the core of every wound healing phase, and healing may be impaired if any of these components is destroyed [3]. In addition, they play a role in angiogenesis, tissue remodelling, and rapid scaffold breakdown [4]. The interaction between the ECM and various cells is very important for proper functioning of the cell [5–8]. This interaction could be direct or indirect. Directly, cellular receptors stimulate the ECM or indirectly, through the structural components of the ECM produced by glycoproteins. Cellular activities directed by these interactions are required for wound healing.Regulation of the wound healing process comprises the interaction of various cell types, namely, neutrophils, lymphocytes, macrophages, and fibroblasts, and regular mediators such as growth factors and cytokines and ECM components (fibronectin (FN); fibrin; collagen; and elastin (EI)); laminin (LMN); proteoglycans (PG); glycosaminoglycans (GAG); matrix metalloproteinases (MMPs); and tissue inhibitor of metalloproteinases (TIMPs) [2].For proper cell survival and gene expression in normal wound healing, the environment needs to be at equilibrium with the activity of growth factors, fibroblast interaction, and mechanical forces to ensure normal tissue remodelling [9]. Fibroblasts produce most of the molecules in the ECM including proteases, integrins, cytokines, and growth factors during tissue repair which are responsible for late phase tissue remodelling and eventually scarring [10, 11]. However, the situation is different in chronic wounds due to the disruption of the regular healing process because of tissue damage, biochemical and cellular imbalances, or an underlying pathological state such as diabetes and venous insufficiency. Venous leg ulcers (VLUs) are amongst the major problems in public health and have become an economic burden in most health care services. It is commonly associated with pain, reduces the quality of life, and is even associated with death. It may also cause tiredness and depression [12–14]. The prevalence of diabetic foot ulcers is approximately 1-2% worldwide [15]; it occurs at any age [16] with an incidence of 3–5% over 65 years [17].Photobiostimulation, or photobiomodulation, is a noninvasive type of treatment that modulates the treatment of wounds through various cellular or biological processes. It is effective in the visible and near infrared (NIR) spectral range. It functions at wavelengths of 500–1 100 nm and a power output of 10–200 mW [18]. The use of photobiomodulation in wound healing has greatly ameliorated various cellular processes affecting different phases of wound healing. Studies have demonstrated the stimulatory effects of photobiomodulation in wounded cell models [19] at 660 nm [20]. Studies have also shown that it enhances diabetic wound healing in both rats and mice [21–23]. Photobiostimulation in the visible and NIR spectral range has been demonstrated to regulate gene expression in human and animal cell cultures, even though its effect was not consistent in all irradiated cells [24]. Studies from different areas showed variations in the gene expression profile of 50 cultures of fibroblasts [25]. Few studies have exploited the relationship of laser irradiation and gene expression of the ECM in fibroblastsin vitro. Due to previous studies which showed an increase in collagen type I (Col-I) in response to laser irradiation at 660 nm [20], this study aimed to determine the effect of laser irradiation at 660 nm on the gene expression profile of the ECM and its cell adhesion molecules.


## 2. Methodology

### 2.1. Cell Culture

This study was performed on human skin fibroblasts isolated from a consenting adult undergoing abdominoplasty (Linksfield, Sandringham, Johannesburg) (University of Johannesburg Academic Ethics Committee Clearance Reference number 01/06). Cells were seeded into 3.4 cm diameter tissue culture flasks at a density of 6 × 105 and routinely cultured according to standard techniques [26]. Different cell models, namely, normal (N), normal wounded (NW), and diabetic wounded (DW), were used. To establish anin vitro diabetic model, 17 mM/L D-glucose was added to the media with a base concentration of 5.6 mM/L D-glucose. Thirty minutes prior to irradiation, a sterile 1 mL disposable pipette was used to scratch the monolayer of cells in a streaking motion (i.e., creating a central scratch (CS)). This creates a cell-free zone on either side of the central scratch [27, 28].


### 2.2. Laser Irradiation

Cells were irradiated with 5 J/cm2 using a continuous wave diode laser emitting at a wavelength of 660 nm (Fremont, CA, USA, RGBlase, TECIRL-100G-650SMA); laser parameters are shown in Table 1. All lasers were supplied and set up by the National Laser Centre (NLC) of the Council for Scientific and Industrial Research (CSIR), South Africa. Nonirradiated (0 J/cm2) normal cells (for irradiated normal and normal wounded) and diabetic wounded cells (for irradiated diabetic wounded cells) served as the control groups. Cells were irradiated from above, with the culture dish lid off, in 1 mL culture media and in the dark to omit nuisance variables suggestive of polychromatic light that would interfere with the laser effect. The power output was measured using a power meter (FieldMate, 0398D05) at bench level prior to each irradiation, and the readings were used to determine the irradiation time. The temperature of the culture media during irradiation was measured every 2 min and remained less than 32°C. Cells were incubated for 48 h, and the profile of genes involved in the ECM and cell adhesion molecules were assessed using a real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) array.Table 1
Laser parameters.
Laser parameters
Wavelength (nm)
660
Wave emission
Continuous wave
Power output (mW)
92.5
Spot size (cm2)
9.1
Output density (mW/cm2)
10.22
Irradiation duration
8 min 9 s
Fluence (J/cm2)
5


### 2.3. RNA Isolation and Purity

Isolation of total RNA from the cells was performed on the Qiagen QIAcube (Whitehead Scientific, Cape Town, South Africa) using the RNeasy Mini Kit (Whitehead Scientific, Cape Town, South Africa, Qiagen, 74104) including QIAshredder homogenizers (Whitehead Scientific, Cape Town, South Africa, Qiagen, 79654). After incubation, cell cultures were detached with TrypLE Express (1 mL/25 cm2) (Life Technologies, Gibco, Invitrogen, 12605-021) and washed with phosphate buffered saline (PBS) to eliminate traces of culture media and then resuspended in 600 μL of a guanidine-thiocyanate-containing buffer (RLT buffer) to disrupt the cells, inactivate RNases, and release cellular contents. Within 30 min, 30 μL of total RNA was eluted and quantified. The concentration of RNA was established using the Quant-iT RNA Assay Kits (Life Technologies, Johannesburg, South Africa, Invitrogen, Q32852) with the Invitrogen Qubit 2.0 fluorometer (Life Technologies, Johannesburg, South Africa). The ratio between absorbance 260 and 280 nm (A260nm/A280nm) was used to estimate the sample purity using a UV/Vis spectrophotometer (Separation Scientific, Johannesburg, South Africa, PerkinElmer, Victor3).


### 2.4. cDNA Synthesis

According to the protocol, a two-step procedure was used to synthesise cDNA using the QuantiTect Reverse Transcription Kit (Whitehead Scientific, Cape Town, South Africa, Qiagen, 205311). Traces of possible contaminating genomic DNA (gDNA) was eliminated from 1μg purified RNA sample using the gDNA Wipeout Buffer for 2 min at 42°C. RNA was then reverse-transcribed using a reverse transcription (RT) master mix. Six microliters of RT master mix was added to the reaction mixture to give a final volume of 20 μL. The mixture was then incubated for 30 min at 42°C and thereafter 3 min at 95°C to terminate the reaction. One microliter of sample was used to estimate the purity as stated earlier. Samples were stored on ice to proceed directly with real-time qPCR or stored at −20°C.


