Ghép Da: Chỉ Định, Ứng Dụng Và Các Nghiên Cứu Hiện Tại (2011)

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chlorhexidine acetate). When the Аcticoat dressing was applied on infected wounds, the
clinical type of the wound remained the same, but the coating on the dressing bottom was
more mucous and thick, and a 4-5-day delay in the wound clearing was observed. Wound
sites in which a pathogenic flora was isolated during the treatment did not clinically change
the wound type. In these rare cases the only clinical sign was the delay of the wound
clearing and epithelization, respectively.
The application of Acticoat™ and Аcticoat Flex for treatment of superficial burns is
demonstrated (Figures 1-15).

Fig. 1. The beginning of treatment
Fig. 2. Result after the 1st dressing

Fig. 3. Result after the 2st dressing – complete epithelization


Fig. 4. Final result – 30th day after the burn

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243

Fig. 5. The beginning of the treatment with Acticoat™ of a left arm with deep dermal flame
burn –



Fig. 6. Wound condition after the 1st
dressing
Fig. 7. Wound condition after the 2nd dressing





Fig. 8. Wound condition after the 3rd dressing –
epithelialized surface with scattered small wounds and
distinguished hyperpigmentation resulted from the
application of the silver dressing

Fig. 9. The 16th day after burn.
Full epithelization with
disappearing
hyperpigmentation

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Fig. 10. Final outcome

Fig. 11. Scalding of the right leg
Fig. 12. Covering the wound with Acticoat
Flex 7


Fig. 13. The result after the 1st dressing on the
7th day of the treatment
Fig. 14. Epithelializing wound covered
with Acticoat Flex


Fig. 15. The 12th day of the treatment – fully
epithelialized wound of the thigh and
progressing wound epithelization of the
lower leg
Fig. 16. Early result (15th day) – removing
of the dressing after moistening. The
wound is almost fully epithelialized

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245
The dressing was applied on 19 children from the second group with deep burns with
various area (from 0.5% to 95%, mean – 15.76%) and various localization. All children
underwent operation. An early surgical excision at one, two or three stages was performed
on 16 children between the 2nd and the 6th day after the burn. Three children were operated
on after the 10th day of the burn. The dressing changes for this group conformed to the times
of surgical treatment. On those patients operated on after the 10th day, the dressing was
applied according to the described standard method. The demarcation and separation of the
devitalized tissue in these patients occurred after the second or third dressing. The burns
were clear and without exudation. The formed necroses were greenish and not dry for
Acticoat™, while those for Acticoat Flex were greenish-brown and drier. Dressing removal
was easy and without bleeding, and a clear wound with a short and fresh granulation was
formed, ready for autografting.
In 5 children from the third group, Acticoat was applied on fresh excised sites of burn
wounds (mean area of 6.75%), where multistage excision was carried out. After the dressing
removal, the excised site was covered with bright yellow coating without smell and
exudation. This coating could be easily removed and the wound area was fresh and ready
for autografting without any additional surgical treatment. Hyperpigmentation of the
wound was not observed.
The dressing was applied on autografts in 5 of the clinically monitored children, who had
above 15% burn and deep burn area ranging from 6% to 72%. Using the standard method,
Acticoat was applied directly on the autografts, which had various areas (300 cm2 to 800
cm2) and was meshed at 1:2 ratio using mesh-graft dermatome (1:4 ratio for one patient
only). The dressing was removed on the 4th day after the operation (in two children on the
5th day) and well-healed autografts with hyperpigmentation, scant smell and without
exudation was observed. The epithelization of the mesh splits was 95%.
In 5 children, the dressing was applied on fresh donor sites situated on the thighs. The
autografts with thickness of 0.2 – 0.5 mm were harvested from these areas with electric
dermatome. The standard way of dressing application was used. After the removal of the
dressing on the 4th day, the donor site had pink colour and was covered with silver scattered
particles. This site did not have any smell, exudate or coating. Epithelization of 95% occurred in
one child on the 4th day. The complete epithelization occurred in the remaining patients within
8 days after the dressing application. Hyperpigmintation of the intact skin was observed in all
patients. The donor sites were monitored up to six months after the epithelization and there
were no signs of hypertrophic scarring according to the Vancouver Scar Scale (VSS).
The following figures demonstrate a clinical case with ActicoatTM application (Figures 17-
28): i) treatment of deep burns localized on the legs; ii) application on excised area; iii)
application on auto- and allograft; iv) application on donor sites.


Fig. 17. Deep burn
Fig. 18. Application of ActicoatTM as a pre-
operative preparation

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246


Fig. 19. Total surgical excision


Fig. 20. Application of ActicoatTM on fresh autograft


Fig. 21. A stable whole autograft on knee and condition of the excised site on the lower leg
after ActicoatTM removal

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247

Fig. 22. A stage of the treatment – healed autografts in the knee area, healed autografts in the
right lower leg; and clinical condition of the excised site on the left lower leg after ActicoatTM
application

Fig. 23. Autografts covered with Acticoat and donor sites on both legs


Fig. 24. Donor sites after the 1st ActicoatTM
dressing
Fig. 25. Epithelialized donor site at the 2nd
dressing and neighbouring donor site after
the 1st dressing

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248

Fig. 26. Wounds covered with stable autografts and fully epithelialized donor sites.
Persistent hyperpigmentation on operated and donor sites due the ActicoatTM use



Fig. 27. Outcome at the end of the treatment and beginning of the prophylaxis of the scars
with silicone sheets


Fig. 28. The patients after the end of the treatment

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249
3.2 Clinical observation of the wounds treated with Dermazin
The application time of the agent was registered until the beginning of stable epithelization
of the wound in the patients with superficial burns. In the deep burns, this period included
the time until the wound clears or the surgical treatment begins. The local treatment
continued further with other agents for both groups of patients, depending on the wound
type. In 32 patients with superficial burns, on which Dermazin was applied, the wounds
were covered with “an abundant amount of fester-like exudate”, which created an
impression of worsened local status (Figure 29). The bacteriological analysis showed that in
most of the cases this exudate was sterile and was caused by the mixing of the wound
exudate and the agent. Forming of “pseudonecrosis”, which was difficult and painful to
remove, was observed (Figure 30). For this reason, the dressing change required general
anaesthesia even in burns with small area. Moist and soft necroses with grey metallic colour,
which were irremovable, were observed in deep-dermal and deep burn wounds (Tonkin &
Wood, 2005). In patients who did not undergo operations, these necroses were slowly and
tangentially detached in due course. In case of local infection, the dressings were wet at the
time of dressing change due to the abundant amount of exudates, while swelling,
reddening, firm wound edges and smell were observed.

Fig. 29. Clinical state of a wound treated with
Dermazin
Fig. 30. Formed “pseudonecrosis“ after the
treatment with Dermazin
3.3 Bacteriological analysis
Acticoat is a dressing with a silver coating and unique physical structure of silver
nanocrystalline, which in a moist environment provides a long-lasting and steady release of
free silver ions and radicals on the surface of a burn wound. Although Acticoat releases 30
times less silver than 0.5 per cent solution of silver nitrate (AgNO3) and SSD, it has a much
quicker and more powerful antibacterial effect. It is an effective agent against Gram-positive
and Gram-negative microorganisms, including aerobic and facultative anaerobic organisms
and fungi. It is effective against all pathogenic microorganisms isolated from burn wounds,
especially Pseudomonas aeroginosa, Staphylococcus aureus – MRSA and VRE (Tredget et
al., 1998). Its antiseptic effect is revealed within 30 minutes of the application and lasts up to
3 days (Wright et al., 1999). The Minimum Inhibitory Concentration (MIC) and the
Minimum Bactericidal Concentration (MBC) of the three silver dressings (Acticoat, AgNO3
and SSD) have been spectrophotometrically studied, determined and compared using five
bacterial strains, which are of clinical interest (Demling & DeSanti, 2002). Acticoat has the
lowest MIC and MBC in comparison with the other two silver dressings. Due to these
laboratory proven results, a preliminary in vitro analysis has not been carried out. The

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250
investigation of the results from the bacteriological analysis after the Acticoat application
indicates that (Figures 31-35):
-
The wounds of 96 children (86%) were without bacterial growth at the beginning of the
treatment with Acticoat and remained unchanged in 95 children until the end of the
treatment.
-
A Gram-positive and Gram-negative flora was isolated in 16 children at the beginning
of the treatment (5 with Staph.aureus-MRSA, 2 with Str.ß haemoliticus and 6 with
Staph.epidermidis).
-
In one child with uncontaminated wound at the beginning of the treatment, the wound
became colonized by Ps. aeroginosa (microbial number was below the critical value of
103, 102) during the treatment course.
-
Bacterial growth was not observed in 109 children (97%) at the end of the treatment.
Contaminated wounds of two children at the beginning of the treatment remained
unchanged even after the third dressing, but the microbial number was below the
critical values (1 child with Ps. aeroginosa 103 and 1 child with Staph.aureus-MRSA
102).


Fig. 31. Bacterial growth distribution during the treatment with Acticoat


Fig. 32. Distribution of the bacteria and bacterial growth at the beginning of the treatment
with Acticoat

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251

Fig. 33. Distribution of the bacteria and bacterial growth at the end of the treatment with
Acticoat


Fig. 34. Distribution of the bacteria at the beginning of the treatment with Acticoat

Fig. 35. Distribution of the bacteria at the end of the treatment with Acticoat

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252
4. Comparative analysis between Acticoat™ and SSD (Dermazin)
4.1 Comparative bacteriological analysis between Acticoat™ and SSD (Dermazin)
A comparative bacteriological analysis between Acticoat™ and SSD (Dermazin) has been
carried out. The following parameters have been monitored:
-
The type of the bacterial flora (wound surface swab) before the Acticoat™ application;
-
The type of the bacterial flora (wound surface swab) after each removal of the dressing.
The dressings were changed every 3 days according to the already described way;
-
The quantity of the bacterial flora (tissue biopsy samples) in gram wound tissue
(microbial number), before and after the application of the agent. The critical value for
development of a local and general infection of the wound is 105 bacteria per gram
tissue (b/gr).
The performed prospective clinical study comprised 111 patients – 68 (61.26%) of them were
treated with Acticoat™ and 43 (38.74%) with Dermazin. The gender distribution is: 67 males
(60.36%) and 44 females (39.64%). The average age of these children was 4.78±4.38 years in
the age range of 9 months – 18 years. The total number of the burns was 265: 151 (56.98%) of
which were treated with Acticoat™ and 114 (43.02%) with Dermazin.
4.1.1 Results
It could be seen from Tables 1 and 2 that the patients included in the sample do not
statistically differ with respect to age, burn area and burn depth. The further analysis shows
that there is no significant difference of gender distribution. The initial similarity of the main
confounding factors gives a good basis for the comparison of the outcome obtained from the
treatments with the two agents.

