Measurements in wound healing withobservations on the effects of topical agentson full thickness dermal incised wounds

D. Theunissen a, B. Seymour b, M. Forder b, S.G. Cox a, H. Rode a,*aDepartment of Paediatric Surgery, Red Cross War Memorial Children’s Hospital, University of Cape Town,

South AfricabDepartment of Anatomical Pathology, Groote Schuur Hospital, NHLS, University of Cape Town, South Africa

b u r n s 4 2 ( 2 0 1 6 ) 5 5 6 – 5 6 3

a r t i c l e i n f o

Article history:

Accepted 19 September 2015

Keywords:

Wound healing

Topical agents

Epithelial growth factor

Porcine model

Computer image analysis

a b s t r a c t

Introduction: A multitude of topical wound treatments are used today. Although it is well

established that the micro-environment of healing wounds can be altered to improve

healing, it is difficult to measure the subtle differences in outcome where therapies are

compared.

Method: We compared wound healing properties between four different topical agents in

surgically incised wounds in a pig model. The four topical agents, 5% Povidone-Iodine

cream, 1% Silver-Sulphadiazine, 2% Mupirocin, and 1% Silver-Sulphadiazine plus 1 mg/100 g

recombinant-human epithelial growth factor (EGF) were randomly assigned to four test

animals each. Test agents were compared to each other and to untreated controls. We

investigated existing and new methodologies of measurement of wound healing: clinical

and histological visual scoring systems, immuno-histochemistry, and computerized image

analysis of the wounds on days 3, 7, and 28.

Results: All agents were found to have improved healing rates with better cellular architec-

ture. Healing was faster, histological appearance resembled normal architecture sooner,

clinical appearance improved, mitotic activity was stimulated and more collagen was

deposited in comparison to the wounds with no agents. EGF-treated wounds showed an

increased rate of epithelisation, but the rate of healing did not correlate well with evaluation

of cosmetic outcome.

Conclusion: Topical agents improve all aspects of wound healing. The addition of a human

recombinant EGF to Silver-Sulphadiazine increases epithelial growth and amounts of

collagen in the regenerating wounds at day 7.

# 2016 Published by Elsevier Ltd and ISBI.

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: www.elsevier.com/locate/burns

1. Introduction

Inert topical agents in the form of creams, lotions and

ointments are often applied to burn wounds to create an

* Corresponding author at: Department of Paediatric Surgery, Red CrossSouth Africa. Tel.: +27 21 6585012; fax: +27 21 685 6632.

E-mail address: heinz.rode@uct.ac.za (H. Rode).

http://dx.doi.org/10.1016/j.burns.2015.09.0140305-4179/# 2016 Published by Elsevier Ltd and ISBI.

environment conducive to wound healing. These agents,

although not pharmacologically active on the intact skin, have

activity when applied to wounds where the stratum corneum

barrier to penetration is absent. Although effective against

organisms causing burn wound infection, they are also

War Memorial Children’s Hospital, Rondebosch, 7700 Cape Town,

Wou nd Al loca�on A Wou nd Al loca�on B Control Forw ard Treatment Forw ard Treatme nt Backwar d Control Backward

Pig Number

Wound tr eatment Wound Alloca� on

Harves�n g Day

1 Povidon e Iodin e A 7 2 Povidon e Iodin e B 28 3 Silver Su lph adiazin e A 7 4 Mupi rocin B 28 5 Povidon e Iodin e A 28 6 Mupi rocin B 7 7 Hebermin A 7 8 Silver Su lph adiazin e B 7 9 Povidon e Iodin e B 7 10 Silver Sulph adiazin e A 28 11 Heberm in B 28 12 Mupi rocin A 7 13 Mupi rocin A 28 14 Heberm in B 7 15 Silver Sulph adiazin e B 28 16 He bermin A 28

Fig. 1 – Animal wound allocation table.

b u r n s 4 2 ( 2 0 1 6 ) 5 5 6 – 5 6 3 557

potentially cytotoxic to regenerating epithelial cells. It is

therefore prudent that the selection of an antibacterial agent is

not only based on its antibacterial effect, but also on its wound

modulation and cytotoxic effects on immature and non-

adherent keratinocytes [1].