### 2.5. Gene Expression Profiling

Real-time qPCR was performed using the SABiosciences RT2 profiler PCR array (Whitehead Scientific, Cape Town, South Africa, PAHS-01321Z) which profiled 84 genes (Table 2). Ninety-two microliters of PCR water (Diethyl Pyrocarbonate, DEPC free) was added to thawed cDNA (19 μL) giving a final volume of 111 μL. One hundred and two microliters of diluted cDNA was added to the ready-to-use 2x SABiosciences RT2 qPCR master mix (330521), and then 1 248 μL of PCR water was added to give a total volume of 2 700 μL. Components were mixed and 25 μL of the experimental cocktail was dispensed into each well of the 96-well plate. The sealed PCR plates were centrifuged at 1000 g (Separation Scientific, Johannesburg, South Africa, Thermo Scientific, Heraeus Labofuge 400) for 1 min to remove any bubbles and run in the preset real-time thermocycler (Anatech, Randburg, South Africa, Stratagene Mx3000p). The thermocycler profile setting was 10 min at 95°C for 1 cycle and 15 s at 95°C and 1 min at 60°C for 40 cycles. The software was also programmed to do a melt or dissociation curve at the end of the run to ensure the amplification of a single product for each gene. The threshold cycle (Ct) values were imported into an Excel spreadsheet (Available from the SABiosciences website: http://www.sabiosciences.com/) which normalised the results against the 5 housekeeping genes (ACTB, B2M, GAPDH, HPRTI, and RPLPO). In addition, the relative gene expression (ΔΔCt) and fold change (2-ΔΔCt) were also calculated. Prior to data analysis, all the Ct values of the controls were examined to ensure proper functioning of the PCR array and preceding steps (positive PCR control, Ct value of 20±2; genomic DNA control, Ct value of >35). A fold change of >1 was reported as fold upregulation and a fold change <1 was reported as fold downregulation.Table 2
Functional genes of the ECM and its adhesion molecules.
Pathway
Genes
Cell adhesion molecules
Transmembrane molecules
CD44, CDH1, HAS1, ICAM1, ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, ITGA7, ITGA8, ITGAL, ITGAM, ITGAV, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, MMP14, MMP15, MMP16, NCAM1, PECAM1, SELE, SELL, SELP, SGCE, SPG7, and VCAM1
Cell-cell adhesion
CD44, CDH1, COL11A1, COL14A1, COL6A2, CTNND1, ICAM1, ITGA8, and VCAM1
Cell-matrix adhesion
ADAMTS13, CD44, ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, ITGA7, ITGA8, ITGAL, ITGAM, ITGAV, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, SGCE, SPP1, and THBS3
Other adhesion molecules
CNTN1, COL12A1, COL15A1, COL16A1, COL5A1, COL6A1, COL7A1, COL8A1, VCAN, CTGF, CTNNA1, CTNNB1, CTNND2, FN1, KAL1, LAMA1, LAMA2, LAMA3, LAMB1, LAMB3, LAMC1, THBS1, THBS2, CLEC3B, TNC, and VTN
Extracellular matrix proteins
Basement membrane constituents
COL4A2, COL7A1, LAMA1, LAMA2, LAMA3, LAMB1, LAMB3, LAMC1, and SPARC
Collagens and ECM structural constituents
COL11A1, COL12A1, COL14A1, COL15A1, COL16A1, COL1A1, COL4A2, COL5A1, COL6A1, COL6A2, COL7A1, COL8A1, FN1, and KAL1
ECM proteases
ADAMTS1, ADAMTS13, ADAMTS8, MMP1, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP2, MMP3, MMP7, MMP8, MMP9, SPG7, and TIMP1
ECM protease inhibitors
COL7A1, KAL1, THBS1, TIMP1, TIMP2, and TIMP3
Other ECM molecules
VCAN, CTGF, ECM1, HAS1, SPP1, TGFBI, THBS2, THBS3, CLEC3B, TNC, and VTN


### 2.6. Statistical Analysis

Experiments were repeated three times (n=3). Student’s t-test was analysed based on the replicate fold change for each gene in both the test and the control groups by the SABiosciences Excel-based data analysis template and reported as significant if P<0.05. Results are represented in Tables 3 to 6.Table 3
Gene expression profile in normal (N) cells irradiated at 660 nm. Total RNA from nonirradiated N cells and irradiated N cells were characterised in triplicate. A fold difference >1 is considered as gene upregulation, while a fold difference <1 is considered as gene downregulation. Fold differences are indicated as upregulation (↑) or downregulation (↓). X denotes the nonsignificant genes in that particular cell model.
Gene symbol
Gene description
Gene ID
Fold difference
Up/downregulation
P value
ADAMTS1
ADAM metallopeptidase with thrombospondin type 1 motif, 1
9510
4.36
↑
0.003
ADAMTS13
ADAM metallopeptidase with thrombospondin type 1 motif, 13
11093
1.32
X
0.259
ADAMTS8
ADAM metallopeptidase with thrombospondin type 1 motif, 8
11095
2.77
↑
0.003
CD44
CD44 molecule (Indian blood group)
960
4.85
↑
0.012
CDH1
Cadherin 1, type 1, E-cadherin (epithelial)
999
0.58
↓
0.019
CLEC3B
C-type lectin domain family 3, member B
7123
1.90
X
0.078
CNTN1
Contactin 1
1272
1.14
X
0.547
COL11A1
Collagen, type XI, alpha 1
1301
1.05
X
0.896
COL12A1
Collagen, type XII, alpha 1
1303
2.21
↑
0.003
COL14A1
Collagen, type XIV, alpha 1
7373
0.56
X
0.116
COL15A1
Collagen, type XV, alpha 1
1306
1.33
X
0.198
COL16A1
Collagen, type XVI, alpha 1
1307
1.57
X
0.074
COL1A1
Collagen, type I, alpha 1
1277
1.88
↑
0.001
COL4A2
Collagen, type IV, alpha 2
1284
1.78
↑
0.022
COL5A1
Collagen, type V, alpha 1
1289
4.23
↑
0.001
COL6A1
Collagen, type VI, alpha 1
1291
2.42
↑
0.010
COL6A2
Collagen, type VI, alpha 2
1292
3.99
↑
0.000
COL7A1
Collagen, type VII, alpha 1
1294
1.13
X
0.658
COL8A1
Collagen, type VIII, alpha 1
1295
3.07
↑
0.000
CTGF
Connective tissue growth factor
1490
0.43
↓
0.002
CTNNA1
Catenin (cadherin-associated protein), alpha 1, 102 kDa
1495
0.73
↓
0.039
CTNNB1
Catenin (cadherin-associated protein), beta 1, 88 kDa
1499
1.27
X
0.153
CTNND1
Catenin (cadherin-associated protein), delta 1
1500
2.21
↑
0.001
CTNND2
Catenin (cadherin-associated protein), delta 2 (neural plakophilin-related arm-repeat protein)
1501
0.09
↓
0.002
ECM1
Extracellular matrix protein 1
1893
0.47
↓
0.000
FN1
Fibronectin 1
2335
2.29
↑
0.005
HAS1
Hyaluronan synthase 1
3036
1.20
X
0.182
ICAM1
Intercellular adhesion molecule 1
3383
0.72
X
0.156
ITGA1
Integrin, alpha 1
3672
1.88
↑
0.002
ITGA2
Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor)
3673
0.99
X
0.650
ITGA3
Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor)
3675
2.98
↑
0.000
ITGA4
Integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)
3676
0.81
X
0.193
ITGA5
Integrin, alpha 5 (fibronectin receptor, alpha polypeptide)
3678
3.26
↑
0.000
ITGA6
Integrin, alpha 6
3655
1.21
X
0.085
ITGA7
Integrin, alpha 7
3679
0.92
X
0.780
ITGA8
Integrin, alpha 8
8516
1.06
X
0.805
ITGAL
Integrin, alpha L (antigen CD11A (p180), lymphocyte function-associated antigen 1; alpha polypeptide)
3683
1.06
X
0.540
ITGAM
Integrin, alpha M (complement component 3 receptor 3 subunit)
3684
1.58
X
0.055
ITGAV
Integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51)
3685
1.17
X
0.331
ITGB1
Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12)
3688
0.92
X
0.459
ITGB2
Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)
3689
0.96
X
0.792
ITGB3
Integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)
3690
1.08
X
0.339
ITGB4
Integrin, beta 4
3691
1.25
X
0.127
ITGB5
Integrin, beta 5
3693
1.05
X
0.672
KAL1
Kallmann syndrome 1 sequence
3730
1.53
↑
0.030
LAMA1
Laminin, alpha 1
284217
0.52
↓
0.001
LAMA2
Laminin, alpha 2
3908
0.52
↓
0.008
LAMA3
Laminin, alpha 33909
3909
2.60
↑
0.023
LAMB1
Laminin, beta 1
3912
0.92
X
0.594
LAMB3
Laminin, beta 3
3914
0.55
X
0.069
LAMC1
Laminin, gamma 1 (formerly LAMB2)
3915
0.83
X
0.063
MMP1
Matrix metallopeptidase 1 (interstitial collagenase)
4312
0.41
↓
0.001
MMP10
Matrix metallopeptidase 10 (stromelysin 2)
4319
0.81
X
0.198
MMP11
Matrix metallopeptidase 11 (stromelysin 3)
4320
1.53
↑
0.015
MMP12
Matrix metallopeptidase 12 (macrophage elastase)
4321
0.48
↓
0.010
MMP13
Matrix metallopeptidase 13 (collagenase 3)
4322
0.42
↓
0.001
MMP14
Matrix metallopeptidase 14 (membrane-inserted)
4323
4.14
↑
0.000
MMP15
Matrix metallopeptidase 15 (membrane-inserted)
4324
1.96
↑
0.000
MMP16
Matrix metallopeptidase 16 (membrane-inserted)
4325
1.11
X
0.186
MMP2
Matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)
4313
3.34
↑
0.000
MMP3
Matrix metallopeptidase 3 (stromelysin 1, progelatinase)
4314
0.23
↓
0.007
MMP7
Matrix metallopeptidase 7 (matrilysin, uterine)
4316
0.62
↓
0.045
MMP8
Matrix metallopeptidase 8 (neutrophil collagenase)
4317
0.75
X
0.112
MMP9
Matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase)
4318
0.39
↓
0.013
NCAM1
Neural cell adhesion molecule 1
4684
1.63
↑
0.027
PECAM1
Platelet/endothelial cell adhesion molecule
5175
0.81
X
0.503
SELE
Selectin E
6401
0.93
X
0.568
SELL
Selectin L
6402
0.99
X
0.961
SELP
Selectin P (granule membrane protein 140 kDa, antigen CD62)
6403
1.35
X
0.469
SGCE
Sarcoglycan, epsilon
8910
0.36
↓
0.004
SPARC
Secreted protein, acidic, cysteine-rich (osteonectin)
6678
3.33
↑
0.000
SPG7
Spastic paraplegia 7 (pure and complicated autosomal recessive)
6687
5.12
↑
0.000
SPP1
Secreted phosphoprotein 1
6696
0.55
↓
0.001
TGFBI
Transforming growth factor, beta-induced, 68 kDa
7045
0.87
X
0.361
THBS1
Thrombospondin 1
7057
1.87
↑
0.006
THBS2
Thrombospondin 2
7058
2.65
↑
0.003
THBS3
Thrombospondin 3
7059
1.20
X
0.235
TIMP1
TIMP metallopeptidase inhibitor 1
7076
1.43
↑
0.035
TIMP2
TIMP metallopeptidase inhibitor 2
7077
1.17
↑
0.025
TIMP3
TIMP metallopeptidase inhibitor 3
7078
0.43
↓
0.008
TNC
Tenascin C
3371
0.49
↓
0.002
VCAM1
Vascular cell adhesion molecule 1
7412
0.38
↓
0.010
VCAN
Versican
1462
0.46
↓
0.008
VTN
Vitronectin
7448
1.12
X
0.585