Parameter
Treated with Acticoat™
Treated with Dermazin
p
N
X
SD
N
X
SD
Age
66
4.65
4.11
43
4.97
4.80
n.s.
Burn area
68
7.65
5.95
43
9.95
7.84
n.s.
Table 1. Comparative analysis of the age and burn area of the patients treated with the
studied agents (X - mean value, SD- standard deviation, N- number, n.s. – not significant)
4.1.2 Initial status of the patients treated with the two agents with respect to bacterial
flora
It is clear from Table 3 that bacterial flora is registered in 17.65% of the patients treated with
Acticoat™ and in 13.95% of those treated with Dermazin. The Fisher’s exact test classifies the
difference with respect to this parameter as non-significant. The ratio of the bacterial findings
deep in the wound was identical to those on the surface at the beginning of the treatment.
4.1.3 The status of the bacterial flora on 4th day after the first dressing
The technology of treatment with the two agents allows comparison of impact only on the
4th day after the first dressing. The bacterial flora decreased from 17.65% at the beginning of

Application of the Nanocrystalline Silver in Treatment of Burn Wounds in Children

253
Degree
Statistics
Agent
Total
Acticoat™
Dermazin
ІІ A
Number
54
38
92
% w.r. degree
58.70
41.30
100.00
% w.r. agent
40.91
43.18
41.82
ІІ B
Number
62
40
102
% w.r. degree
60.78
39.22
100.00
% w.r. agent
46.97
45.45
46.36
ІІІ
Number
16
10
26
% w.r. degree
61.54
38.46
100.00
% w.r. agent
12.12
11.36
11.82
Total
Number
132
88
220
% w.r. degree
60.00
40.00
100.00
% w.r. agent
100.00
100.00
100.00
Table 2. The sample participants distribution with respect to the burn degree and agent
(w.r. - with respect to)

Bacterial
flora
on the surface
Statistics
Agent
Total
Acticoat™
Dermazin
Absence
Number
56
37
93
% w.r. flora
60.22
39.78
100.00
% w.r. agent
82.35
86.05
83.78
Presence
Number
12
6
18
% w.r. flora
66.67
33.33
100.00
% w.r. agent
17.65
13.95
16.22
Total
Number
68
43
111
% w.r. flora
61.26
38.74
100.00
% w.r. agent
100.00
100.00
100.00
Table 3. The sample participants distribution with respect to the agent and existence of
bacterial flora (w.r. - with respect to)
the treatment up to 16.18% on the 4th day for Acticoat™, while an increase from 13.95 up
to 25.58% was observed in the patients treated with Dermazin. The change is significant,
but it is in favour of the treatment with Acticoat™. On the 4th day of the treatment the
ratio of the bacterial findings deep in the wound was completely identical to that on the
surface. The dynamics of the change of the bacterial findings for the two agents on the 1st
and 4th (Figure 36).
The dynamics of the change of the bacterial flora on the surface, treated with the two agents,
showed that from 4 bacterial findings of Staph.aureus - MRSA at the beginning of the
treatment with Acticoat™ only one was isolated on the 12th day, whereas from 3 bacterial
findings of Pseudomonas aeroginosa two were present, while the other initial bacterial
findings were killed.

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254


Fig. 36. The dynamics of the bacteria change for the two agents on the 1st and 4th day
In the treatment with Dermazin, an increase in the present bacterial findings or appearance
of new ones in the course of treatment up to the 4th day were observed. This is most
pronounced for Acinetobacter and Pseudomonas aeroginosa with respect to quantity. There
was a decrease in Staph.aureus - MRSA from 5 to 3 findings and complete clearing of
Str.beta haemoliticus (from 1 to 0), but the sterility as a whole aggravated for this agent on
the 4th day (Figures 37-39).
The results of the deep analysis of the bacterial flora also showed the advantages of the
treatment with Acticoat™ (Figures 40-42). The patients treated with Acticoat™ have worse
bacteriological status in the beginning and significantly better dynamics on the 4th day of the
treatment, while a worsening of the sterility in the deepness is observed for the patients
treated with Dermazin. In one patient treated with Acticoat™, bacterial flora was observed
only on the surface, but not in the deepness on the 8th and 12th day.


Fig. 37. Bacterial growth distribution during the treatment with Dermazin

Application of the Nanocrystalline Silver in Treatment of Burn Wounds in Children

255

Fig. 38. Distribution of the bacteria at the beginning of the treatment with Dermazin


Fig. 39. Distribution of the bacteria at the end of the treatment with Dermazin


Fig. 40. Bacterial growth distribution during the treatment with Acticoat

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256


Fig. 41. Distribution of the bacteria at the beginning of the treatment with Acticoat


Fig. 42. Distribution of the bacteria at the end of the treatment with Acticoat
4.2 Comparative analysis of the patients treated with the two agents with respect to
clearing time, epithelization and hospital stay
The results unambiguously illustrated that Acticoat™ possesses significantly better
therapeutic effect in comparison with Dermazin (Figures 43-56; Table 4). It was clear that the
burns treated with Acticoat™ had significantly shorter clearing/ epithelization time and
hospital stay (Table 4).

Application of the Nanocrystalline Silver in Treatment of Burn Wounds in Children

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Fig. 43. Distribution of the wound clearing time


Fig. 44. Distribution of the wound epithelization time


Fig. 45. Distribution of the hospital stay

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258

Fig. 46. Mean wound clearing time, mean epithelization time and mean hospital stay

Parameter
Treated with Acticoat™
Treated with Dermazin
р
N
X
SD
N
X
SD
Clearing day
66
8.92
3.16
43
14.93
2.20
<0.001
Epithelization
time (days)
60
12.98
4.02
39
19.79
3.02
<0.001
Hospital
stay
(days)
66
16.95
6.19
42
26.90
6.91
<0.001
Table 4. Comparative analysis of the patients treated with the studied agents with respect to
clearing time, epithelization time and hospital stay
The performed comparative statistical analysis (t-test) reveals that the differences of
application of Acticoat™ and Dermazin were statistically significant. The obtained p-value
for the three tests (comparison of the clearing time, epithelization time and hospital stay for
the two agents) was p<0.01. The Mann-Whitney test was applied for the statistical analysis
of the bacterial growth (microbial number) and the obtained value (p=<0.01) implied that
the difference of application of Acticoat™ and Dermazin for the treatment of the monitored
patients was statistically significant. This analysis showed statistically significant differences
between the application of Acticoat™ and Dermazin with respect to the clearing time,
epithelization time, hospital stay and microbial growth.
The comparative clinical and microbiological analysis between the two silver agents revealed
that treatment with Acticoat™ achieves better efficacy in killing superficial and deep bacterial
flora and yielded faster wound clearing, epithelization and shorter hospital stay.
4.3 Side effects
Three children of the observed contingent treated with Acticoat™ developed rash, which
was transient and faded quickly. The most probable reason for the rash was due to the

Application of the Nanocrystalline Silver in Treatment of Burn Wounds in Children

259
occlusive effect of the dressing caused by the polyurethane foil. Toxic effects were not
observed. The hyperpigmentation of the intact skin was transient and disappeared within 14
days after the end of the treatment or dressing change. In the children of the control group
with Dermazin no toxic and allergic reactions were observed. Cases of argyria were not
observed.
4.4 Comparative cost analysis between Acticoat™ and SSD (Dermazin)
The comparative cost analysis was based on the treatment of a burn wound of second
degree (IIAB) with 2000 cm2 area. One surgeon, one anaesthesiologist, one surgical nurse,
one anaesthetic nurse and one hospital attendant were involved for application of both
agents. Table 5 shows the cost comparison of both agents.


Acticoat™
Dermazin
Labour cost per dressing ( L)
€ 13.36
€ 13.36
Materials per dressing (M)
€ 311.65
€ 93.60
Total Cost per dressing (L+M)
€ 325.01
€ 106.96
Dressing numbers (n)
4
20
Total (Euros) - (L+M)*n
€ 1300.04
€ 2139.20
Table 5. Comparative cost (in Euros) analysis of the treatment of a burn (2000 sm2) of second
(IIAB) degree for both agents
The cost comparative analysis demonstrated that the treatment of a second degree (IIAB)
burn wound (2000 cm2 area) with Dermazin was more expensive by approximately € 839
(65% more expensive) than the treatment with Acticoat.
5. Discussion
General concepts and techniques for treatment of all wounds are applied regardless of the
mechanism causing the wound. The key to high quality and proper local treatment is the
correct and complete assessment of the burn wound. This includes etiology, localization,
area (cm2), depth, exudation (quantity, type, colour), smell, appearance of the wound
surface, appearance of the intact skin, presence of pain, and prior treatment method. The
treatment goals are determined on the basis of proper assessment preceding the treatment.
The burn wound changes dynamically depending on inner (release inflammatory
mediators, bacterial proliferation) and outer (dehydration, hypotension and cooling) factors.
Thus, the applied local agent is subjected to permanent control (both objective and
subjective). The perfect dressing for children should provide optimal protection, reparation
and minimization of the pain (Khundkar et al. 2010).
Silver-impregnated Acticoat™ dressing has been used in the Clinic of Burns at the
“Pirogov” Emergency Hospital since 2004 and has gradually prevailed as the standard
dressing in the treatment of burn wounds and post-traumatic defects. The clinical studies,
conducted in the Clinic of Burns in Children in the second half of 2009 and 2010,
demonstrate the advantages of topical treatment with nanocrystalline silver and was
concluded that the agent was efficient in all cases and was easily tolerated. The less frequent
and atraumatic dressing changes make this agent a preferable one for children. The clinical