Central to in vivo and in vitro studies of wounds and wound

healing is the correct choice of an experimental animal model

and need for accurate methods to evaluate the effect of topical

agents on wound healing [2–4].

There are many methods used for the analysis of wounds

and the healing process. Parameters used for measuring

outcome should be unambiguous and measurements should

be accurate and reproducible. Studies in pig models have been

used extensively to investigate the mechanisms of wound

healing. Clinical evaluation is usually subjective and not

quantitative, resulting in unacceptable levels of inter- and

intra-observer variation. Similarly lacking are clear histologi-

cal correlates of what would be considered good healing

characteristics of a wound. The need for objective measure-

ments becomes more urgent to evaluate and compare

treatment options in view of the large numbers of topical

agents currently in use.

Previous measurement systems were developed, ranging

from visual scoring systems to measurement of biological and

chemical wound constituents. The structural and ultra-

structural elements of healing wounds remain difficult to

measure, although immuno-histochemistry and scanning

electron microscopy allows some quantification and simple

morphometric measurements [5].

With current advances in computer technology, image

analysis offers dynamic functional imaging, linking multiple

data sources to provide composite quantitative systems.

The aim of this porcine study was to explore two aspects:

comparing healing of wounds treated with four different

topical agents, and the use and value of computerized image

analysis along with existing scoring systems to evaluate

wound healing.

2. Materials and methods

The experimentation was performed in an established animal

research facility and the study was approved by the Animal

Research Review Committee at the University of Cape Town

[Project Number 97/024]. Sixteen female pigs of between 15

and 20 kg were used. The animals were stabled in clean

individual pens throughout the period of experimentation and

received regular feeds and water ad libitum. The pigs were

sedated before induction of anaesthesia. Inhalation anaesthe-

sia with halothane was used during all procedures. Post-

operative analgesia was provided on a scale that would be

appropriate for humans, subjected to the same procedure.

Intravenous buprenorphene hydrochloride (Temgesic) was

administered at a dose of 0.004 mg/kg 12 hourly for the first

48 h post operative and by intramuscular injection thereafter.

The dorsum of the pig was shaved and no washing or prepping

with anti-septic solution was done prior to surgery to prevent

any carry over effect from the agent to influence the outcome.

After induction, a random assignment was made to one of

the treatment agents by an independent assistant and a study

number was marked on the ear with a marker pen. A plastic

template was used to mark 16 paravertebral wounds of

2 cm � 2 cm on the dorsum of the animal for treatment of the

test agent(s). They were positioned either towards the head or

the tail of the animal. Sixteen additional paravertebral

wounds, using the same method, were then marked in a

similar way towards the other end of the animal. These

wounds were left untreated and served as controls. Animal

allocation and wound orientation is depicted in Fig. 1. To

exclude the effect that regional placement of wounding might

have on healing properties, two of the four animals assigned to

a specific treatment had the test wounds towards the tail and

the control wounds towards the head. This order was reversed

for the other 2 animals. Full thickness wound were created by

using a scalpel blade to excise, in a square fashion, the skin of

each area down to the fascial plane. The level of excision

included the panniculus carnosus thereby eliminating the

effect of the latter on wound healing/contracture. All wounds

created were clinically at the same depth.

The four topical agents, randomly assigned to 16 test

animals, were 5% Povidone-Iodine cream, 1% Silver-Sulpha-

diazine, 2% Mupirocin, and 1% Silver-Sulphadiazine plus

1 mg/100 g recombinant-human epithelial growth factor

(Hebermin–Heber Biotech, S.A., Havana, Cuba). The test agents

(approximately 10 ml per site and filling the whole wound),

were then applied to the freshly created wounds and

replenished daily without disturbing the healing process. No

occlusive dressings were used, while the control wounds were

left untreated.

Table 1 – Clinical assessment sheet encompassing thevisual analogue scale and a clinical assessment score.