## 2.1. Cell Culture

This study was performed on human skin fibroblasts isolated from a consenting adult undergoing abdominoplasty (Linksfield, Sandringham, Johannesburg) (University of Johannesburg Academic Ethics Committee Clearance Reference number 01/06). Cells were seeded into 3.4 cm diameter tissue culture flasks at a density of 6 × 105 and routinely cultured according to standard techniques [26]. Different cell models, namely, normal (N), normal wounded (NW), and diabetic wounded (DW), were used. To establish anin vitro diabetic model, 17 mM/L D-glucose was added to the media with a base concentration of 5.6 mM/L D-glucose. Thirty minutes prior to irradiation, a sterile 1 mL disposable pipette was used to scratch the monolayer of cells in a streaking motion (i.e., creating a central scratch (CS)). This creates a cell-free zone on either side of the central scratch [27, 28].


## 2.2. Laser Irradiation

Cells were irradiated with 5 J/cm2 using a continuous wave diode laser emitting at a wavelength of 660 nm (Fremont, CA, USA, RGBlase, TECIRL-100G-650SMA); laser parameters are shown in Table 1. All lasers were supplied and set up by the National Laser Centre (NLC) of the Council for Scientific and Industrial Research (CSIR), South Africa. Nonirradiated (0 J/cm2) normal cells (for irradiated normal and normal wounded) and diabetic wounded cells (for irradiated diabetic wounded cells) served as the control groups. Cells were irradiated from above, with the culture dish lid off, in 1 mL culture media and in the dark to omit nuisance variables suggestive of polychromatic light that would interfere with the laser effect. The power output was measured using a power meter (FieldMate, 0398D05) at bench level prior to each irradiation, and the readings were used to determine the irradiation time. The temperature of the culture media during irradiation was measured every 2 min and remained less than 32°C. Cells were incubated for 48 h, and the profile of genes involved in the ECM and cell adhesion molecules were assessed using a real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) array.Table 1
Laser parameters.
Laser parameters
Wavelength (nm)
660
Wave emission
Continuous wave
Power output (mW)
92.5
Spot size (cm2)
9.1
Output density (mW/cm2)
10.22
Irradiation duration
8 min 9 s
Fluence (J/cm2)
5


## 2.3. RNA Isolation and Purity

Isolation of total RNA from the cells was performed on the Qiagen QIAcube (Whitehead Scientific, Cape Town, South Africa) using the RNeasy Mini Kit (Whitehead Scientific, Cape Town, South Africa, Qiagen, 74104) including QIAshredder homogenizers (Whitehead Scientific, Cape Town, South Africa, Qiagen, 79654). After incubation, cell cultures were detached with TrypLE Express (1 mL/25 cm2) (Life Technologies, Gibco, Invitrogen, 12605-021) and washed with phosphate buffered saline (PBS) to eliminate traces of culture media and then resuspended in 600 μL of a guanidine-thiocyanate-containing buffer (RLT buffer) to disrupt the cells, inactivate RNases, and release cellular contents. Within 30 min, 30 μL of total RNA was eluted and quantified. The concentration of RNA was established using the Quant-iT RNA Assay Kits (Life Technologies, Johannesburg, South Africa, Invitrogen, Q32852) with the Invitrogen Qubit 2.0 fluorometer (Life Technologies, Johannesburg, South Africa). The ratio between absorbance 260 and 280 nm (A260nm/A280nm) was used to estimate the sample purity using a UV/Vis spectrophotometer (Separation Scientific, Johannesburg, South Africa, PerkinElmer, Victor3).


## 2.4. cDNA Synthesis

According to the protocol, a two-step procedure was used to synthesise cDNA using the QuantiTect Reverse Transcription Kit (Whitehead Scientific, Cape Town, South Africa, Qiagen, 205311). Traces of possible contaminating genomic DNA (gDNA) was eliminated from 1μg purified RNA sample using the gDNA Wipeout Buffer for 2 min at 42°C. RNA was then reverse-transcribed using a reverse transcription (RT) master mix. Six microliters of RT master mix was added to the reaction mixture to give a final volume of 20 μL. The mixture was then incubated for 30 min at 42°C and thereafter 3 min at 95°C to terminate the reaction. One microliter of sample was used to estimate the purity as stated earlier. Samples were stored on ice to proceed directly with real-time qPCR or stored at −20°C.


## 2.5. Gene Expression Profiling

Real-time qPCR was performed using the SABiosciences RT2 profiler PCR array (Whitehead Scientific, Cape Town, South Africa, PAHS-01321Z) which profiled 84 genes (Table 2). Ninety-two microliters of PCR water (Diethyl Pyrocarbonate, DEPC free) was added to thawed cDNA (19 μL) giving a final volume of 111 μL. One hundred and two microliters of diluted cDNA was added to the ready-to-use 2x SABiosciences RT2 qPCR master mix (330521), and then 1 248 μL of PCR water was added to give a total volume of 2 700 μL. Components were mixed and 25 μL of the experimental cocktail was dispensed into each well of the 96-well plate. The sealed PCR plates were centrifuged at 1000 g (Separation Scientific, Johannesburg, South Africa, Thermo Scientific, Heraeus Labofuge 400) for 1 min to remove any bubbles and run in the preset real-time thermocycler (Anatech, Randburg, South Africa, Stratagene Mx3000p). The thermocycler profile setting was 10 min at 95°C for 1 cycle and 15 s at 95°C and 1 min at 60°C for 40 cycles. The software was also programmed to do a melt or dissociation curve at the end of the run to ensure the amplification of a single product for each gene. The threshold cycle (Ct) values were imported into an Excel spreadsheet (Available from the SABiosciences website: http://www.sabiosciences.com/) which normalised the results against the 5 housekeeping genes (ACTB, B2M, GAPDH, HPRTI, and RPLPO). In addition, the relative gene expression (ΔΔCt) and fold change (2-ΔΔCt) were also calculated. Prior to data analysis, all the Ct values of the controls were examined to ensure proper functioning of the PCR array and preceding steps (positive PCR control, Ct value of 20±2; genomic DNA control, Ct value of >35). A fold change of >1 was reported as fold upregulation and a fold change <1 was reported as fold downregulation.Table 2
Functional genes of the ECM and its adhesion molecules.
Pathway
Genes
Cell adhesion molecules
Transmembrane molecules
CD44, CDH1, HAS1, ICAM1, ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, ITGA7, ITGA8, ITGAL, ITGAM, ITGAV, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, MMP14, MMP15, MMP16, NCAM1, PECAM1, SELE, SELL, SELP, SGCE, SPG7, and VCAM1
Cell-cell adhesion
CD44, CDH1, COL11A1, COL14A1, COL6A2, CTNND1, ICAM1, ITGA8, and VCAM1
Cell-matrix adhesion
ADAMTS13, CD44, ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, ITGA7, ITGA8, ITGAL, ITGAM, ITGAV, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, SGCE, SPP1, and THBS3
Other adhesion molecules
CNTN1, COL12A1, COL15A1, COL16A1, COL5A1, COL6A1, COL7A1, COL8A1, VCAN, CTGF, CTNNA1, CTNNB1, CTNND2, FN1, KAL1, LAMA1, LAMA2, LAMA3, LAMB1, LAMB3, LAMC1, THBS1, THBS2, CLEC3B, TNC, and VTN
Extracellular matrix proteins
Basement membrane constituents
COL4A2, COL7A1, LAMA1, LAMA2, LAMA3, LAMB1, LAMB3, LAMC1, and SPARC
Collagens and ECM structural constituents
COL11A1, COL12A1, COL14A1, COL15A1, COL16A1, COL1A1, COL4A2, COL5A1, COL6A1, COL6A2, COL7A1, COL8A1, FN1, and KAL1
ECM proteases
ADAMTS1, ADAMTS13, ADAMTS8, MMP1, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP2, MMP3, MMP7, MMP8, MMP9, SPG7, and TIMP1
ECM protease inhibitors
COL7A1, KAL1, THBS1, TIMP1, TIMP2, and TIMP3
Other ECM molecules
VCAN, CTGF, ECM1, HAS1, SPP1, TGFBI, THBS2, THBS3, CLEC3B, TNC, and VTN