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260
observations of the use of Acticoat ™ and the comparative analysis with Dermazin show
that the time of dressing changes is reduced by 50%, since the dressing change is every 3rd
day, in contrast to the Dermazin dressing, which has to be changed every 12 hours. This
yields a reduced use of sedative and narcotic agents applied in dressings as well as less
stress in dressing change, respectively (Probert & Burston, 2005).
Prior to the development of this type of dressing, it was considered that moist environment
contributes to increased risk of infection. The clinical observations of many authors (Ang et
al., 2003; Fong & Wood, 2006; Wright, et al. 2002) as well as our own studies reject such
concern. Dermazin and other types of SSD extract water from the wound surface as a result
of the cream hyperosmolarity, which requires frequent change. All the advantages, which
the moist environment has for the healing process, pose the question of maintaining the
moisture (Fong, 2005). The moistening of Acticoat has double effect – to maintain the moist
environment in the wound and to release the antimicrobial power of nanocrystalline silver.
The results (clinical and bacteriological) of polyethylene foil application, as an instrument
for moisture maintaining, are very good. Everyday moistening by irrigation or change of the
upper gauze layers is unpractical and more difficult to control. Use of Intrasite gel and
Allevyn is expensive and economically disadvantageous, therefore, semi-occlusive dressings
are utilized. Each modern dressing should possess the property of moisture transmission
and control. The complete occlusion may create an environment for the development of
facultative pathogenic microorganisms, causing infection. For this reason, the Moisture
Vapour Transmission Rate (MVTR) is an important parameter for each modern dressing. A
value of MVTR = 600-3000 for the polyethylene foil is determined, and a complete occlusion
occurs at value <500. Excellent moisture maintaining until the next dressing change was
observed in the monitored clinical contingent and maceration was not observed. The typical
imbibition was temporal and disappeared immediately after the change of the dressing type
or after the treatment was discontinued. In cases of infected wounds with abundant
exudation, an additional moistening is not necessary because the dressing has absorption
property when it comes in contact with the wound exudates.
The clinical observations on the contingent studied confirm these properties of Acticoat. The
comparative study carried out in the clinic shows that the mean time for the wound clearing
is 8.92 days for Acticoat, whereas for Dermazin it is longer – 14.93 days. Correspondingly,
the epithelization time is shorter for Acticoat (mean =12.98 days), while for Dermazin the
mean epithelization time is 19.79 days. This parameter is particularly essential in the
treatment of second-degree (IIA and IIB) burns, which epidemically have the highest
incidence rate, and in the treatment of the so-called “mixed burns”, in which the epidermis
is completely damaged while the dermis is damaged at various levels of thickness. Contrary
to deep burns, mixed burns have the capacity of spontaneous healing in case of proper
treatment. The incidence rate of these burns (having in mind a total damaged body area) is
high, although it has not specially been reported in the literature. While the treatment
method for the first- and third-degree burns is clearly determined, the algorithm for this
type of superficial burns is a dilemma (Storm-Versloot et al., 2010). In this case, the active
surgical behaviour has some disadvantages, since there are not exact demarcations of the
areas, which will epithelize in time and those which will not close non-operatively. Good
and fast epithelization of these border burns is very important in widespread burns with
vast areas of deep burn, where the shortened epithelization time contributes to the quicker
decrease in the total burn area and to the increase in the area of possible donor sites (Wright
et al., 2002). The shortened wound epithelization time diminishes the risk of development of

Application of the Nanocrystalline Silver in Treatment of Burn Wounds in Children

261
bacteraemia and septic condition, which are a major cause of death in burns. Thus, the
necessity of antibiotic therapy is reduced (Tonkin & Wood, 2005). Earlier wound clearing
and closing diminishes the risk of development of hypertrophic scars and joint contractures
on the one hand, and contributes to the child’s faster recovery, on the other. An optimum
microenvironment and absence of cytotoxic factors are essential for the phase of
epithelization of the wound healing process. In-vitro studies show that Acticoat is toxic to
both keratinocytes and fibroblasts, suppressing proliferation at the same time (Andrew et
al., 2005; Burd et al., 2007; Paddle-Ledinek et al., 2006). The monitored patients’ clinical
results do not demonstrate such an in-vitro effect. This is confirmed by other in vivo studies.
Apart from burn wounds, the anti-inflammatory, antibacterial and epithelotonic effect of the
dressing is utilized in wounds covered with fresh autografts and donor sites (Demling &
DeSanti, 2002; Olson et al., 2000; Silver, 2007). A faster stabilization and re- epithelization in
comparison with other types of antiseptic and epithelotonic dressings was observed. Well-
healed autografts and epithelization of the mesh splits (95%), in case of Acticoat application on
5 children with fresh autografts, was observed after the removing of the first dressing. In
application on donor sites (5 children) almost full epithelization (96%) was detected in one
child, and in the remaining 4 children full epithelization was observed on the 8th day. The
donor sites were monitored up to six months and no signs of hypertrophic scarring appeared.
The bacteriological observations demonstrate the powerful antibacterial effect of Acticoat.
The rate and degree of killing of microbes is higher than those of the other silver agents used
so far, which is due to the specific physical-chemical structure (Dunn & Edwards-Jones,
2004). After 30 minutes, nanocrystalline silver reduces the number of viable bacteria to very
low levels, while the other dressings achieve this effect after 2 hours (Wright et al., 1999; Yin,
2003; Burrell, 2003; Heggers et al., 2005; Lansdown, 2006). Pure silver ions and radicals have
been shown to provide protection against a broad spectrum of bacteria (more than 150
types), viruses and fungi and especially antibiotic-resistant bacteria. This effect is prolonged
and lasts up to 3 days after the dressing application. Acticoat has preventive antibacterial
function, too. Applied on wounds, which have no bacterial growth, it prevents
contamination. The dressing reduces the risk of wound contamination, being a highly
efficient agent for the prevention of general bacterial infection, especially in widespread
burns (Singh et al., 2007; Nadworny et al, 2010). This is confirmed by the microbiological
studies carried out (Hadjiiski & Argirova, 2008). Wounds without bacterial growth at the
beginning of the treatment remained uncontaminated until the end of the treatment, and
only in one child local infection (below the critical values) was observed during the
treatment course. From the initially contaminated wounds (in 16 children), bacterial flora
with non-significant quantitative values was isolated only in 3 children at the end of the
treatment. Wherever a local infection was isolated, regardless of its type, the microbial
numbers remained below the critical values and signs of general infection were not
observed (Figures 31-35). The antimicrobial preventive activity described is essential for the
successful surgical treatment of widespread and deep burns. The bacterial monitoring of the
children to whom Acticoat was applied on excised sites, autografts and donor sites (total –
15) showed a negative bacterial growth. The results of the microbiological analysis are
assessed as very good in 97%, good in 2% and unsatisfactory in 1% of the cases. The use of
Acticoat does not lead to the development of resistant bacterial strains. The distinct agent
action -superficial and in depth - against Ps.aeroginosa and St.aureus MRSA, which is
demonstrated by the obtained results, make the dressing preferable for burn wounds (Chae
et al., 2010). Thus, the need of antibiotic therapy is reduced, leading to a decreased risk of

Skin Grafts – Indications, Applications and Current Research

262
the development of multi-resistant strains. A fungal infection was not isolated in the studied
children. The Acticoat™ low adherent property allows a less painful dressing change, which
makes it a preferable dressing for children (Tancheva, 2008). The long-lasting activity of the
dressing reduces the total number of dressing changes. This yields better comfort, mobility
and ability of the patients to participate in rehabilitation programmes. In the analysis of
silver serum concentrations, haematological and biochemical parameters of the patients of
the studied clinical contingent, treated with nanocrystalline silver, no indication for toxicity
was observed (Moiemen et al., 2011; White, 2010). Due to lack of toxic effect, it is suitable for
application even on very young children, unlike Dermazin (Rustogi et al., 2005). Side effects
were not observed in the youngest patient, who was 9 months old. Silver is deposited
mostly in the skin, oral mucosa, gums, kidneys, liver and cornea. Excretion is done mostly
by biliary way. Monitoring of the plasma levels is recommended in burns over 10% and
burns of second (IIB) and third (III) degree, and especially in patients with abnormal liver
and kidney function (Landsdown, 2006).
6. Conclusion
The obtained clinical results, microbiologically and statistically verified, as well as the
conducted comparative study between Acticoat and Dermazin, provide reasons to consider
the Acticoat silver dressing as highly efficient for application in superficial burns of second
(IIA and IIB) degree, pre-surgical treatment of patients, prophylaxis and treatment of
infection in burns, treatment of donor sites, excised sites and autografts.
The presented properties of Acticoat allow us to define it as one of the leading agents for
local treatment of burns. During the short time of its use in our clinic, it has been recognized
as a preferable agent for wound treatment. The ease of use, powerful antibacterial effect,
lower frequency of dressing change, variety of sizes, good moulding, shortened time of
wound clearing and epithelization, and painless dressing change are the advantages of
Acticoat™ over the other silver agents used so far. Acticoat™ is significantly more cost
effective than the other frequently used agent Dermazin.
7. References
Andrew, P. et al. ( 2005). Evaluation of Cytotoxicity and Antimicrobial Activity of Acticoat®
Burn Dressing for Management of Microbial Contamination in Cultured Skin
Substitutes Grafted to Athymic Mice, J Burn Care Rehabil, 26, pp.238–246
Ang, E. et al. (2003). Pain Control in a Randomized, Controlled, Clinical Trial Comparing
Moist Exposed Burn Ointment and Conventional Methods in Patients with Partial-
Thickness Burns, J Burn Care Rehabil, 2003, Sep-Oct; 24(5), pp.289-96
Atiyeh, B.S.; Dibo, S.A. & Hayek, S.N. (2009). Wound Cleansing, Topical Antiseptics and
Wound Healing, International Wound Journal, 6(6), pp.420-430
Burd, A. et al. (2007). A comparative Study of the Cytotoxicity of Silver-based Dressings in
Monolayer Cell, Tissue Explant, and Animal Models, Wound Repair Regen.; 15(1),
pp. 94-104
Burrell, R.E. (2003). A Scientific Perspective on the Use of Topical Silver Preparations.
Ostomy Wound Manage, 49 (Suppl.): 19
Burrell, R.E.; Wright, J.B.; Heggers, J.P. & Davis,G.J. (1999). Efficacy of silver coated
dressings as barriers in a rodent burn sepsis model. Wounds,11(4), pp.64-71

Application of the Nanocrystalline Silver in Treatment of Burn Wounds in Children