Visual analogue scale: Poorj-10-j-9-j-8-j-7-j-6-j-5-j-4-j-3-j-2-j-1-

jExcellent

Clinical assessment score

A Colour (compared to surrounding skin)

Perfect 1

Slight mismatch 2

Obvious mismatch 3

Gross mismatch 4

B Contour

Flush with surrounding skin 1

Slightly proud/indented 2

Hypertrophic 3

Keloid 4

C Distortion

None 1

Mild 2

Moderate 3

Severe 4

D Texture

Normal 1

Just palpable 2

Firm 3

Hard 4

E Surface

Matte 1

Shiny 2

Score range 5–18

b u r n s 4 2 ( 2 0 1 6 ) 5 5 6 – 5 6 3558

Each pig was subjected to the following surgical proce-

dures: Punch biopsy on day 3, excisional biopsies on day 7 and

on day 28. On day three after wounding, 10 wounds from each

animal were biopsied (five from the treatment wounds and

five from the control wounds). A punch biopsy instrument,

3 mm in diameter, was used to take a biopsy of the base of the

wound. The wounds that were biopsied were marked with a

permanent marker pen and were excluded from further

analysis.

Two of the four animals assigned to one of the four

treatment groups, were anaesthetized on day seven after

wounding. Twenty wounds were surgically excised with

excision margin deep to the base of the wound and with

surrounding skin intact (ten treated wounds and ten control

wounds). The remaining two animals, assigned to this specific

treatment group, were anaesthetized on day 28 and the wounds

harvested in the same manner. This process was used for all 4

treatment groups. No visible contamination or self-mutilation

of the wounds occurred during the 28 day period. All animals

were sacrificed at the end of the experimental procedure.

3. Analysis

Results were only analysed once all data had been acquired and

the slides assessed by an experienced histopathologist blinded

to the treatment agent. Investigators were blinded to the

treatment agents. Wounds were evaluated by four different

methods: Visual score, histology, immune-histochemistry and

computerized image analysis. Statistical analysis was done

using Student’s t-test, chi-square, ANOVA with Bonferroni test,

and correlation statistics where appropriate. p-Values of <0.05

were interpreted as statistically significant.

3.1. Visual score

A clinical assessment score was previously developed for the

clinical and histological evaluation of healed wounds in

humans [6].

In this study it was used for the assessment of re-

epithelialization in the porcine model. The 5 parameter score,

with a total numerical value of 18, compared the re-epithelia-

lized wound with the surrounding normal skin on day 28

(Table 1). Each of 4 parameters (colour, contour, distortion and

texture was given a score of between 1 and 4, increasing values

indicating increasing levels of poor healing. Whether a wound

was matte or shiny was recorded, the former scoring 1 and the

latter 2. The wounds were independently scored by 3 observers

and a consensus number agreed upon.

An overall assessment was also made and indicated on a

visual analogue scale as a vertical mark on a 10 cm line, 0

indicating an excellent wound and 10 indicating a poor wound.

This score, expressed in centimetres to one decimal place, was

then added to the sum of the individual parameter scores to give

an overall score for each wound (Clinical Assessment Sheet).

3.2. Histology

Histologic assessment of the wounds, performed by a

histopathologist blinded to the treatment agent, involved

quantification of the major architectural abnormalities that

are known to occur in the healing wound. Routine

Haematoxylin and Eosin [H&E] stain were done on day 28

biopsy wounds. A semi-quantitative scale was used where a

score of 0–4 was given according to features of density,

mitotic activity and epithelial growth (Table 2). Epidermis

was characterized by a simple 3-point scale, depending on

the degree of restoration of the rete ridges. Papillary and

reticular dermis was scored in comparison to normal skin.

Parameters were collagen fibre alignment, density and

maturity. This scale has been previously validated [7] and

has been shown to be a reliable, consistent tool in histologic

assessment of human wounds (Histologic Assessment

Scale). The length of epithelial regrowth from the wound

edge to the tip of the epithelial tongue was also measured,

The tip of the epithelial tongue was defined as the most

distal point of confluent epithelial cells and measured along

the basal layer of epithelium.

3.3. Immuno-histochemistry

The MIB1 stain, an indicator of proliferative cell activity, was

done on biopsy specimens from day 7. The slides were

assessed by an experienced histopathologist blinded to the

treatment agent. A semi-quantitative scale was used where a

score of 0–4 was given according to features of staining

density, mitotic activity and epithelial growth. Computerized

image analysis was also used to validate the visual results.