## 2.6. Statistical Analysis

Experiments were repeated three times (n=3). Student’s t-test was analysed based on the replicate fold change for each gene in both the test and the control groups by the SABiosciences Excel-based data analysis template and reported as significant if P<0.05. Results are represented in Tables 3 to 6.Table 3
Gene expression profile in normal (N) cells irradiated at 660 nm. Total RNA from nonirradiated N cells and irradiated N cells were characterised in triplicate. A fold difference >1 is considered as gene upregulation, while a fold difference <1 is considered as gene downregulation. Fold differences are indicated as upregulation (↑) or downregulation (↓). X denotes the nonsignificant genes in that particular cell model.
Gene symbol
Gene description
Gene ID
Fold difference
Up/downregulation
P value
ADAMTS1
ADAM metallopeptidase with thrombospondin type 1 motif, 1
9510
4.36
↑
0.003
ADAMTS13
ADAM metallopeptidase with thrombospondin type 1 motif, 13
11093
1.32
X
0.259
ADAMTS8
ADAM metallopeptidase with thrombospondin type 1 motif, 8
11095
2.77
↑
0.003
CD44
CD44 molecule (Indian blood group)
960
4.85
↑
0.012
CDH1
Cadherin 1, type 1, E-cadherin (epithelial)
999
0.58
↓
0.019
CLEC3B
C-type lectin domain family 3, member B
7123
1.90
X
0.078
CNTN1
Contactin 1
1272
1.14
X
0.547
COL11A1
Collagen, type XI, alpha 1
1301
1.05
X
0.896
COL12A1
Collagen, type XII, alpha 1
1303
2.21
↑
0.003
COL14A1
Collagen, type XIV, alpha 1
7373
0.56
X
0.116
COL15A1
Collagen, type XV, alpha 1
1306
1.33
X
0.198
COL16A1
Collagen, type XVI, alpha 1
1307
1.57
X
0.074
COL1A1
Collagen, type I, alpha 1
1277
1.88
↑
0.001
COL4A2
Collagen, type IV, alpha 2
1284
1.78
↑
0.022
COL5A1
Collagen, type V, alpha 1
1289
4.23
↑
0.001
COL6A1
Collagen, type VI, alpha 1
1291
2.42
↑
0.010
COL6A2
Collagen, type VI, alpha 2
1292
3.99
↑
0.000
COL7A1
Collagen, type VII, alpha 1
1294
1.13
X
0.658
COL8A1
Collagen, type VIII, alpha 1
1295
3.07
↑
0.000
CTGF
Connective tissue growth factor
1490
0.43
↓
0.002
CTNNA1
Catenin (cadherin-associated protein), alpha 1, 102 kDa
1495
0.73
↓
0.039
CTNNB1
Catenin (cadherin-associated protein), beta 1, 88 kDa
1499
1.27
X
0.153
CTNND1
Catenin (cadherin-associated protein), delta 1
1500
2.21
↑
0.001
CTNND2
Catenin (cadherin-associated protein), delta 2 (neural plakophilin-related arm-repeat protein)
1501
0.09
↓
0.002
ECM1
Extracellular matrix protein 1
1893
0.47
↓
0.000
FN1
Fibronectin 1
2335
2.29
↑
0.005
HAS1
Hyaluronan synthase 1
3036
1.20
X
0.182
ICAM1
Intercellular adhesion molecule 1
3383
0.72
X
0.156
ITGA1
Integrin, alpha 1
3672
1.88
↑
0.002
ITGA2
Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor)
3673
0.99
X
0.650
ITGA3
Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor)
3675
2.98
↑
0.000
ITGA4
Integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)
3676
0.81
X
0.193
ITGA5
Integrin, alpha 5 (fibronectin receptor, alpha polypeptide)
3678
3.26
↑
0.000
ITGA6
Integrin, alpha 6
3655
1.21
X
0.085
ITGA7
Integrin, alpha 7
3679
0.92
X
0.780
ITGA8
Integrin, alpha 8
8516
1.06
X
0.805
ITGAL
Integrin, alpha L (antigen CD11A (p180), lymphocyte function-associated antigen 1; alpha polypeptide)
3683
1.06
X
0.540
ITGAM
Integrin, alpha M (complement component 3 receptor 3 subunit)
3684
1.58
X
0.055
ITGAV
Integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51)
3685
1.17
X
0.331
ITGB1
Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12)
3688
0.92
X
0.459
ITGB2
Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)
3689
0.96
X
0.792
ITGB3
Integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)
3690
1.08
X
0.339
ITGB4
Integrin, beta 4
3691
1.25
X
0.127
ITGB5
Integrin, beta 5
3693
1.05
X
0.672
KAL1
Kallmann syndrome 1 sequence
3730
1.53
↑
0.030
LAMA1
Laminin, alpha 1
284217
0.52
↓
0.001
LAMA2
Laminin, alpha 2
3908
0.52
↓
0.008
LAMA3
Laminin, alpha 33909
3909
2.60
↑
0.023
LAMB1
Laminin, beta 1
3912
0.92
X
0.594
LAMB3
Laminin, beta 3
3914
0.55
X
0.069
LAMC1
Laminin, gamma 1 (formerly LAMB2)
3915
0.83
X
0.063
MMP1
Matrix metallopeptidase 1 (interstitial collagenase)
4312
0.41
↓
0.001
MMP10
Matrix metallopeptidase 10 (stromelysin 2)
4319
0.81
X
0.198
MMP11
Matrix metallopeptidase 11 (stromelysin 3)
4320
1.53
↑
0.015
MMP12
Matrix metallopeptidase 12 (macrophage elastase)
4321
0.48
↓
0.010
MMP13
Matrix metallopeptidase 13 (collagenase 3)
4322
0.42
↓
0.001
MMP14
Matrix metallopeptidase 14 (membrane-inserted)
4323
4.14
↑
0.000
MMP15
Matrix metallopeptidase 15 (membrane-inserted)
4324
1.96
↑
0.000
MMP16
Matrix metallopeptidase 16 (membrane-inserted)
4325
1.11
X
0.186
MMP2
Matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)
4313
3.34
↑
0.000
MMP3
Matrix metallopeptidase 3 (stromelysin 1, progelatinase)
4314
0.23
↓
0.007
MMP7
Matrix metallopeptidase 7 (matrilysin, uterine)
4316
0.62
↓
0.045
MMP8
Matrix metallopeptidase 8 (neutrophil collagenase)
4317
0.75
X
0.112
MMP9
Matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase)
4318
0.39
↓
0.013
NCAM1
Neural cell adhesion molecule 1
4684
1.63
↑
0.027
PECAM1
Platelet/endothelial cell adhesion molecule
5175
0.81
X
0.503
SELE
Selectin E
6401
0.93
X
0.568
SELL
Selectin L
6402
0.99
X
0.961
SELP
Selectin P (granule membrane protein 140 kDa, antigen CD62)
6403
1.35
X
0.469
SGCE
Sarcoglycan, epsilon
8910
0.36
↓
0.004
SPARC
Secreted protein, acidic, cysteine-rich (osteonectin)
6678
3.33
↑
0.000
SPG7
Spastic paraplegia 7 (pure and complicated autosomal recessive)
6687
5.12
↑
0.000
SPP1
Secreted phosphoprotein 1
6696
0.55
↓
0.001
TGFBI
Transforming growth factor, beta-induced, 68 kDa
7045
0.87
X
0.361
THBS1
Thrombospondin 1
7057
1.87
↑
0.006
THBS2
Thrombospondin 2
7058
2.65
↑
0.003
THBS3
Thrombospondin 3
7059
1.20
X
0.235
TIMP1
TIMP metallopeptidase inhibitor 1
7076
1.43
↑
0.035
TIMP2
TIMP metallopeptidase inhibitor 2
7077
1.17
↑
0.025
TIMP3
TIMP metallopeptidase inhibitor 3
7078
0.43
↓
0.008
TNC
Tenascin C
3371
0.49
↓
0.002
VCAM1
Vascular cell adhesion molecule 1
7412
0.38
↓
0.010
VCAN
Versican
1462
0.46
↓
0.008
VTN
Vitronectin
7448
1.12
X
0.585