263
Cavanagh, M.H.; Burrell, R.E. & Nadworny, P.L. (2010). Evaluating Antimicrobial Efficacy
of New Commercially Available Silver Dressings. International Wound Journal, Oct.,
7(5), pp.394-405
Chae, J. et al. (2010). Comparison of the Efficacies of Silver-containing Dressing Materials for
Treating a Full-thickness Rodent Wound Infected by Methicillin-resistant
Staphylococcus Aureus: P1563, Clinical Microbiology & Infection, 16 Sup 2:S454, April
2010
Cuttle, L. et al. (2007). A Retrospective Cohort Study of Acticoat™ versus Silvazine™ in a
Paediatric Population, Burns, Sep. 2007, 33 (6), pp.701-7
Demling, R.H. & De Santi, L.( 2001). Effects of Silver on Wound Management, Wounds, 13
(Supp A), pp.5-15
Demling, R.H. & De Santi, L.( 2002). The Rate of Re-epithelialization Across Meshed Skin
Grafts is Increased With Exposure to Silver, Burns, 28(3), pp.264-6
Dunn, K. & Edwards-Jones, V. (2004). The Role of Acticoat™ with Nanocrystalline Silver in
the Management of Burns, Burns, 30, Suppl. 1, SlLS9, pp. S1-S9
Fong, J.; Wood, F. & Fowler, B. (2005). A Silver Coated Dressing Reduces the Incidence of
Early Burn Wound Cellulites and Associated Costs of Inpatient Treatment:
Comparative Patient Care Audits, Burns, 31, pp.562-567
Fong, J. & Wood, F. (2006). Nanocrystalline Silver Dressing in Wound Management: A
Review, International Journal of Nanomedicine, 1(4), pp.441-449
Fong, J. (2005). The Use of Silver Products in the Management of Burn Wounds: Change in
Practice for the Burn unit at Royal Perth Hospital, Primary Intention, 13(4), pp. S16-
S22
Gravante, G. et al. (2009). Nanocrystalline Silver: A Systematic Review of Randomized Trials
Conducted on Burned Patients and an Evidence-Based Assessment of Potential
Advantages Over Older Silver Formulations, Annals of Plastic Surgery, 63(2), pp.201-
205, August 2009
Hadjiiski, O. & Argirova, M. (2008). Antiseptic agents containing silver components,
Handbook on Surgery with Atlas, Vol. VII - Burns and Frostbite (Hadjijski O. Editor),
pp. 86-97 (in Bulgarian)
Heggers, J. et al. (2005). Therapeutic Efficacy of Three Silver Dressings in an Infected Animal
Model, J Burn Care Rehabil,.26, pp. 53–56
Huang, Y. et al. (2007). A Randomized Comparative Trial between Acticoat and SD-Ag in
the Treatment of Residual Burn Wounds, Including Safety Analysis, Burns, Vol. 33
(2), pp.161-166
Khundkar, R.; Malic, C. & Burge, T. (2010). Use of Acticoat Dressings in Burns: What Is the
Evidence? Burns, 36(6), pp.751-8
Klasen, H. (2000). A Historical Review of the Use of Silver in the Treatment of Burns. II.
Renewed Interest for Silver, Burns, 26, pp.131-138
Landsdown, A.B. (2006). Silver In Health Care: Antimicrobial Effects and Safety in Use,
Curr. Probl. Dermatol, 33, pp.17-34
Leaper, D.J. (2006). Silver Dressings: Their Role in Wound Management, Int Wound J., 2006,
3, pp.282-294
Moiemen, N.S. et al. (2011). Acticoat Dressings and Major Burns: Systemic Silver
Absorption, Burns, 37 (1), pp.27-35

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Nadworny, P.L. et al. (2010). Does Nanocrystalline Silver Have a Transferable Effect?,
Wound Repair Regen., 18(2), pp.254-65
Olson, M.E. et al. (2000). Healing of Porcine Donor Sites Covered With Silver-coated
Dressings, Eur. J. Surg, 166(6), pp. 486-9
Orvington, L. (2001). Nanocrystalline Silver: Where the Old And Familiar Meets a New
Frontier, Wounds, 13(Suppl B), pp.5-10
Orvington, L. (2001). The Role of Silver Technology in Wound Healing: Part 2 Why Is
Nanocrystalline Silver Superior?, Wounds, A Compendium Clinical Research and
Practice, 13(2)(Suppl.B), pp.5-10
Paddle-Ledinek, J.E.; Nasa, Z. & Cleland, H.J. (2006). Effect of Different Wound Dressings on
Cell Viability and Proliferation, Plast Reconstr Surg, 117(7 Suppl), pp.110S-118S,
discussion 119S-120S.
Probert, R. & Burston, S. (2005). Wounds with a Silver Lining, Primary Intention, 13(4), pp.
10-S14
Rustogi, R. et al. (2005). The Use of Acticoat™ in Neonatal Burns, Burns, 31(7), pp.878-882
Singh, V. et al. (2007). The Pathogenesis of Burn Wound Conversion, Annals of Plastic
Surgery, 59(1), pp.109-115
Silver, G.M. et al. (2007). A Silver-Coated Antimicrobial Barrier Dressing Used
Postoperatively on Meshed Autografts: A Dressing Comparison Study, J Burn Care
Res, 28, pp.715-719.
Storm-Versloot, M.N. et al. (2010). Topical Silver for Preventing Wound Infection, Cochrane
Database Syst Rev, 17;(3), CD006478.
Tancheva, D. (2008). Burns and Frostbite, In: Handbook on Surgery with Atlas, Vol. VII - Burns
and Frostbite (Hadjijski O. Editor), pp.222-231, “Prof. M. Drinov” Academic
Publishing House (in Bulgarian)
Tonkin, C. & Wood, F. (2005). Nanocrystalline Silver Reduces the Need for Antibiotic
Therapy in Burn Wounds, Primary Intention, 13(4), pp.163-168
Tredget, E. et al. (1998). A Matched-Pair, Randomised Study Evaluating the Efficacy and
Safety of Acticoat Silvercoated Dressing for the Treatment of Burn Wounds, J Burn
Care Rehabil, 19(6), pp.531-537
White, R. (2010). Silver-containing Dressings: Availability Concerns, Ostomy Wound Manage,
56(8), pp.6-8
Wright, J.B. et al. (2002). Early Healing Events in a Porcine Model of Contaminated Wounds:
Effects of Nanocrystalline Silver on Matrix Metalloproteinases, Cell Apoptosis, and
Healing, Wound Repair Regen, 10, pp.141-51
Wright, J.B. et al. (1999). Efficacy of Topical Silver Against Fungal Burn Wound Pathogens,
Am J Inf Cont, 27, pp.344-50.
Wright, J.B.; Hansen, C.E. & Burrell, R.E. (1998). The Comparative Efficacy of Two
Antimicrobial Barrier Dressings: In-vitro Examination of Two Controlled Release of
Silver Dressings, Wounds, 10, pp.179-188
Yin, H.G. (2003). Antimicrobial Effects of Acticoat Nanocrystalline Silver-Coated Dressing,
John A. Boswick - Burn and Wound Care Symposium, Maul, Hawaii, February 2003.
Yin, H.Q.; Langford, R. & Burrell, R.E. (1999). Comparative Evaluation of the Antimicrobial
Activity of Acticoat Antimicrobial Barrier Dressing, J. Burn Care & Rehabil, 20,
pp.195-200

18
How Does Human Amniotic Membrane
Help Major Burn Patients Who Need
Skin Grafting: New Experiences
Ali Akbar Mohammadi1 and
Mohammad Kazem Mohammadi2
1Associate Professor of Surgery, Shiraz Burn Research Center,
Division of Plastic Surgery, Department of Surgery,
Shiraz University of Medical Sciences, Shiraz
2Faculty of Science, Ahvaz Branch, Islamic Azad
University, Ahvaz
Iran
1. Introduction
Burns are among the most common traumas in developing countries, which consume large
amounts of medical resources. It is important to find an appropriate material for dressing of
burn wounds that improves healing and is readily available, easily applicable, and
economical and also be protective from infection and desiccation, and facilitate healing. [1]
Most burn injuries happen in people with lower socio-economic class. Therefore, reducing the
expenses of burn treatment is an important goal alongside healing as the primary aim. [2]
Amniotic membrane has been used since 1910 with variable success as a material for burn
injury coverage. [3] It has the following advantages:

readily available in sufficient quantity

application not associated with immunological problems

large size

simple to prepare and sterilize

no allergic reactions

up to 15% reduction of water losses in wounds

histological structure similar to that of skin [4,5]
The disadvantage of the use of amniotic membrane is that there is some risk of viral
infection transmission, e.g. hepatitis, syphilis and AIDS. [4]. Two varieties of amniotic
membrane are mainly used:

in Toto (amnion + chorion) on deep burns

amnion alone (epithelium + basal membrane) on superficial burns[3]
The amnion is a thin semi-transparent tissue forming the innermost layer of the fetal
membrane. As fresh amnion carries contamination risk and disease transmission, amnion is
collected from placentae of selected and screened donors. Various preservation methods
have been introduced, including cryopreservation in liquid nitrogen, preservation in silver

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266
nitrate, storage in antibiotics solution, glycerol-preserved sheets, dried sheets and gamma-
irradiated sheets. [6]
It has been claimed to be one of the most effective biological skin substitutes used in burn
wounds, with efficiency of maintaining low bacterial counts. It also has advantages of
reducing loss of protein, electrolytes and fluids, decreasing the risk of infection, minimizing
pain, acceleration of wound healing and good handling properties. [6]
Amniotic membrane is readily available and does not present immunological problems and
allergic responses. It also reduces water loss.
The risk of the transmission of some viral infections has to be taken into account. Bacterial
examinations performed in 20 patients with burn wounds covered with amniotic membrane
showed low or no bacterial colonization of the burn surface. It is concluded that amniotic
membrane should be more widely used in this particular aspect of burn treatment. [4]
Human
amniotic
membrane
has
several
interesting
physiologic
and
histologic
characteristics which are the basis of its increased use in burn patients. It also has a unique
combination of properties, including the facilitation of migration of epithelial cells,
reinforcement of basal cellular adhesion and encouragement of epithelium differentiation.
Amniotic membrane is able to modulate stromal scarring and has anti-inflammatory activity
and is primarily used for covering partial thickness burns until complete healing [7].
Amnion is primarily used for covering partial-thickness burns until complete healing.
The most important features of amniotic membrane can be divided to 4 categories:

Rapid adherence to the wound bed.