The percentage surface area stained positive by MIBI was

Table 2 – Histologic Assessment Scale.

Epidermis

Normal 0

Some restoration of rete ridges 1

No restoration of rete ridges 2

Dermis

Collagen fibre orientation

Normal basket weave pattern 0

<25% abnormal 1

26–50% abnormal 2

51–75% abnormal 3

76–100% abnormal 4

Keloid-like fibre orientation 5

Collagen fibre density

Normal fibre bundle density 0

<25% abnormal 1

26–50% abnormal 2

51–75% abnormal 3

76–100% abnormal 4

Keloid-like fibres 5

Collagen fibre maturity

Normal fibre maturity 0

<25% abnormal 1

26–50% abnormal 2

51–75% abnormal 3

76–100% abnormal 4

Keloid-like fibres 5

Score range: 0–17

Table 3 – Visual Score wounds (day 28) All agents hadenhanced results compared with untreated controlwounds ( p < 0.01).

Visual analoguescore

Clinical score Total

Average Range Average Range

Silver

Sulphadiazine

3.1 (2.1–3.6) 15 (13.6–16.5) 144.2

Povidone Iodine 2.22 (1–2.7) 13.6 (11–16.3) 136.2

Mupirocin 2.8 (1.9–3.7) 14.7 (12.9–16.7) 147.9

Hebermin 2.7 (1.5–4) 11.9 (12.2–17) 150.1

Silver

Sulphadiazine

control

3.8 (2.2–5) 17.1 (13.2–18) 164.9

Povidone Iodine

control

3.65 (2.1–4.3) 16.7 (16.1–18.1) 165.7

Mupirocin control 3.83 (2.4–5) 17 (14.4–18) 170.9

Hebermin control 3.49 (2.6–4.2) 16.4 (15–17.8) 171.9

b u r n s 4 2 ( 2 0 1 6 ) 5 5 6 – 5 6 3 559

measured, and correlated with the results of the visual

scoring.

3.4. Computerized image analysis

Computerized image analysis was used to measure the rate of

re-epithelialization from the wound edge towards the centre

of the wound as well as the maximum thickness of the new

epithelial layer sampled at the wound edge. The excision

biopsies of wounds on day 7 were used to prepare slides with

routine H&E staining, which was then digitized and analysed

using computerized imaging software.

Computerized image analysis was also used to determine

collagen content of wounds biopsied on day 3, day 7 and day

28. In previous studies it was shown that this method

(calculating total collagen content by measuring percentage

surface area of collagen on Sirius Red stained biopsy samples),

correlate well with direct measurement of hydroxyproline

[8–10].

Table 4 – Histologic Assessment Scale (day 28) treatment agenwounds ( p < 0.01).

Epidermis Collagen orientat

Povidone Iodine 5 18

Mupirocin 7 19

Hebermin 7 15

Silver Sulphadiazine 11 18

Povidone Iodine control 9 9

Mupirocin control 11 21

Hebermin control 10 21

Silver Sulphadiazine control 10 22

4. Results

4.1. Visual score

The visual score of wounds on day 28 showed all treatment

agents had enhanced results compared with their untreated

control wounds ( p < 0.01) (Table 3). The best result was

achieved with Povidone Iodine (136.2), followed by Silver

Sulphadiazine (144.2), Mupirocin (148), and Hebermin (150.1),

although differences between treatment groups were not

significant ( p = 0.11).

4.2. Histology

The histological assessment scores at 28 days also showed

that treatment agents had improved results compared with

their untreated control wounds ( p < 0.01) (Table 4). The best

results were achieved with Povidone Iodine (74), followed by

Mupirocin (77), Silver Sulphadiazine (79), and Hebermin (79).

Differences between treatment groups were not significant

( p = 0.72).

4.3. Immuno-histochemistry

Immuno-histochemistry is an important tool in the measure-

ment of healing parameters. Day 7 biopsy specimens stained

ts had improved results compared with untreated control

ion Collagen density Collagen maturity Total

20 31 74

19 32 77

24 33 79

18 32 79

20 32 80

21 31 84

20 33 84

24 31 87

Fig. 2 – MIB 1 stains for mitosis on day 7 showing increased

mitotic activity in the upper papillary dermis with

proliferation of fibroblasts and epithelial cell growth.