## 3. Results

Irradiation of N cells with 660 nm resulted in the significant upregulation of 29 genes and downregulation of 19 genes (Table3). Irradiation of NW cells with 660 nm resulted in the significant upregulation of 32 genes and downregulation of 6 genes (Table 4). Irradiation of DW cells with 660 nm resulted in the upregulation of 18 genes and downregulation of 31 genes (Table 5). A summary of the results is presented in Table 6.Table 4
Gene expression profile in normal wounded (NW) cells irradiated at 660 nm. Total RNA from nonirradiated N cells and irradiated NW cells were characterised in triplicate. A fold difference >1 is considered as gene upregulation, while a fold difference <1 is considered as gene downregulation. Fold differences are indicated as upregulation (↑) or downregulation (↓). X denotes the nonsignificant genes in that particular cell model.
Gene symbol
Gene description
Gene  ID
Fold difference
Up/downregulation
P value
ADAMTS1
ADAM metallopeptidase with thrombospondin type 1 motif, 1
9510
1.86
↑
0.045
ADAMTS13
ADAM metallopeptidase with thrombospondin type 1 motif, 13
11093
2.39
↑
0.003
ADAMTS8
ADAM metallopeptidase with thrombospondin type 1 motif, 8
11095
1.85
↑
0.001
CD44
CD44 molecule (Indian blood group)
960
1.25
X
0.356
CDH1
Cadherin 1, type 1, E-cadherin (epithelial)
999
1.23
X
0.205
CLEC3B
C-type lectin domain family 3, member B
7123
1.15
X
0.299
CNTN1
Contactin 1
1272
1.25
X
0.068
COL11A1
Collagen, type XI, alpha 1
1301
1.04
X
0.854
COL12A1
Collagen, type XII, alpha 1
1303
1.69
↑
0.004
COL14A1
Collagen, type XIV, alpha 1
7373
0.81
X
0.370
COL15A1
Collagen, type XV, alpha 1
1306
1.20
X
0.216
COL16A1
Collagen, type XVI, alpha 1
1307
1.05
X
0.749
COL1A1
Collagen, type I, alpha 1
1277
1.45
↑
0.006
COL4A2
Collagen, type IV, alpha 2
1284
1.44
X
0.069
COL5A1
Collagen, type V, alpha 1
1289
1.61
↑
0.005
COL6A1
Collagen, type VI, alpha 1
1291
1.27
X
0.170
COL6A2
Collagen, type VI, alpha 2
1292
1.36
X
0.105
COL7A1
Collagen, type VII, alpha 1
1294
1.42
↑
0.005
COL8A1
Collagen, type VIII, alpha 1
1295
1.36
↑
0.011
CTGF
Connective tissue growth factor
1490
0.95
X
0.284
CTNNA1
Catenin (cadherin-associated protein), alpha 1, 102 kDa
1495
1.03
X
0.720
CTNNB1
Catenin (cadherin-associated protein), beta 1, 88 kDa
1499
0.95
X
0.700
CTNND1
Catenin (cadherin-associated protein), delta 1
1500
1.36
X
0.105
CTNND2
Catenin (cadherin-associated protein), delta 2 (neural plakophilin-related arm-repeat protein)
1501
0.63
↓
0.019
ECM1
Extracellular matrix protein 1
1893
0.75
↓
0.017
FN1
Fibronectin 1
2335
1.63
↑
0.012
HAS1
Hyaluronan synthase 1
3036
2.00
↑
0.005
ICAM1
Intercellular adhesion molecule 1
3383
0.78
X
0.139
ITGA1
Integrin, alpha 1
3672
1.57
↑
0.005
ITGA2
Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor)
3673
0.86
X
0.073
ITGA3
Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor)
3675
1.33
X
0.121
ITGA4
Integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)
3676
1.19
X
0.057
ITGA5
Integrin, alpha 5 (fibronectin receptor, alpha polypeptide)
3678
1.46
↑
0.021
ITGA6
Integrin, alpha 6
3655
1.55
↑
0.016
ITGA7
Integrin, alpha 7
3679
0.90
X
0.462
ITGA8
Integrin, alpha 8
8516
2.94
↑
0.012
ITGAL
Integrin, alpha L (antigen CD11A (p180), lymphocyte function-associated antigen 1; alpha polypeptide) 3683
3683
2.04
↑
0.030
ITGAM
Integrin, alpha M (complement component 3 receptor 3 subunit) 3684
3684
1.59
↑
0.027
ITGAV
Integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51)
3685
1.43
↑
0.013
ITGB1
Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12)
3688
1.05
X
0.652
ITGB2
Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)
3689
1.17
X
0.259
ITGB3
Integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)
3690
1.53
↑
0.007
ITGB4
Integrin, beta 4
3691
1.42
X
0.062
ITGB5
Integrin, beta 5
3693
1.07
X
0.539
KAL1
Kallmann syndrome 1 sequence
3730
1.41
↑
0.006
LAMA1
Laminin, alpha 1
284217
0.81
X
0.096
LAMA2
Laminin, alpha 2
3908
1.01
X
0.921
LAMA3
Laminin, alpha 33909
3909
1.33
X
0.546
LAMB1
Laminin, beta 1
3912
1.18
X
0.113
LAMB3
Laminin, beta 3
3914
0.87
X
0.273
LAMC1
Laminin, gamma 1 (formerly LAMB2)
3915
0.94
X
0.419
MMP1
Matrix metallopeptidase 1 (interstitial collagenase)
4312
0.53
↓
0.011
MMP10
Matrix metallopeptidase 10 (stromelysin 2)
4319
1.06
X
0.456
MMP11
Matrix metallopeptidase 11 (stromelysin 3)
4320
1.60
↑
0.009
MMP12
Matrix metallopeptidase 12 (macrophage elastase)
4321
0.75
X
0.019
MMP13
Matrix metallopeptidase 13 (collagenase 3)
4322
0.78
X
0.075
MMP14
Matrix metallopeptidase 14 (membrane-inserted)
4323
0.97
X
0.909
MMP15
Matrix metallopeptidase 15 (membrane-inserted)
4324
2.66
↑
0.000
MMP16
Matrix metallopeptidase 16 (membrane-inserted)
4325
1.00
X
0.963
MMP2
Matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)
4313
1.52
↑
0.004
MMP3
Matrix metallopeptidase 3 (stromelysin 1, progelatinase)
4314
0.33
↓
0.005
MMP7
Matrix metallopeptidase 7 (matrilysin, uterine)
4316
1.15
X
0.499
MMP8
Matrix metallopeptidase 8 (neutrophil collagenase)
4317
1.53
↑
0.049
MMP9
Matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase)
4318
1.02
X
0.803
NCAM1
Neural cell adhesion molecule 1
4684
1.09
X
0.475
PECAM1
Platelet/endothelial cell adhesion molecule
5175
2.24
↑
0.009
SELE
Selectin E
6401
1.39
↑
0.041
SELL
Selectin L
6402
1.08
X
0.597
SELP
Selectin P (granule membrane protein 140 kDa, antigen CD62)
6403
1.47
↑
0.032
SGCE
Sarcoglycan, epsilon
8910
0.73
↓
0.034
SPARC
Secreted protein, acidic, cysteine-rich (osteonectin)
6678
1.59
↑
0.001
SPG7
Spastic paraplegia 7 (pure and complicated autosomal recessive)
6687
1.28
↑
0.042
SPP1
Secreted phosphoprotein 1
6696
0.84
X
0.118
TGFBI
Transforming growth factor, beta-induced, 68 kDa
7045
1.10
X
0.317
THBS1
Thrombospondin 1
7057
1.59
↑
0.008
THBS2
Thrombospondin 2
7058
2.04
↑
0.004
THBS3
Thrombospondin 3
7059
0.89
X
0.349
TIMP1
TIMP metallopeptidase inhibitor 1
7076
1.31
X
0.073
TIMP2
TIMP metallopeptidase inhibitor 2
7077
1.07
X
0.244
TIMP3
TIMP metallopeptidase inhibitor 3
7078
0.89
X
0.340
TNC
Tenascin C
3371
0.73
↓
0.005
VCAM1
Vascular cell adhesion molecule 1
7412
0.61
X
0.065
VCAN
Versican
1462
0.87
X