Increased angiogenesis.

Inhibition of protease activity and PMN infiltration.

Rapid re-epithelialization and promotion of wound healing.
These characteristics has guided researchers to use this magic & cheep membrane as an
adjunct to burn patients management and there are numerous researches on its benefits in
acute burn wound management. We want to explain our new experiences in amniotic
membrane administration on skin graft taking and also their early & late advantages &
disadvantages in this group of burn patients .
2. Amniotic membrane as a skin graft fixator, and graft take accelerator to
Increase overall graft take in non-complicated wounds
Amniotic membrane immediately adheres to the grafted areas & fixes skin graft to their
bed, which eliminates suturing on extremities and diminishes the need for suturing on other
body parts. It also eliminates painful suture removal, accelerates graft take and decreases
traumatic graft loss induced by suture removal.
The revascularization of a skin graft depends on immobilization of the graft on the wound
bed. In cases where circular dressings are needed, the graft may be displaced under the
dressing, leading to partial graft loss and several methods of graft fixation have been
described [7,8,9]. A common technique is to use stitches but this is time consuming and the
stitches need to be removed after graft take. Placing surgical drapes over the graft is another
method but these must be removed on the second or third day when the grafts are still not
well vascularised, and this can lead to graft failure. Fixation with skin staples was
introduced as a fast and reliable method for graft fixation; [11]however it is expensive and
stressful for the patient as these staples must be removed which can be painful and

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Burn Patients Who Need Skin Grafting: New Experiences

267
frightening, particularly for children. Forgotten staples are another troublesome
complication. [12] (Fig 1a ,b)





Fig. 1a. Incidental radiologic finding of multiple asymptomatic forgotten staples
Another technique uses steri-strip micro porous tapes for fixation, but the surrounding skin
must be completely healthy to retain the tapes. [8] Other methods applying different
materials such as honey [13] or cyanoacrylate [14] have been designed. We would like to
present a method of graft fixation pioneered at our burn center and found convenient for
both patient and surgeon, using amniotic membrane as fixator. This is particularly
appropriate in children and also extremity burns. The skin graft is inserted on the wound
bed, then covered with amniotic membrane (Fig. 2) and the dressing is applied.
The membrane adheres to the grafted area in a few minutes. There is no need for stitches or
staples in extremities [15] or just a few anchoring sutures may be needed in some other body
parts. [16]
Several studies have shown that the application of amniotic membrane as a graft fixator is
accompanied by rapid re-epithelialization and healing [2,16,17] .Amniotic membrane has
also been found an effective biological dressing for burns, as it diminishes the loss of
plasma, fluid, protein and heat . [17], [18] ,[19] .

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268






















Fig. 1b. Painful forgotten staples removed 1 year after skin grafting.

How Does Human Amniotic Membrane Help Major
Burn Patients Who Need Skin Grafting: New Experiences

269










Fig. 2. Applying amniotic membrane for skin graft fixation.
In one study we evaluated the effect of amniotic membrane on rate of graft take. The
patients were divided into two groups: 54 limbs in amnion group and 54 limbs in control
group. The mean success rate of graft take was about 97 % and mean graft take duration
was about 7 days in amnion group (Fig 3) versus 89 % and 14 days in control group
respectively. Also this difference was statistically significant.
Our results show that amniotic membrane as skin graft fixator has no negative impact on
graft take, but also significantly reduces the duration of complete graft take which is very
important for both patient and health care system. Therefore, we recommend amniotic
membrane as a skin graft fixator, and graft take accelerator to eliminate or diminish the need
for painful and traumatizing postoperative dressing changes and thereby help to decrease
the cost and also need for possible regrafting .

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270

Fig. 3. The graft fixed with amniotic membrane, 5 days after procedure
3. Acceleration of donor site healing
Donor site morbidity including delayed and non-healing wounds and hypertrophic scar
formation are major problems in skin grafted burn patients. Amniotic membrane dressing
of skin graft donor sites ,accelerates epithelialization and results in early wound closure,
thereby, significantly reducing the risk of complications and eliminates postoperative
dressing change.
Amnion have been used to cover clean partial-thickness wounds and donor sites, and
applied as a temporary dressing for freshly excised burns.
It has advantages such as pain relief, prevention of infection, maintenance of a moist
environment to promote healing, good adherence to wounds, and simple handling [2, 11-
14]. Laboratory investigations have revealed that the basement membrane (BM) of amnion
shares major BM components with human skin, and the BM zone resembles human skin,
morphologically [17]. The epithelial side of denuded amnion has been shown to support the
proliferation, spreading, and differentiation of corneal, mucousa and bronchial epithelial
cells [18-20]. Furthermore, the stroma of amnion can serve as a dermal matrix in which
fibroblasts show good adherence and proliferation [21, 22].
After cleansing the donor bed of blood, the amniotic membrane is applied (Fig 4) & then
covered with Vaseline gauze simple dressing. The amniotic membrane adheres to the
wound bed and peels off, when the wound is completely epithelialized.
We studied 50 patients which had 2 similar donor sites of both lower exterimities. The
mean healing time was about 6 days in amnion group and 13 days in control group, and
pain was significantly lower in amnion group.
The desirable properties include rapid adherence, transparency, decreased patient
discomfort and decrease in infection. One of the most important observations was that,

How Does Human Amniotic Membrane Help Major
Burn Patients Who Need Skin Grafting: New Experiences

271
amniotic membrane was used as a single application dressing. In all cases, it remained over
the donor bed until healing was complete. This characteristic (single application)
significantly reduces patient discomfort and costs by elimination the need for multiple
dressing changes.


Fig. 4. Amniotic membrane dressing of donor site
Our clinical experience with amniotic membrane moved us to use this product widely for
donor sites, especially in children.
4. Increased graft take in old infected granulation tissue
The standard treatment today for deep partial-thickness and full-thickness burns is early
excision and grafting (E&G) but this technique may not be possible in centers with poor
medical facilities. In these centers burn physicians should wait and see until the superficial
burns heal primarily or granulation tissue appears in the base of the wound.
One problem in delayed grafting is the appearance of granulation tissue which, in addition
to microbial colonization, reduces graft take and increases complications, mortality, hospital
stay and costs. [20- 32]
Old granulation tissue is a prevalent problem in developing countries. Management of these
wound is very difficult, the result of skin grafting is poor and still there is no consensus
about the best treatment modality of these wounds.
The problem which now arises is how to apply skin grafts on chronic granulation tissue and
controversy continues on the best management.
Some surgeons favor application of graft after removal of granulation tissue while
others favor application of grafts directly on the granulation tissue. We used amniotic
membrane to manage these wounds in a clinical randomized control trial and observed
good results. (not published)

Skin Grafts – Indications, Applications and Current Research

272
90 adult patients with old burn wounds ( more than two weeks after granulation tissue
appearance) and infected (according to the surgeon diagnosis and positive tissue cultures)
were included in this study. (Fig 5).


Fig. 5. Old granulation tissue in a burn patient (these wounds are shinny and easily
removed with minimal digital pressure )
The most frequent bacterial isolate from burn wound culture was staphylococcus followed
by pseudomonas. The most common cause of chronicity of burn wound was delay in
referring of patient to burn center.
The extremities were randomly divided into two groups. Debridement of granulation tissue
was performed in both groups. In group A, the graft surface was covered with amnion and
in Group B( control group )skin grafting was performed in the conventional fashion.
Graft take was assessed on 14th post-grafting day.
The graft take was 90.13 % in amnion group and 65.61 % in control group that was
statistically significant .(p value < 0.05)
5. Decreased hypertrophic scar and itching
Hypertrophic scar and itching are disabling complications in burn patients. We studied &
saw interesting result with applying amniotic membrane on skin grafted areas in
diminishing hypertrophic scar & itching. This may be explained by accelerated wound
healing & epithelialization.

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Burn Patients Who Need Skin Grafting: New Experiences

273
Hypertrophic scarring after injuries, and especially burns, is a great concerns for patients
and a challenging problem for clinicians.
Hypertrophic scars may cause significant functional and cosmetic impairment, and pruritus
causes discomfort for the patient, and is responsible for decrease in quality of life. Pruritus
causes discomfort and decreases quality of life .Hypertrophic scars result from general
failure of normal wound healing processes. After burn injury, hypertrophic scars typically,
appear on the trunk and extremities. [33]
Many factors such as race, age, genetic factors, hormone levels, atopy and immunologic
responses of the individual patient appear to play a role. The type of injury, wound size and
depth, anatomic region and mechanical tension on the wound are important as well. Also,
complicating factors such as bacterial colonization and infection of the wound seem to
induce hypertrophic scarring [34]. To predict the development of a hypertrophic scar in a
burn wound, the time interval between burn and and healing is the most important factor
and is closely related to depth and size of the wound [35].
Treatment of the hypertrophic scar still is a challenging issue due to the lack of effective
treatments. Various treatments are currently available including surgical excision, steroid
injection, radiation, laser therapy, silicone gels, calcium channel blockers and pressure
therapy. Although management of hypertrophic scars has advanced in the past years, the
lesions remain difficult to prevent and treat. Burn hypertrophic scars are often widespread
and not linear . Although extensive research has led to an increase of knowledge in the
pathophysiologic processes of wound healing and the formation of scars, but still there is no
consensus regarding the best treatment to reduce or prevent hypertrophic scarring.
Recurrences remain common and patient satisfaction is variable .
Optimal treatment of the burn wound is of eminent importance for wound healing and the
prevention of hypertrophic scar formation. Deitch et al demonstrated that wound closure
should be achieved within three weeks to reduce the risk for hypertrophic scar development
[36]. Timing of grafting is still under debate, both for survival of the patients as well as the
incidence and quality of hypertrophic scar. Adequate topical wound treatment allows for
wound healing with controlled inflammation and should be applied to obtain fast wound
closure. The autologous split thickness skin graft is still the mainstay of burn wound
surgery. Although autologous split thickness skin graft may result in faster wound closure,
it may not prevent hypertrophic scar formation in the operated area.
Several studies have shown that the application of amniotic membrane as a biological
dressing in the management of burns is accompanied by rapid re-epithelialization and
healing. Amniotic membrane has been found an effective biological dressing for burns, as it
diminishes the oozing of plasma, bacterial count, and fluid, protein and heat loss [15-17].
In another study, we evaluated the effect of amniotic membrane as an adjunct in split-
thickness skin graft on itching and hypertrophic scar formation in burn patients.(not
published) This double blind randomized clinical controlled trial showed 59.25 % decreased
itching and 64.81 % less hypertrophic scar formation in the skin grafted area covered with
amniotic membrane in comparison to areas which managed by conventional skin grafting
method.
This study showed that using amniotic membrane as an adjunct in split-thickness skin
grafting significantly reduces post-burn itching and hypertrophic scar formation. It seems
that additional research still is required to determine the best possible treatments. Since
there is no optimum treatment option at present, amniotic membrane can be used as a novel