Arrows indicate mitotic figures.Fig. 3 – H&E stained histology of wounds on day 7,

digitalized and analyzed using computerized imaging

software.

b u r n s 4 2 ( 2 0 1 6 ) 5 5 6 – 5 6 3560

with MIB1 showed increased mitotic activity in the upper

papillary dermis, with proliferation of fibroblasts and epithe-

lial cell growth (Fig. 2 and Table 5). All agents tested appear to

stimulate regeneration in the granulation tissue as compared

to untreated control wounds ( p < 0.01). Hebermin, containing

Epidermal Growth Factor, had the most pronounced effect,

scoring 7, followed by Silver Sulphadiazine (10), Mupirocin

(10), and Povidone Iodine (12). This effect approached

statistical significance ( p = 0.06).

These results showed good correlation with the image

analysis method of measuring percentage surface area stained

positive. Hebermin again had the most effect (4.1%), followed

by Silver Sulphadiazine (3.58%), Mupirocin (3.38%), and

Povidone Iodine (3.14%).

4.4. Computerized image analysis

The distance of re-epithelisation was measured from the

wound edge along the wound surface to the end of new

epidermis tissue on day 7 (Fig. 3 and Table 6). There was a

significant difference in the distance between treatment

groups ( p < 0.01) (Fig. 4), and all groups showed a higher rate

of epithelisation when compared to untreated control wounds

( p < 0.01). The distance of epithelisation correlated with the

Table 5 – The MIB1 Score on day 7 for the various agentsAll agents stimulate regeneration as compared to un-treated control wounds ( p < 0.01).

Assessmentscore

Percentagesurface area

stained positive

Hebermin 7 4.1

Silver Sulphadiazine 10 3.58

Mupirocin 10 3.38

Povidone Iodine 12 3.14

Control 21 2.81

surface area covered by the new epidermal layer, with

Hebermin showing the fastest healing rate (distan-

ce = 1902.78 mm), followed by Povidone Iodine (1723.22 mm),

Silver Sulphadiazine (1401.22 mm), and Mupirocin

(1179.36 mm).

The thickness of the new epidermal layer was measured at

the wound edge, believed to be the germinal centre from

where new epidermal cells originate before migration along

the wound surface (Fig. 5 and Table 6). A thicker layer of

epidermis was measured in wounds from treatment groups

Hebermin (120.56 mm, range 49.77–210.91) and Mupirocin

(130.08 m, range 61.2–223.8) when compared with Silver

Sulphadiazine (96.19 mm, range 41.5–138.47) and Povidone

Iodine (101.68 mm, range 55.16–221.12) ( p < 0.01). These two

groups were the only groups significantly different from the

untreated control wounds ( p < 0.01). Mupirocin had the

thickest epidermal layer (223.8 mm), followed by Povidone

Iodine (221.1 mm), Hebermin (210.9 mm), Silver Sulphadiazine

(138.47 mm) and control (203.2 mm).

Collagen content, measured as percentage surface area

stained positively by Sirius Red was done on biopsy wounds on

day 3, day 7 and day 28. In biopsies from day 3, collagen formed

2.7% of the granulation tissue (Table 7). This increased to 41.4%

Table 6 – Epithelial growth measured in mm from woundedge on day 7 All groups showed a higher rate ofepithelisation when compared to untreated controlwounds ( p < 0.01).

Distance (mm) Thickness (mm)

Hebermin 1902.78 (971.28–2327.6) 120.56 (49.77–210.91)

Povidone Iodine 1723.22 (405–2458.0) 101.68 (55.16–221.12)

Silver

Sulphadiazine

1401.22 (660 50–1803.7) 96.19 (41.5–138.47)

Mupirocin 1179.36 (776.85–1802.6) 130.08 (61.21–223.81)

Control 945.18 (620.41–1639.1) 103.14 (53.28–203.2)

Fig. 4 – Epithelial growth measured in mm from wound edge on day 7 in the various treatment groups.