0.252
VTN
Vitronectin
7448
0.62
↓
0.024Table 5
Gene expression profile in diabetic wounded (DW) cells irradiated at 660 nm. Total RNA from nonirradiated DW cells and irradiated DW cells were characterised in triplicate. A fold difference >1 is considered as gene upregulation, while a fold difference <1 is considered as gene downregulation. Fold differences are indicated as upregulation (↑) or downregulation (↓). X denotes the nonsignificant genes in that particular cell model.
Gene symbol
Gene description
Gene ID
Fold difference
Up/downregulation
P value
ADAMTS1
ADAM metallopeptidase with thrombospondin type 1 motif, 1
9510
0.50
↓
0.003
ADAMTS13
ADAM metallopeptidase with thrombospondin type 1 motif, 13
11093
0.91
X
0.589
ADAMTS8
ADAM metallopeptidase with thrombospondin type 1 motif, 8
11095
2.75
↑
0.005
CD44
CD44 molecule (Indian blood group)
960
0.53
↓
0.049
CDH1
Cadherin 1, type 1, E-cadherin (epithelial)
999
2.12
↑
0.051
CLEC3B
C-type lectin domain family 3, member B
7123
1.14
X
0.749
CNTN1
Contactin 1
1272
2.19
↑
0.041
COL11A1
Collagen, type XI, alpha 1
1301
3.68
↑
0.002
COL12A1
Collagen, type XII, alpha 1
1303
0.37
X
0.003
COL14A1
Collagen, type XIV, alpha 1
7373
1.24
↑
0.023
COL15A1
Collagen, type XV, alpha 1
1306
0.67
X
0.082
COL16A1
Collagen, type XVI, alpha 1
1307
0.61
↓
0.001
COL1A1
Collagen, type I, alpha 1
1277
0.87
X
0.498
COL4A2
Collagen, type IV, alpha 2
1284
0.99
X
0.966
COL5A1
Collagen, type V, alpha 1
1289
0.56
↓
0.036
COL6A1
Collagen, type VI, alpha 1
1291
0.64
↓
0.015
COL6A2
Collagen, type VI, alpha 2
1292
0.58
↓
0.014
COL7A1
Collagen, type VII, alpha 1
1294
0.36
↓
0.017
COL8A1
Collagen, type VIII, alpha 1
1295
0.71
X
0.271
CTGF
Connective tissue growth factor
1490
1.10
X
0.521
CTNNA1
Catenin (cadherin-associated protein), alpha 1, 102 kDa
1495
0.83
X
0.340
CTNNB1
Catenin (cadherin-associated protein), beta 1, 88 kDa
1499
0.64
X
0.084
CTNND1
Catenin (cadherin-associated protein), delta 1
1500
0.36
↓
0.005
CTNND2
Catenin (cadherin-associated protein), delta 2 (neural plakophilin-related arm-repeat protein)
1501
2.72
↑
0.052
ECM1
Extracellular matrix protein 1
1893
0.74
X
0.076
FN1
Fibronectin 1
2335
0.58
↓
0.008
HAS1
Hyaluronan synthase 1
3036
0.87
X
0.544
ICAM1
Intercellular adhesion molecule 1
3383
0.97
X
0.843
ITGA1
Integrin, alpha 1
3672
0.97
X
0.721
ITGA2
Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor)
3673
0.52
↓
0.015
ITGA3
Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor)
3675
0.50
↓
0.010
ITGA4
Integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)
3676
0.86
X
0.459
ITGA5
Integrin, alpha 5 (fibronectin receptor, alpha polypeptide)
3678
0.54
↓
0.004
ITGA6
Integrin, alpha 6
3655
0.76
↓
0.012
ITGA7
Integrin, alpha 7
3679
1.11
X
0.513
ITGA8
Integrin, alpha 8
8516
2.75
↑
0.051
ITGAL
Integrin, alpha L (antigen CD11A (p180), lymphocyte function-associated antigen 1; alpha polypeptide) 3683
3683
1.89
↑
0.003
ITGAM
Integrin, alpha M (complement component 3 receptor 3 subunit)
3684
1.77
X
0.065
ITGAV
Integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51)
3685
0.50
↓
0.002
ITGB1
Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12)
3688
0.53
↓
0.005
ITGB2
Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)
3689
1.58
X
0.083
ITGB3
Integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)
3690
0.58
↓
0.006
ITGB4
Integrin, beta 4
3691
1.87
↑
0.041
ITGB5
Integrin, beta 5
3693
0.97
X
0.879
KAL1
Kallmann syndrome 1 sequence
3730
0.41
↓
0.001
LAMA1
Laminin, alpha 1
284217
0.60
↓
0.014
LAMA2
Laminin, alpha 2
3908
1.17
X
0.396
LAMA3
Laminin, alpha 3
3909
2.25
↑
0.031
LAMB1
Laminin, beta 1
3912
0.95
X
0.609
LAMB3
Laminin, beta 3
3914
0.61
↓
0.010
LAMC1
Laminin, gamma 1 (formerly LAMB2)
3915
0.54
↓
0.008
MMP1
Matrix metallopeptidase 1 (interstitial collagenase)
4312
0.38
↓
0.001
MMP10
Matrix metallopeptidase 10 (stromelysin 2)
4319
1.28
X
0.271
MMP11
Matrix metallopeptidase 11 (stromelysin 3)
4320
1.71
↑
0.025
MMP12
Matrix metallopeptidase 12 (macrophage elastase)
4321
0.52
↓
0.005
MMP13
Matrix metallopeptidase 13 (collagenase 3)
4322
1.89
↑
0.034
MMP14
Matrix metallopeptidase 14 (membrane-inserted)
4323
0.44
↓
0.016
MMP15
Matrix metallopeptidase 15 (membrane-inserted)
4324
1.24
X
0.157
MMP16
Matrix metallopeptidase 16 (membrane-inserted)
4325
0.44
↓
0.003
MMP2
Matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)
4313
0.54
↓
0.002
MMP3
Matrix metallopeptidase 3 (stromelysin 1, progelatinase)
4314
2.42
↑
0.009
MMP7
Matrix metallopeptidase 7 (matrilysin, uterine)
4316
1.86
↑
0.024
MMP8
Matrix metallopeptidase 8 (neutrophil collagenase)
4317
0.63
↓
0.029
MMP9
Matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase)
4318
1.47
↑
0.009
NCAM1
Neural cell adhesion molecule 1
4684
0.97
X
0.811
PECAM1
Platelet/endothelial cell adhesion molecule
5175
3.66
X
0.213
SELE
Selectin E
6401
1.50
X
0.078
SELL
Selectin L
6402
2.36
↑
0.013
SELP
Selectin P (granule membrane protein 140 kDa, antigen CD62)
6403
1.90
X
0.249
SGCE
Sarcoglycan, epsilon
8910
0.93
X
0.657
SPARC
Secreted protein, acidic, cysteine-rich (osteonectin)
6678
0.66
↓
0.054
SPG7
Spastic paraplegia 7 (pure and complicated autosomal recessive)
6687
0.48
↓
0.005
SPP1
Secreted phosphoprotein 1
6696
0.58
↓
0.009
TGFBI
Transforming growth factor, beta-induced, 68 kDa
7045
0.79
X
0.099
THBS1
Thrombospondin 1
7057
0.35
↓
0.001
THBS2
Thrombospondin 2
7058
0.81
X
0.088
THBS3
Thrombospondin 3
7059
1.03
X
0.794
TIMP1
TIMP metallopeptidase inhibitor 1
7076
1.47
↑
0.046
TIMP2
TIMP metallopeptidase inhibitor 2
7077
0.93
X
0.788
TIMP3
TIMP metallopeptidase inhibitor 3
7078
1.05
X
0.825
TNC
Tenascin C
3371
1.18
X
0.492
VCAM1
Vascular cell adhesion molecule 1
7412
1.79
↑
0.002
VCAN
Versican
1462
0.96
X
0.775
VTN
Vitronectin
7448
1.20
X
0.240Table 6
Summary of the various genes significantly expressed and their pathways in normal (N), normal wounded (NW), and diabetic wounded (DW) cells. Fold differences are indicated as upregulation (↑) or downregulation (↓). X denotes the nonsignificant genes in that particular cell model.
Gene symbol
Gene description
Gene pathway
N
NW
DW
ADAMTS1