Skin Grafts – Indications, Applications and Current Research

274
modality in prevention rather than treatment of hypertrophic scar formation and itching in
burn patients.
6. Conclusion
According to our experiences, amniotic membrane can strongly participate in skin grafted
areas to improve skin graft take and decrease post graft complications.
7. Acknowledgment
I am grateful to the Shiraz burn research center staffs, ghotbeddin burn hospital physicians,
caregivers and also countless burn patients for their invaluable helps, Hamed Ghoddusi
Johari ,M.D for contribution in our research , (effect of amniotic membrane on skin graft
take) and Mandana Kiasat Dolatabadi , M.D (effect of amniotic membrane on old infected
granulation tissue ) as their thesis for degree on general of surgery and Shima Eskandari
(effect of amniotic membrane on hyper trophic scare formation and itching ) as her thesis for
degree of MD, Mr. Iman Ahrari ,medical student for their help in edition , Miss Zahra
Moradi and Mrs. Masumeh Kherad for their special contribution in our researches, Mrs
Zahra Jahedmah and Mrs. Mahbubeh Nazari for taking photographs .
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[4] Hadjiiski O,Anatassov N. Amniotic membranes for temporary burn coverage. Annals of
Burns and Fire Disasters. 1996; 10: 2-7
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[6] SukariHalim A, Khoo T.L, Mohd. YussofSh.J.Biologic and synthetic skin substitutes: An
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19
Influence of Microorganisms on the
Healing of Skin Grafts from
Chronic Venous Leg Wounds
Tine Yding Wolff1, Thomas Bjarnsholt2,3,
Klaus Kirketerp-Møller4 and Trine Rolighed Thomsen1,5
1Life Science Division, The Danish Technological Institute
2University of Copenhagen, Faculty Of Health Sciences, Department
of International Health, Immunology and Microbiology
3Rigshospitalet, Department for Clinical Microbiology
4Orthopedic Department, Hvidovre University Hospital
5Department of Biotechnology, Chemistry and Environmental
EngineeringAalborg University
Denmark
1. Introduction
Chronic wounds are associated with a significant morbidity and continue to affect an
increasing number of patients. In developed countries an estimated 1-2% of the population
will suffer from a chronic wound at some point in their lifetime. Chronic wound treatment is
associated with considerable expenses and in the United States alone more than 25 billion
dollars are spent on treatment annually (Percival et al., 2011).
Although it is well known that infection is involved in making the wound chronic and
unable to heal, the process for identifying bacterial flora within wounds is not standardized
(Frankel et al., 2009). The microorganisms associated with chronic wounds are usually
investigated by traditional culture–dependent methods by taking a swab or biopsy from the
wound and applying it as inoculate for various microbial cultures. The introduction of
molecular biological methods and improved sampling techniques has illustrated that the
culture-dependent methods presumably underestimate the microorganisms present in the
wound. This is often due to a combination of inadequate growth conditions and
colonization with slow, unculturable, fastidious or anaerobic bacteria growing in biofilms
(Price et al., 2010).
It is well known that naked bacterial DNA can persist in a sample for a while after the
infection has been eliminated, and the risk of detecting DNA from dead (non-viable)
bacteria is a recognized bias when using molecular methods. The commercially available kit
Molysis (Molzym, Bremen, Germany) degrades the naked bacterial DNA residing in a
sample, thereby, improving the detection of viable and hence more clinically relevant
bacteria in the subsequent molecular analysis. RNA has a much shorter half-life than DNA
making it suitable as a measure of microbiological activity in a sample. RNA extracted from

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a sample can be converted into cDNA and used as a target in polymerase chain reaction
(PCR) or the RNA can be targeted directly within the cell for instance by fluorescence in situ
hybridization (FISH).
The exact role and function of microorganisms in chronic wounds is not fully understood,
but bacteria seem to have an important influence on the prognosis of the wound. In a study
by Høgsberg et al. (in press) the bacterial flora was examined in chronic wound beds
determined for split-skin grafting using conventional culture methods. In the study the
presence of Pseudomonas aeruginosa was found to be particularly important. If P. aeruginosa
was present, only 33% of the grafted chronic wounds healed successfully in contrast to 77%
when P. aeruginosa was not detected. This emphasizes the importance of a careful sampling
technique, characterization of the wound and clinical correlation.
In this chapter we describe techniques to characterize the microorganisms from a swab or
skin grafting biopsy from chronic wounds. New and previously obtained results are
presented and discussed.
2. Molecular methods for studying the microbial flora in chronic wounds
Various methods have been applied for the study of microorganisms in chronic wounds and
each method has its advantages and disadvantages. In most clinical settings the gold
standard is traditional culture-based methods. The weakness of cultivation is that it can take
several days to obtain a final identification, and that fastidious and slow-growing
microorganisms in the sample might be overlooked. However, cultivation usually provides
additional information such as phenotypic resistance patterns that are highly relevant from
a clinical perspective. Molecular diagnostic approaches on the other hand offer a more rapid
and deep identification but have other limitations. In the following section some of the well-
established molecular techniques are described as well as a few of the latest methods in the
field.
2.1 16S rDNA cycle
Culture-independent identification of bacteria can be carried out by performing a ribosomal
RNA (rRNA) gene cycle e.g. (Nielsen et al., 2008; Thomsen et al., 2010). This method is
based on the fact that the composition of the gene encoding the bacterial 16S rRNA (16S
rDNA) is unique for each bacterial species. Total DNA is extracted from a sample and the
16S rDNA is amplified nearly in its full length by PCR using broad-range primers. If the
sample is polymicrobial the resulting PCR product will consist of amplicons with varying
sequences making it unsuitable for direct sequencing. The individual amplicons need to be
separated before sequencing, and construction of clone libraries and denaturant gradient gel
electrophoresis (DGGE) are two different ways of achieving that (Andersen et al., 2007;
Muyzer et al., 1993; Thomsen et al., 2010).
To construct clone libraries the amplicons are ligated into plasmid vectors and cloned into E.
coli cells. The cells are spread on agar plates and grown over night, and each colony
appearing on the plate represents one amplicon. The vectors are then extracted from each
individual colony, and the 16S rDNA amplicons are sequenced. By performing BLAST
(Basic Local Alignment Search Tool) searches in public databases (Altschul, 1997), the
obtained sequences can be assigned to specific bacteria, and an improved phylogenetic
resolution can be obtained by performing a phylogenetic analysis on basis of the sequences.

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In DGGE the amplicons are separated by providing them with a GC-rich primer tail and
loading them on a polyacrylamide gel containing a gradually increasing concentration of
chemical denaturant. The unique base composition of each amplicon results in individual
melting points meaning that the amplicon can only move through the gel until a certain
point, where the double stranded structure is fully melted and only held together by the GC
tail. Upon staining, each band on the gel theoretically represents a unique 16S rDNA
amplicon which can be excised and extracted from the gel and sequenced. Construction of
clone libraries and DGGE are relatively labour-intensive methods with many individual
steps and it takes several days to obtain the sequences that eventually reveal the bacteria
present in a sample. This makes the methods unsuitable for diagnostic purposes where a
rapid answer is required, whereas, they are more relevant as a research tool. The sequence
information provided by the clone libraries and the DGGE can be used to make more
specific analyses on the original sample, such as FISH or quantitative PCR (qPCR).
2.2 qPCR / RT-qPCR
qPCR is a culture-independent method for determining the number of a specific gene in a
sample. qPCR basically proceeds like a regular PCR but has the advantage that the
formation of PCR product can be followed as it progresses, making laborious post-PCR
steps such as gel electrophoresis unnecessary. In qPCR the formation of PCR product is
usually detected by one of two techniques: addition of a dye that fluoresces upon binding to
double stranded DNA, or by application of a sequence-specific probe that fluoresces only
when the target DNA is present. In both cases, the fluorescence is measured by a detector in
the PCR machine after each PCR cycle. The intensity of the fluorescence progresses
exponentially as does the PCR product formation because the increase in fluorescence is
proportional to the increasing amount of target sequence. By analyzing standards with
known concentrations of the target sequence, it is possible to quantify the initial amount of
target DNA in the sample. In order to focus only on the active bacteria the RNA can be
extracted from the sample, converted into cDNA and used as target in qPCR in a method
called reverse transcriptase qPCR RT-qPCR).
2.3 FISH
DNA-FISH and peptide nucleic acid (PNA)-FISH rapidly identify microorganisms in
complex samples and provide information about number, morphology and spatial
distribution. The FISH technique is based on fluorescent probes of varying specificity that
are complementary to a ribosomal RNA sequence, and the target cells can be visualized
using fluorescence microscopy. A detailed procedure has been published by Amann (1995)
and Stender (2003).
2.4 Pyrosequencing
Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis"
principle and it is likely to replace the relatively labour-intensive procedure of constructing
clone libraries. It differs from the classic Sanger sequencing, in that it relies on the detection
of pyrophosphate release upon nucleotide incorporation, rather than chain termination with
dideoxynucleotides (Ronaghi et al., 1998). This deep sequencing technique has been used to
evaluate the diversity of microbial populations where thousands of reads/sequences can be