b u r n s 4 2 ( 2 0 1 6 ) 5 5 6 – 5 6 3 561

on day 7 and 68% by day 28. Collagen content was similar for

all wounds on day 3, with Mupirocin 2.63%, Povidone Iodine

2.87%, Silver Sulphadiazine 2.91% and Hebermin 3.05%

( p = 0.8). There was also no difference between treated and

untreated wounds ( p = 0.062). By day 7, collagen content

increased in the treated group to average 42.51% (41.9–43.27%)

while in the untreated wounds there was significantly less

collagen 40.39% (39.9–40.93%), p < 0.01. The difference in

collagen content between treated and untreated wounds

Fig. 5 – Maximum thickness of new epithelial layer

sampled at the wound edge.

was even more pronounced at day 28, with average collagen

content 70.27% (67.94–70.55%) and 65.73% (65.29–66.9%)

respectively ( p < 0.01). Between treatment groups, Heber-

min-treated wounds had the most collagen by day 7 (43.27%),

followed by Silver Sulphadiazine (42.46%), Mupirocin (42.41%),

and Povidone Iodine (41.90%) with no statistical significance

between these groups ( p = 0.85). By day 28 Hebermin wounds

again had the most collagen (72.30%), followed by Silver

Suphadiazine (70.55%), Mupirocin (70.29%), and Povidone

Iodine (67.94%). There was no statistical significance between

groups ( p = 0.27). The tensile strength of the wounds was not

measured.

5. Discussion

We were asked by the South African Government’s Department

of Health to test the efficacy of a topical agent containing a

recombinant epithelial growth factor (EGF) in wound healing.

The Centre for Genetic Engineering and Biotechnology (CIGB) in

Havana (Cuba) developed a hydrophilic topical cream with

potent mytogenic properties stimulating cellular proliferation.

The product, Hebermin is a cream containing 10 mg/g of Rh-EGF

in a 1% Silver Sulphadiazine base. The cream was tested in a

series of clinical trials in Cuba on superficial burn wounds in

adults and children with good preliminary results; accelerated

wound healing, epithelialisation and epidermal regeneration.

Epithelial growth factor has extensively been studied in human

and porcine models and shown to accelerate the healing of

partial thickness wound and donor sites. It is commercially

available in approximately 20 countries [4,11–13].

In this study three commonly used topical agents for burn

care and the combination of Silver-Sulfadiazine containing

the EGF was investigated in a porcine model. The porcine

model was chosen, because of anatomical, physiological and

healing processes which are very similar to the human

scenario. The model was also large enough to allow compar-

isons between treated and non-treated wounds on the same

Table 7 – Percentage collagen content of granulation tissue as measured by Computerized Image Analysis on days 3, 7 and28.

Day 3(%, Range)

Day 7(%, Range)

Day 28(%, Range)

Hebermin 3.05 (1.16–3.87) 43. 27 (38.18–48.1) 72.30 (70.6–77.71)

Silver Sulphadiazine 2.91 (1.05–3.99) 42.46 (38.26–45.47) 70.55 (61.79–74.66)

Mupirocin 2.63 (1.29–3.75) 42.41 (39.32–44.47) 70.29 (63.78–74.16)

Povidone Iodine 2.87 (1.69–3.89) 41.90 (38.8–45.3)1 67.94 (62.94–71.7)

Mupirocin control 2.60 (1.26–3.85) 39.91 (36.98–44.4) 66.90 (61.95–71.01)

Hebermin control 2.59 (1.09–3.94) 40.93 (37.57–45.84) 66.13 (61.28–72.62)

Povidone Iodine control 2.31 (1.08–3.81) 40.69 (37.58–45.48) 65.77 (61.42–71.95)

Silver Sulphadiazine control 2.34 (1.42–3.71) 40.04 (40.75–44.68) 65.29 (61.36–68.13)

b u r n s 4 2 ( 2 0 1 6 ) 5 5 6 – 5 6 3562

animal. Four methods were used to assess the effects on

healing; a visual score, tissue histology, immune-chemistry

and computerized image analysis. In this study, the wounds

treated with different test agents differed in the rate of

epithelisation, clinical healing properties and indices of

wound maturity. All were shown to improve wound healing

as compared to untreated control wounds. Silver Sulphadia-

zine in combination with EGF had 2 specific beneficial effects:

increasing cell proliferation and epithelial growth as mea-

sured on day 7 [2,3,13–17]. There were no statistically

significant differences between the 4 topical agents as

measured by the other investigative modalities.