ADAM metallopeptidase with thrombospondin type 1 motif, 1
ECM protease
↑
X
X
ADAMTS13
ADAM metallopeptidase with thrombospondin type 1 motif, 13
ECM protease, cell-matrix adhesion
X
↑
X
ADAMTS8
ADAM metallopeptidase with thrombospondin type 1 motif, 8
ECM protease
↑
↑
↑
CD44
CD44 molecule (Indian blood group)
Transmembrane, cell-cell adhesion
↑
X
↓
CDH1
Cadherin 1, type 1, E-cadherin (epithelial)
Transmembrane, cell-cell adhesion
↓
X
↓
CLEC3B
C-type lectin domain family 3, member B
Other adhesion and ECM molecules
↑
X
X
CNTN1
Contactin 1
Other adhesion molecules
X
X
↑
COL11A1
Collagen, type XI, alpha 1
Cell-cell adhesion, collagen, and ECM structure constituent
X
X
↑
COL12A1
Collagen, type XII, alpha 1
Other adhesion molecules, collagen, and ECM structure constituent
↑
↑
↓
COL14A1
Collagen, type XIV, alpha 1
Cell-cell adhesion, collagen, and ECM structure constituent
X
X
↑
COL16A1
Collagen, type XVI, alpha 1
Other adhesion molecules, collagen, and ECM structure constituent
X
X
↓
COL1A1
Collagen, type I, alpha 1
Collagen and ECM structure constituent
↑
↑
X
COL4A2
Collagen, type IV, alpha 2
Basement membrane constituent and ECM structure constituent
↑
X
X
COL5A1
Collagen, type V, alpha 1
Other adhesion molecules, collagen, and ECM structure constituent
↑
↑
↓
COL6A1
Collagen, type VI, alpha 1
Other adhesion molecules, collagen, and ECM structure constituent
↑
X
↓
COL6A2
Collagen, type VI, alpha 2
Cell-cell adhesion, collagen, and ECM structure constituent
↑
X
↓
COL7A1
Collagen, type VII, alpha 1
Other adhesion molecules, basement membrane constituent, collagen, ECM structure constituent, and ECM protease inhibitor
X
X
↓
COL8A1
Collagen, type VIII, alpha 1
Other adhesion molecules, collagen, and ECM structure constituent
↑
↑
X
CTGF
Connective tissue growth factor
Other adhesion and ECM molecules
↓
X
X
CTNNA1
Catenin (cadherin-associated protein), alpha 1, 102 kDa
Other adhesion molecules
↓
X
X
CTNND1
Catenin (cadherin-associated protein), delta 1
Cell-cell adhesion
↑
X
↓
CTNND2
Catenin (cadherin-associated protein), delta 2 (neural plakophilin-related arm-repeat protein)
Other adhesion molecules
↓
↓
↑
ECM1
Extracellular matrix protein 1
Other ECM molecules
↓
↓
X
FN1
Fibronectin 1
Other adhesion molecules, collagen, and ECM structure constituent
↑
↑
↓
HAS1
Hyaluronan synthase 1
Transmembrane and other ECM molecules
X
↑
X
ITGA1
Integrin, alpha 1
Transmembrane
↑
↑
X
ITGA2
Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor)
Transmembrane
X
X
↓
ITGA3
Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor)
Transmembrane
↑
X
↓
ITGA5
Integrin, alpha 5 (fibronectin receptor, alpha polypeptide)
Transmembrane
↑
↑
↓
ITGA6
Integrin, alpha 6
Transmembrane
X
↑
↓
ITGA8
Integrin, alpha 8
Transmembrane and cell-cell adhesion
X
↑
↑
ITGAL
Integrin, alpha L (antigen CD11A (p180), lymphocyte function-associated antigen 1; alpha polypeptide)
Transmembrane
X
↑
↑
ITGAM
Integrin, alpha M (complement component 3 receptor 3 subunit)
Transmembrane
X
↑
X
ITGAV
Integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51)
Transmembrane
X
↑
↓
ITGB1
Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 including MDF2, MSK12)
Transmembrane
X
X
↓
ITGB3
Integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)
Transmembrane
X
↑
↓
ITGB4
Integrin, beta 4
Transmembrane
X
X
↑
KAL1
Kallmann syndrome 1 sequence
Other adhesion molecules, collagen, ECM structure constituent, and ECM protease inhibitor
↑
↑
↓
LAMA1
Laminin, alpha 1
Other adhesion molecules and basement membrane constituent
↓
X
X
LAMA2
Laminin, alpha 2
Other adhesion molecules and basement membrane constituent
↓
X
X
LAMA3
Laminin, alpha 33909
Other adhesion molecules and basement membrane constituent
↑
X
↑
LAMB3
Laminin, beta 3
Other adhesion molecules and basement membrane constituent
X
X
↓
LAMC1
Laminin, gamma 1 (formerly LAMB2)
Other adhesion molecules and basement membrane constituent
X
X
↓
MMP1
Matrix metallopeptidase 1 (interstitial collagenase)
ECM protease
↓
↓
↓
MMP11
Matrix metallopeptidase 11 (stromelysin 3)
ECM protease
↑
↑
↑
MMP12
Matrix metallopeptidase 12 (macrophage elastase)
ECM protease
↓
↓
↓
MMP13
Matrix metallopeptidase 13 (collagenase 3)
ECM protease
↓
X
↑
MMP14
Matrix metallopeptidase 14 (membrane-inserted)
Transmembrane and ECM protease
↑
X
↓
MMP15
Matrix metallopeptidase 15 (membrane-inserted)
Transmembrane and ECM protease
↑
↑
X
MMP16
Matrix metallopeptidase 16 (membrane-inserted)
Transmembrane and ECM protease
X
X
↓
MMP2
Matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, and 72 kDa type IV collagenase)
ECM protease
↑
↑
↓
MMP3
Matrix metallopeptidase 3 (stromelysin 1 and progelatinase)
ECM protease
↓
↓
↑
MMP7
Matrix metallopeptidase 7 (matrilysin and uterine)
ECM protease
↓
X
↑
MMP8
Matrix metallopeptidase 8 (neutrophil collagenase)
ECM protease
X
↑
↓
MMP9
Matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, and 92 kDa type IV collagenase)
ECM protease
↓
X
↑
NCAM1
Neural cell adhesion molecule 1
Transmembrane
↑
X
X
PECAM1
Platelet/endothelial cell adhesion molecule
Transmembrane
X
↑
X
SELE
Selectin E
Transmembrane
X
↑
X
SELL
Selectin L
Transmembrane
X
X
↑
SELP
Selectin P (granule membrane protein 140 kDa and antigen CD62)
Transmembrane
X
↑
X
SGCE
Sarcoglycan, epsilon
Transmembrane
↓
↓
X
SPARC
Secreted protein, acidic, and cysteine-rich (osteonectin)
Basement membrane constituent
↑
↑
X
SPG7
Spastic paraplegia 7 (pure and complicated autosomal recessive)
Transmembrane and ECM protease
↑
↑
↓
SPP1
Secreted phosphoprotein 1
Cell-matrix adhesion and other ECM molecules
↓
X
↓
THBS1
Thrombospondin 1
Other adhesion molecules and ECM protease inhibitor
↑
↑
↓
THBS2
Thrombospondin 2
Other adhesion and ECM molecules
↑
↑
X
TIMP1
TIMP metallopeptidase inhibitor 1
ECM protease + inhibitor
↑
X
↑
TIMP2
TIMP metallopeptidase inhibitor 2
ECM protease inhibitor
↑
X
X
TIMP3
TIMP metallopeptidase inhibitor 3
ECM protease inhibitor
↓
X
X
TNC
Tenascin C
Other adhesion molecules
↓
↓
X
VCAM1
Vascular cell adhesion molecule 1
Transmembrane and cell-cell adhesion
↓
X
↑
VCAN
Versican
Other adhesion and ECM molecules
↓
X
X
VTN
Vitronectin
Other adhesion and ECM molecules
X
↓
X