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obtained from each sample (Andersson et al., 2007; Bogaert et al.; Dowd et al., 2008; Price et
al., 2009; Price et al., 2010; Smith et al., 2010; Stewart et al., 2010; Wolcott et al., 2009).
2.5 Ibis T-5000
The Ibis T5000 Biosensor system combines automated sample preparation, PCR
amplification, electrospray ionization mass spectrometry, and information management to
rapidly characterize known and unknown organisms. The system is based on base ratios
(not base sequences) and can provide test results in six to seven hours (Ecker et al., 2008).
The method was recently identified as the molecular system most likely to fulfill the
requirements of routine diagnosis in orthopedic surgery (Costerton et al., 2011).
2.6 Method evaluation
All the mentioned methods are somehow biased and application of multiple methods is
generally recommended for research studies. For example, the nucleic acid extraction
efficiency is crucial for all methods except FISH, where sample fixation is important. Primer
specificity, differential amplification efficiency and incomplete databases for bioinformatic
search are other possible biases. In addition, it is important to be critical to molecular
findings based on DNA. Previous studies have shown that naked bacterial DNA can reside
in a sample for a long time after an infection has been eradicated fx Pichlmaier et al. (2008).
The PCR techniques cannot distinguish between DNA from active and dead bacteria and
this raises the question of the clinical relevance of the findings. Targeting RNA instead of
DNA (for instance using FISH or RT-qPCR) could help exclude irrelevant findings and focus
only on the active bacteria.
3. Results
In a study by Thomsen and coworkers (2010) the bacterial flora in different types of wound
material from 14 skin graft operations was examined. Routine cultivation as well as a panel
of DNA-based methods were performed and the results compared. Cultivation showed
polymicrobial growth in all but one wound, and S. aureus, P. aeruginosa, K. oxytoca and
enterococci were identified most often, whereas no anaerobic species were found. In most of
the samples the finding of S. aureus and P. aeruginosa by cultivation was confirmed by the
molecular methods. However, for all samples DNA from additional species was identified,
including anaerobic bacteria. Among others DNA from Alcaligenes sp., Anaerococcus sp., E.
faecalis and Stenotrophomonas sp. was identified.
Furthermore, the study comprised an examination of the spatial location of S. aureus and P.
aeruginosa within two wounds using species specific qPCR. This showed a great variation in
the distribution of bacteria at different locations in the same wound. As an example, the
number of P. aeruginosa varied with three orders of magnitude for two biopsies taken from
the same wound indicating that the bacteria grow locally in micro colonies (biofilms)
(Figure 1). This tendency to proliferate in biofilms is illustrated in Figure 2, where a biopsy
from a skin graft operation is examined for the presence of P. aeruginosa using PNA-FISH. In
a recent analysis we examined the bacterial diversity in four samples (referred to as A-D)
taken from the same wound by standard cultivation techniques as well as construction of
clone libraries, PNA FISH and qPCR on basis of DNA and RNA (Table 1). Cultivation
identified three bacterial species: Staphylococcus pettenkoferi, Enterobacter cloacae and

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281
Stentrophomonas maltophilia. These findings were confirmed in one or more of the clone
libraries (S. pettenkoferi DNA was found in clone library A and C, S. maltophilia DNA in C
and D and E. cloacae DNA in D). DNA from additional bacteria was found in all four clone
libraries of which Propionibacterium sp., Massilia timonae, Brevibacterium pityocampae,
Streptococcus mitis, Achromobacter sp. and Acinetobacter sp. were the most abundant. The
presence of Propionibacterium acnes, a common inhabitant of the human skin, was confirmed
and quantitated with species specific qPCR.







Fig. 1. Quantitative PCR data obtained from two wounds (wound E and F, respectively) for
S. aureus and P. aeruginosa (copies/ng DNA ±standard error of the mean, n=3). Cultivation
and DGGE results are mentioned as well + : Samples were taken under skin graft operations
at the center (C), and at approximately 3, 6, 9, and 12 o’clock around the wounds’ periphery.
From Thomsen (2010).
Some of the 16S rDNA sequences from clone library A, C and D matched Staphylococcus sp.
upon BLAST search.
To further annotate these bacteria, RT-qPCR assays targeting S. aureus femA mRNA and
Staphylococcus sp. 16S rRNA were applied. The S. aureus specific qPCR was negative for all
four samples whereas the Staphylococcus sp. assay was positive, indicating that the clones
matching Staphylococcus sp. originated from coagulase-negative staphylococci (CoNS).
mRNA from P. acnes and Staphylococcus sp. was detected in the four wound samples
indicating that these bacteria were active at the time of sampling. This partially corresponds
with the cultivation results where Staphylococcus pettenkoferi was observed, whereas P. acnes
was not. P. acnes is considered to be a fastidious bacterium that requires anaerobic growth
conditions and has a slow growth rate. This might explain why it was not detected by
cultivation.

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Fig. 2. P. aeruginosa identified in a biopsy from a skin graft operation by a specific PNA FISH
probe (red stain). Host cells were stained with DAPI (blue).
P. aeruginosa and S. aureus are common in chronic wound infections but in this specifically
examined case not found by cultivation, or in any of the clone libraries or by species specific
qPCR. When comparing the results of our recent analysis to those obtained by Thomsen and
coworkers (2010) it is seen that in both cases DNA from Brevibacterium sp., Anaerococcus sp.,
Achromobacter sp. and Alcaligenes faecalis was identified using the clone library approach but
not by cultivation. Furthermore, both studies show that molecular methods identify
additional bacteria compared to cultivation and that the bacterial composition differs
depending on the location within the individual wound.

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283
Sample
Cultivation Clone libraries (genomic DNA)
Parentheses numbers of clones
qPCR
(genomic
DNA)
RT-qPCR
(cDNA)
A
Staphylococc
us
pettenkoferi,
Enterobacter
cloacae,
Stentrophom
onas
maltophilia
Brevibacterium pityocampae (7),
Staphylococcus sp. (8), Propionibacterium sp.
(19), Massilia timonae (1), Staphylococcus
epidermidis (1), Xanthomonas sp. (1),
Xanthomonadaceae sp. (2), Stenotrophomonas
sp. (3), Herbaspirillum sp. (1) and 12
uncultured bacteria best matching: Ralstonia
solanacearum (1), Massilia timonae (5),
Propionibacterium sp. (1), Propionibacterium
acnes (3) and Acidobacterium sp. (2).
1200 P.
acnes
CFU/g
tissue



Positive for
P. acnes


Positive for
Staphylococc
us sp.
B
Propionibacterium sp. (23), Streptococcus
mitis (4), Achromobacter sp. (2), Kocuria sp.
(3), Micrococcus sp. (2), 12 uncultured
bacteria best matching: Ralstonia
solanacearum (3), Anaerococcus sp. (2),
Kocuria sp. (4), Propionibacterium sp. (1),
Achromobacter xylosoxidans (1) and
Neisseriales sp. (1).
1200 P.
acnes
CFU/g
tissue
Positive for
P. acnes

Positive for
Staphylococc
us sp.
C
Acinetobacter sp. (4), Staphylococcus sp. (1),
Stenotrophomonas maltophilia (4),
Stenotrophomonas sp. (5), Propionibacterium
sp. (4), Alcaligenes faecalis (1), Micrococcus
sp. (2), Mesorhizobium sp. (1), Enterobacter
hormachei (1) and 22 uncultured bacteria
best matching: Propionibacterium acnes (3),
Enterbacter hormachei (4), Anaeromyxobacter
sp. (2), Lactobacillus iners (7), Ralstonia
solanacearum (2), Corynebacterium accolens
(3), Bradhyrizobium sp. (1).
1700 P.
acnes
CFU/g
tissue


Positive for
P. acnes

Positive for
Staphylococc
us sp.
D
Achromobacter denitrificans (5), Achromobacter
sp. (4), Enterobacter hormachei (7),
Stenotrophomonas maltophilia (6), Enterobacter
sp. (1), Enterobacter cloacae (1),
Propionibacterium sp. (1), Staphylococcus sp.
RNA (1) and 25 uncultured bacteria best
matching: Enterobacter hormachei (12),
Enterobacter cloacae (6), Streptococcus
alactolyticus (4), Enterococcus ludwigii (2) and
Massilia sp. (1).
1000 P.
acnes RNA
CFU/g
tissue


No P. acnes
detected

Positive for
Staphylococc
us sp.

Table 1. Comparison of 4 separate biopsies from one wound investigated by cultivation,
clone libraries, qPCR and RT-qPCR. Genomic DNA and RNA extracted from the four
samples were examined using qPCR and RT-qPCR assays specific for Propionibacterium
acnes, Staphylococcus sp., P. aeruginosa and S. aureus. RT-qPCR was only used to prove the
presence of active bacteria and not to quantitate the bacteria.

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4. Discussion
4.1 Microbial diversity
Over the years the microbial flora in chronic wounds has been target for thorough
investigation and the advancing culture-independent techniques become more and more
common as research tools. In such studies the molecular methods generally tend to reveal a
much greater microbial diversity than the traditional cultivation methods. The study by
Thomsen and coworkers (2010) is an example of this, where culture-independent methods
revealed multiple species in each investigated wound. All of the wounds contained S.
aureus, and P. aeruginosa was also frequently found, but aside from this, rather different
floras were identified for the individual wounds. Furthermore, the molecular biological
methods detected a varied anaerobic flora in some of the wounds and pathogenic species
not found previously in chronic venous leg ulcers were identified. No anaerobes or new
species were detected with culture methods. This corresponds well with the results of our
recent examination of four biopsies taken from the same wound. Only aerobic bacteria were
identified by cultivation whereas DNA and RNA from the anaerobic P. acnes were found by
molecular methods. Furthermore, the molecular methods indicated varying polymicrobial
communities in all four samples. Surprisingly, S. aureus and P. aeruginosa were not identified
neither by cultivation or the cultivation independent methods in this specific wound.
The examination of wound flora has mainly focused on bacterial diversity, but a study from
Dowd and collagues (2010) indicated that fungi might also be important in chronic wound
infections. In their retrospective study, 915 chronic wounds were examined using molecular
methods and fungal DNA was identified in 208 samples (corresponding to 23% of the
investigated samples). DNA from a total of 48 different fungal species was identified of
which yeasts of the Candida genus were the most abundant. In a retrospective analysis of the
DNA resulting from the wounds included in the Thomsen (2010) study, qPCR with
universal fungal primers and probe revealed fungal DNA in one of 14 samples.
In a recent study by Smith (2010) pyrosequencing was used to identify bacterial populations
in 49 pressure ulcers. Diversity estimators were utilized and wound community
compositions analyzed in relation to metadata such as age, race, gender, and comorbidities.
Pressure ulcers are shown to be polymicrobial of nature with no single bacterium
exclusively colonizing the wounds. The microbial community among such ulcers is highly
variable and typically contains 3-10 primary populations. Up to hundreds of different
species can be present in each wound; however, many in trace amounts only. There are no
clearly significant differences in the microbial ecology of pressure ulcers in relation to
metadata except when considering diabetes. The microbial populations and composition in
the pressure ulcers of diabetics may be significantly different from the communities in non-
diabetics. However in this particular work, the location of the pressure ulcer was not
recorded. As diabetes tends to facilitate pressure ulcers on lower extremities due to
neuropathy, the variability could reflect variations in location of the ulcer rather than
diabetes itself.
The biofilm mode of growth seems important in chronic wounds with many different
species present and it is generally accepted that the microbial composition of the entire
wound is not represented in only one wound biopsy. Using FISH, we detected bacteria in
biofilms in a biopsy from a skin graft operation (see Fig. 2) and this might explain how the
bacteria survive inside the wound bed. In chronic wounds individual micro colonies might
exist that only consist of single species, and it is very likely that mono- and polymicrobial