Various investigators have used clinical criteria in asses-

sing wounds. A visual score for semi-quantitative assessment

of surgical scars was developed by Beausang et al. [6] and was

found to be a sensitive instrument in scar assessment,

allowing validated quantification with a strong correlation

between the macroscopic and microscopic appearance of the

wounds. [18] The visual score of wounds on day 28 showed all

treatment agents had enhanced results compared with their

untreated control wounds ( p < 0.01), although differences

between treatment groups were not significant ( p = 0.11).

Histopathology also played an important role as an

instrument for more objective measurements in wound

healing. Apart from all the histo-pathologic characteristics

assessed the quality and rate of re-epithelialization was of

great importance. The length of the regenerating epithelial

tongue was measured along the basal layer of the epithelium

as this represents the proliferation and migration of epithelial

cells in all directions as a marker of epithelial restoration a

crucial factor in the early closure of a wound [19] The

histological assessment scores at 28 days showed that

treatment agents had improved results compared with their

untreated control wounds ( p < 0.01). Differences between

treatment groups were not significant ( p = 0.72). The effects of

EGF were seen in the increased rate of epithelisation on day 7,

indicating its potential as a topical agent where rapid healing

is required. Epithelial growth factor stimulates a mitogenic

response with a concomitant increase in DNA, RNA protein

and hyaluronic acid synthesis, and promotes the rate of cell

migration [20,21].

Computerized wound image analysis is an objective,

reproducible and accurate method to interpret, both qualita-

tive and quantitatively the macro-morphological changes

occurring in a wound during the healing process. The

computerized data is converted into images for analysis,

quantification and presentation and increases the reproduc-

ibility of wound images and result markedly. It is more

comprehensive than simple morphometric measurements

and less subjective than conventional histopathologic scoring

systems. Computerized Image Analysis was used in this study

to measure the rate of re-epithelisation. We believe that the

images obtained gave an accurate reproducible analysis of the

findings of epithelial ingrowths from the wound edge towards

the centre of the wound [22].

MIB1 monoclonal antibody is a promising tool for deter-

mining cell proliferation on routine histologic material and

strongly relates to histologic and mitotic index. Mitoses are

also stimulated in wounds treated with EGF, as seen in the

higher score in the MIB1 stained slides. This stimulation of

healing is most apparent when the effects of Hebermin are

compared with Silver-Sulphadiazine, where the only differ-

ence between the two agents is the effect of the addition of EGF

to the cream. The same results were observed in a human

study demonstrating an accelerated rate of epidermal regen-

eration on donor sites with a combination of Silver Sulfadia-

zine containing epidermal growth factor (10 mg/ml) compared

with donor sites treated with Silver Sulfadiazine alone [11].

Sirius Red staining is a method for quantitative morpho-

metric determination of collagen fibre measurement. EGF also

had an effect on collagen production, with marginally higher

collagen content in wounds treated with Hebermin at day 7

and day 28. The tensile strength of the wounds were not

measured. This would suggest a novel function for the use of

EGF topical agents where increased rate of healing is required.

6. Conclusion

It is important to determine the best topical agent for the

treatment of wounds. Although it is recognized that a burn

wound heals differently from incisional wounds, this model

and using various topical agents, allowed for a road map to

observe the healing process in a full thickness wound. Agents

differ in their clinical healing properties, rate of epithelialisa-

tion and indices of wound maturity and these results may aid

in developing strategies to improve the healing process. All

agents tested in this animal model improved wound healing.

The wounds healed faster, histological appearance resembled

normal architecture sooner, clinical appearance improved,

mitotic activity was stimulated and more collagen was

deposited in comparison to the wounds with no agents. The

b u r n s 4 2 ( 2 0 1 6 ) 5 5 6 – 5 6 3 563

only measured value of adding EGF to a topical anti-bacterial

agent is increased cell proliferation and epithelial growth as

measured on day 7.

r e f e r e n c e s

[1] Fraser JF, Cuttle L, Kempf M, Kimble RM. Cytotoxicity oftopical antimicrobial agents used in burn wounds inAustralasia. ANZ J Surg 2004;74(3):139–42.