## 4. Discussion and Conclusion

ECM components are very useful in different aspects of wound healing. The ECM interacts with various cells and growth factors in cell proliferation, influencing migration, cell differentiation, and regulating several biological responses [1, 5, 6, 29, 30]. The effects shown by different ECM components depend on the stage of the wound and are determined by the interactions between the cells and growth factors [29]. There is great need for gene expression profiling in the ECM following laser irradiation to be exploited. In this study, 84 genes related to the ECM were studied in various models. Photoirradiation was shown to stimulate gene expression 48 h after incubation in irradiated N, NW, and DW cells as compared to their respective controls. The genes, either up- or downregulated, are functionally grouped depending on their pathways in the ECM.In the present study, four main CAM families were mediated following irradiation at 660 nm. They include cadherins, integrins, selectins, and immunoglobulin CAM (Ig-CAM). The cadherin family are mainly calcium-dependent glycoproteins containing an extracellular domain, a transmembrane domain, and an intracellular domain [31]. Cadherins and integrins form the main cell-surface transmembrane receptors and are involved in modulating cell-cell and cell-matrix adhesion. They function in various cellular events, namely, cell migration, proliferation, survival, differentiation, and modulation of gene expression profiling [32, 33]. In irradiated N cells, CTNND2 was upregulated and CDH1, CTNNA1, and CTNND1 were downregulated at 660 nm; irradiated NW cells showed downregulation of CTNND2; and DW cells showed an upregulation of CDH1 and CTNND2, while CTNND1 was downregulated.Integrins are the main receptor family in charge of interactions in the ECM and consist of two noncovalentα and β subunits; the specific combination of the subunits determines the degree of cell signalling [34]. In irradiated N cells, ITGA1, ITGA3, ITGA5, and ITGAM were upregulated; in irradiated NW cells, ITGA1, ITGA5, ITGA6, ITGA8, ITGAL, ITGAM, ITGAV, and ITGB3 were upregulated; and in irradiated DW cells, ITGA8, ITGAL, and ITGB3 were upregulated, while ITGA2, ITGA3, ITGA5, ITGA6, ITGB1, and ITGB4 were downregulated.Selectins consist of an extracellular domain with a calcium-dependent lectin domain, an epidermal growth factor domain, and a hydrophobic transmembrane domain [35, 36]. Selectins expressed in response to laser irradiation in N cells included CLEC3B, while NW cells showed an upregulation in SELE, SELL, and SELP and in DW cells there was an upregulation in SELL.Ig-CAM contains an extracellular domain with FN repeats, a transmembrane domain, and an intracellular domain [31, 35]. These domains bind with proteins of the ECM, namely, collagen, LMN, and FN, as well as certain integral cell-surface proteins [31]. Members of the Ig-CAM family expressed in response to LILI were CD44, FN1, NCAM1, PECAM1, SGCE, THBS1, THBS2, SPP1, VTN, VCAM1, and CNTN1. In irradiated N cells, CD44, FN1, NCAM1, THBS1, and THBS2 were upregulated, while SGCE, VCAM1, and SPP1 were downregulated following irradiation at 660 nm as compared to nonirradiated N cells. In irradiated NW cells, FN1, PECAM1, THBS1, and THBS2 were upregulated, while SGCE and VTN were downregulated at 660 nm as compared to nonirradiated N cells. In irradiated DW cells, CNTN1 and VCAM1 were upregulated, while CD44, FN1, THBS1, and SPP1 were downregulated at 660 nm as compared to nonirradiated DW cells. Other adhesion molecules expressed in response to LILI were HAS1, VCAN, TNC, KAL1, and CTGF. In this study, KAL1 was upregulated while TNC, VCAN, and CTGF were downregulated in irradiated N cells. In irradiated NW cells, HAS1 and KAL1 were upregulated and TNC was downregulated. In irradiated DW cells, KAL1 was upregulated at 660 nm as compared to nonirradiated DW cells.ECM proteins including collagen, LMN, EI, proteoglycans, and FN have both adhesive and structural functions. The ECM maintains skin integrity and homeostasis and interacts with several structural and extracellular proteins. Collagen is encoded for by more than 42 genes [29, 37]. Some of the collagen molecules are formed through the interaction between FN and integrins [38, 39]. Collagens are extracellular proteins produced mainly by fibroblasts, divided into two main classes, namely, the nonfibril forming (collagens types IV, VI, VII, and XI) and the fibril forming collagens distinguished by their triple helix (collagens types I, II, III, V, and XI). Their main function is to maintain the structural integrity of various tissues and to strengthen and reorganise the ECM [40]. In this study, COL1A1, COL4A2, COL5A1, COL6A1, COL6A2, COL8A1, and COL12A1 were upregulated in irradiated N cells. In irradiated NW cells, COL1A1, COL5A1, COL7A1, COL8A1, and COL12A1 were upregulated, while in DW cells COL11A1 and COL14A1 were upregulated and COL5A1, COL6A1, COL6A2, COL7A1, COL12A1, and COL16A1 were downregulated.LMNs are basement membrane proteins made up of three nonidentical chains. They are associated with cell adhesion, differentiation, migration, matrix organisation, and signal transduction. LAMA1 was upregulated in irradiated N cells, while LAMA2 and LAMA3 were downregulated. In irradiated DW cells LAMA3 was upregulated and LAMA1, LAMB3, and LAMC1 were downregulated. Other matrix associated proteins, such as secreted protein, acidic, and cysteine-rich (encoded for by SPARC), spastic paraplegin 7 (encoded for by SPG7), and extracellular matrix protein 1, were also evaluated. SPARC is associated with cell structure organisation, cell migration, and ECM synthesis [41, 42]. SPG7 is involved in the breakdown of incorrectly folded proteins intracellular motility, membrane trafficking, and organelle biogenesis [43]. ECM1 is part of a cluster of genes involved in epidermal differentiation. Irradiation of N and NW cells to 660 nm resulted in an upregulation of SPARC and SPG7 and a downregulation in ECM1. Analysis of the gene profile of irradiated DW cells revealed a significant downregulation of SPARC and SPG7. Significantly increased gene expression of the constituents of the basement membrane was observed at 660 nm in N and NW cells, while DW cells showed a decrease in gene regulation, with most of the genes downregulated.MMPs are metalloproteases involved in the degradation of the ECM and can be affected in normal or pathological tissue remodelling and wound healing with different substrates, mostly collagen. MMPs are inhibited by TIMPs. ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) is a family of 19 peptidases that are involved in the processing of procollagen, connective tissue organization, and cell migration [44–48]. The gene profile for ECM proteases and inhibitors in response to irradiation at 660 nm revealed an upregulation in MMP2, MMP11, MMP14, MMP15, ADAMTS1, ADAMTS8, TIMP1, TIMP2, and TIMP3 and a downregulation in MMP1, MMP3, MMP7, MMP9, MMP12, and MMP13 in N cells. Irradiated NW cells showed an upregulation of MMP2, MMP8, MMP11, MMP15, ADAMTS1, ADAMTS8, and ADAMTS13, while MMP1, MMP3, and MMP12 showed a downregulation as compared to nonirradiated N cells. In irradiated DW cells, MMP3, MMP7, MMP9, MMP11, MMP13, ADAMTS8, and TIMP1 were upregulated, while ADAMTS1, MMP1, MMP2, MMP12, MMP14, and MMP16 were downregulated as compared to nonirradiated DW cells.These results demonstrated changes in gene expression within the different irradiated N, NW, and DW cell models. The genetic profile seen in the N cell model is a normal response of fibroblast cells to laser irradiation at 660 nm with 5 J/cm2. On the other hand, cells in the NW and DW models have been stressed and compromised in some way, and the genetic profile seen in these cells is a response of wounded/stressed fibroblast cells to laser irradiation at 660 nm. Mechanical modulation of these cells would increase upregulation of ECM components, ECM-specific receptors, and enhanced expression of several cytokines and growth factors in a time-dependent manner [49–51]. The present study showed that DW cells had a significantly downregulated gene expression profile as compared to N and NW cells when irradiated at 660 nm. The downregulation of most of the genes in DW cells is probably due to the dysfunctioning of the ECM exhibited in chronic wounds as a result of hyperglycaemia. Also in chronic wounds, the inflammatory phase is normally delayed, which promotes increased levels of proteases such as MMPs, causing destruction of the ECM, and damages growth factors as well as receptors essential in the healing process. This also results in a lack of integrins which bind to FN to enhance migration, and hence the decrease in migration [52, 53]. Furthermore MMP3, MMP7, MMP9, MMP11, and MMP13 were upregulated in DW cells irradiated at 660 nm. This is possibly due to the fact that degraded collagen molecules do not interact properly enabling a disorganised and weak ECM, increasing the levels of some MMPs in chronic wounds [54]. This corresponds with the decrease in Col-I seen in these cells [21]. However, there was a significant upregulation of some of the collagens and other essential ECM proteins, which is in line with the increase in collagen seen in irradiated DW cells [20].In conclusion, photobiomodulation at a wavelength of 660 nm enhances gene expression of proteins involved in the ECM. The profile is dependent on the culture conditions and stressors placed on the cells. Increased glucose concentration in the culture media was associated with impaired gene regulation, which could be accountable for the poor response of these cells seen in wound healing. Previous studies have not exploited the role of LILI in gene expression of proteins in the ECM using fibroblast cellsin vitro. This study was able to show the gene profile in normal and diabetic wound healingin vitro.The results also confirm the very important role exhibited by cell adhesion molecules (CAMs), integrins, ECM proteins, proteases, and inhibitors in wound healing. Therefore LILI mediated gene expression in wounded fibroblasts through paracrine and autocrine interactions to enhance wound healing. Further work on the molecular advances of gene modulation and their receptors will elucidate the therapeutic importance of LILI.



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*Source: 604518-2014-06-23.xml*