[2] Ansell DM, Holden KA, Hardman MJ. Animal models ofwound repair: are they cutting it? Exp Dermatol2012;21(8):581–5.

[3] Davidson JM. Animal models for wound repair. ArchDermatol Res 1998;290(Suppl.):S1–1.

[4] Sullivan TP, Eaglstein WH, Davis SC, Mertz P. The pig as amodel for human wound healing. Wound Repair Regen2001;9(2):66–76.

[5] Cuttle L, Kempf M, Phillips GE, Mill J, Hayes MT, Fraser JF,et al. A porcine deep dermal partial thickness burn modelwith hypertrophic scarring. Burns 2006;32(7):806–20.

[6] Beausang E, Floyd H, Dunn KW, Orton CI, Ferguson MW. Anew quantitative scale for clinical scar assessment. PlastReconstr Surg 1998;102(6):1954–61.

[7] Jia S, Zhao Y, Law M, Galiano RD, Mustoe TA. The effect ofcollagenase on ischemic wound healing: results of an invivo study. Ostomy Wound Manage 2011;57(8):20–6.

[8] James J, Bosch KS, Aronson DC, Houtkooper JM. Sirius redhistophotometry and spectrophotometry of sections inthe assessment of the collagen content of liver tissueand its application in growing rat liver. Liver 1990;10(1):1–5.

[9] Jimenez W, Pares A, Caballeria J, Heredia D, Bruguera M,Torres M, et al. Measurement of fibrosis in needle liverbiopsies: evaluation of a colorimetric method. Hepatology1985;5(5):815–8.

[10] Lopez-De Leon A, Rojkind M. A simple micromethod forcollagen and total protein determination in formalin-fixedparaffin-embedded sections. J Histochem Cytochem1985;33(8):737–43.

[11] Brown GL, Nanney LB, Griffen J, Cramer AB, Yancey JM,Curtsinger 3rd LJ, et al. Enhancement of wound healingby topical treatment with epidermal growth factor. NEngl J Med 1989;321(2):76–9.

[12] Chvapil M, Gaines JA, Gilman T. Lanolin and epidermalgrowth factor in healing of partial-thickness pig wounds. JBurn Care Rehabil 1988;9(3):279–84.

[13] Nanney LB. Epidermal and dermal effects of epidermalgrowth factor during wound repair. J Invest Dermatol1990;94(5):624–9.

[14] Branski LK, Mittermayr R, Herndon DN, Norbury WB,Masters OE, Hofmann M, et al. A porcine model of full-thickness burn, excision and skin autografting. Burns2008;34(8):1119–27.

[15] Mawafy M, Cassens RG. Microscopic structure of pig skin. JAnim Sci 1975;41(5):1281–90.

[16] Meyer W, Schwarz R, Neurand K. The skin of domesticmammals as a model for the human skin, with specialreference to the domestic pig. Curr Probl Dermatol1978;7:39–52.

[17] Wang XQ, Kravchuk O, Kimble RM. A retrospective reviewof burn dressings on a porcine burn model. Burns2010;36(5):680–7.

[18] Wang XQ, et al. The evaluation of a clinical scar scale forporcine burn scars. Burns 2009;35(4):538–46.

[19] Ca H. The effect of a nanocrystalline silver dressing,Acticoat TM on wound healing in full-thickness decisionalwounds in a porcine model. University of theWitwatersrand; 2014.

[20] Barrandon Y, Green H. Cell migration is essential forsustained growth of keratinocyte colonies: the roles oftransforming growth factor-alpha and epidermal growthfactor. Cell 1987;50(7):1131–7.

[21] Brown GL, Curtsinger 3rd L, Brightwell JR, Ackerman DM,Tobin GR, Polk Jr HC, et al. Enhancement of epidermalregeneration by biosynthetic epidermal growth factor. J ExpMed 1986;163(5):1319–24.

[22] Engstrom NHF, Hellgren L, Johansson T, Nordin B, Vincent J,Wahlberg A. Computerized wound image analysis. In:Pathogenesis of wound and biomaterial-associatedinfections. 1990. p. 189–92.