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285
biofilms can be found in the same ulcer, but the importance and relevance of this has yet to
be established (Kirketerp-Moller et al., 2008; Burmølle et al., 2010). There are also biofilms in
healing wounds, and other factors like host response, virulence and antibiotic resistance
seem important for the fate of the wound.
4.2 Heterogeneous distribution
A chronic wound is a heterogeneous environment that varies at different locations with
regard to the accessibility of oxygen and the flow of fluid. Furthermore, external influences
such as the application of a wound dressing can cause local changes in the wound. This
means that different microorganisms have more or less favorable growth conditions at
different locations which often results in a heterogeneous microbiological distribution
throughout the wound (Price et al., 2010). Wolcott (2009) concluded that individual wounds
have distinct ecological footprints, and within the individual wounds there can be both
significant site specific differences and relative uniformity in the bacterial ecology.
The heterogeneous distribution of microorganisms in chronic wounds has been described in
several studies and the clinical relevance of this is discussed (e.g. by Fazli et al., 2009;
Kirketerp-Moller et al., 2008¸Wolcott et al., 2009). Using qPCR and FISH it has been
illustrated that the number of the pathogens S. aureus and P. aeruginosa varied depending on
the location and depth of the wound examined (Fig. 1) (Thomsen et al., 2010). S. aureus was
primarily located close to the wound surface while P. aeruginosa was primarily located
deeper in the wound (Fazli et al., 2009). Some bacterial species were present all over the
chronic venous leg ulcer while some were only present in parts of the wounds (Thomsen et
al., 2010).
The macroscale spatial variation in wound microbiota was also investigated by Price et al.
(2010). A total of 31 curette samples were collected at the leading edge, opposing leading
edge, and/or center of 13 chronic wounds. Bacterial community composition was
characterized using a combination of 16S rDNA-based pyrosequencing and various other
methods. A total of 58 bacterial families and 91 bacterial genera were characterized among
the 13 wounds. While substantial macroscale spatial variation was observed among the
wounds, bacterial communities at different sites within individual wounds were
significantly more similar than those in different wounds (p=0.001). Our recent study of four
biopsies from one wound showed a similar tendency regarding the spatial heterogeneity.
The clone libraries constructed for each sample had sequences in common, for instance P.
acnes that appeared in all libraries, but also contained bacterial sequences that were unique
to the particular location. Even though the results are based on only one wound they
support the prevalent opinion that a careful adjustment of sample sites may improve the
quality of wound microbiota studies. However, the significant similarity in bacterial
communities from different sites within individual wounds indicates that studies failing to
control for sampling site should not be disregarded based solely on this criterion. Ideally a
composite sample from multiple sites across the surface and in the depth of individual
wounds may provide the most robust characterization of wound microbiota (Price et al.,
2010); however, this is often not possible clinically.
The introduction of next-generation sequencing methods such as pyrosequencing has made
it possible to achieve huge amounts of information about the microbial composition in a
sample in a relatively short time. However the large amount of data generated by the new
methods gives rise to new challenges considering data handling and especially

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interpretation. As stated in the review by Rogers and coworkers (2010), different species in a
polymicrobial infection can interact and thereby result in a different pathogenesis than the
individual species would cause. This could mean that microorganisms that are usually
considered non-pathogenic might play an important role in a polymicrobial community. For
the time being it cannot be concluded if the new molecular methods are helpful to the
clinicians or if all the extra information only complicates the overall picture. More studies
have to be carried out to elucidate this.
The uneven distribution of bacteria in the wounds is highly relevant for the clinician and
how and when the sample is taken highly influences the outcome of the diagnostic analysis.
The ideal solution would be to examine the entire wound and identify every single
pathogen, but again this is not possible nor does it provide the full answer. New questions
would arise: Which bacterial strain or even subgroup is important and what influence do
they have on the healing of the wound? Evaluating the result of a culture-independent
analysis is after all still a paramount challenge for the clinician.
4.3 Consequences
4.3.1 Optimal sampling and diagnosis
No diagnostic methods can compensate for the three-dimensional uneven distribution of
microorganisms in chronic ulcers. Several studies have indicated that the microbial flora
varies at different locations in a wound (fx Kirketerp-Moller et al, 2008; Fazli et al, 2009,
Thomsen et al., 2010), emphasizing the importance of adequate sampling techniques and
ideally the use of multiple swabs or biopsies when examining wounds.
In situations where infected tissue is removed with the purpose of attaching a skin graft,
instead of focusing on the microbial flora in the removed tissue it might be more relevant to
focus on the remaining tissue on which the graft is to be attached (Bitsch et al., 2005).
Microbial growth reduces the chance of the skin graft healing, and therefore examining the
debrided wound before placing the graft, for instance by using a swab, might be of value.
The surface of the chronic wound is likely to host commensal flora, and it is more likely that
an in-depth residing bacterium is more pathogenic than a superficial one.
When designing a protocol for sampling we propose the following to be considered: 1.
Revise the ulcer before sampling. 2: Swap a large area or take a big or multiple biopsies.
Melendez et al. (2009) developed a panel of qPCR assays targeting 14 common, clinically
relevant pathogens for rapid identification of bacteria directly from tissue samples. Thirty-
nine tissue samples from 29 chronic wounds were evaluated and the results compared with
those obtained by culture. As revealed by culture and PCR, the most common organisms
were methicillin-resistant Staphylococcus aureus (MRSA) followed by Streptococcus agalactiae
(Group B Streptococcus) and P. aeruginosa. Under optimal conditions, the turnaround time for
PCR results is only 4-6 h. Furthermore, qPCR is an inexpensive approach that easily can be
introduced into clinical practice for detection of organisms directly from tissue samples.
However, the downside to qPCR is that the primers and probe dictate the specificity and
therefore there is a risk of overlooking species. For instance the qPCR assays reported by
Melendez et al. (2009) only monitored aerobic pathogens but other studies have indicated
that it is highly relevant to include assays for anaerobic pathogens and probably also fungi.
The anaerobic and also slow-growing pathogen P. acnes was fx detected in the investigated
wound presented in this study. In addition to the DNA identified in the clone libraries RNA
from P. acnes was also identified indicating that the bacterium had been active very recently.

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Which method is the optimal to examine the microbial flora of a chronic wound is a subject
of discussion but in order to get as much information as possible the sample could be
examined using a combination of traditional cultivation methods and molecular methods
especially for research purposes.
4.3.3 The role of microorganisms in chronic wounds
Non-healing ulcers of mixed origin could heal if the underlying, but in many cases, still
unknown cause was resolved. One of these factors is bacterial biofilm. But what is the
difference between the biofilm in the healing group and in the non-healing group? Right
now it is uncertain which microorganisms are the real important and truly pathogenic ones
in chronic wounds, so should we treat for all of them? The most abundant microorganisms
are not necessarily the most important in the wound from a clinical perspective.
Furthermore, the pathogenicity between different strains and phenotypes differ, and they
probably differ over time within the same phenotype. The most abundant bacteria found by
traditional methods might just be the one with the most optimal growth conditions. Other
factors like virulence genes, communication between the bacteria, the physical environment
for the microorganisms and the host-biofilm interaction are also important. Hemolytic
streptococci for instance are more virulent than certain other bacteria and a relatively small
load of these are needed to initiate the infection of a wound. The beta-haemolytic
Streptococcus and Staphylococcus aureus are examples of species that are highly virulent and
co-exist very well with other species. So far it is unknown whether the virulence of a certain
species is dependent upon another. Cell–cell communication in bacteria is accomplished
through the exchange of chemical signal molecules called autoinducers. This process, called
quorum sensing (QS), allows bacteria to monitor their environment for the presence of other
bacteria and to respond to fluctuations in the number and/or species present by altering
particular behaviors (Miller & Bassler, 2001). The N-acyl homoserine lactone QS signal
molecule in P. aeruginosa will trigger the production of virulence factors such as
rhamnolipids that have been shown to eliminate neutrophils in vitro (Van Gennip et al.,
2009). Adding detection of known virulence genes to the molecular methods would be
helpful in the process of interpretation.
Ideally, each wound should be carefully evaluated and in addition to traditional biofilm-
based wound care strategies, an antimicrobial/antibiofilm treatment program with
individualized therapeutic approaches should be identified for each patient's respective
wound microflora (Smith et al., 2010; Wolcott et al., 2009). However, we do not have the
tools for interpretation yet. First some questions should be asked when studying chronic
wounds: What are the microorganisms doing to prevent healing of the wound? Can markers
of the microbial community be identified that predict a change in infection dynamics and
clinical outcomes? Would it be sufficient to search only for the presence of Pseudomonas?
Can these new strategies directly characterize the impact of antimicrobial therapies,
allowing treatment efficacy to be both assessed and optimized (Rogers et al., 2010)?
5. Conclusion
Longitudinal studies correlating multiple analyses of the microorganisms present in a
chronic wound to patient metadata will increase our understanding of the problems that are
caused by the microorganisms. The questions researchers should ask are: What role does

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every single bacterial and fungal species have in the ulcer? Which role does the biofilm
formation play and is it the same for all species? Which virulence factors are the most
important, and does QS play a role? The understanding of the exact function of the
microorganisms in chronic wounds will make the use of molecular methods as a diagnostic
tool highly relevant in the future. Only by obtaining thorough knowledge of this we will be
able to develop sufficient treatment strategies for each individual ulcer. We find it
premature to implement the new culture independent methods into daily clinical practice as
it will drive the clinician to act on the results. The result of that could very well be the
application of additional antibiotics because “if the bug is there, it must do harm”. For now
we do not know that, but we need to find out. Until then, the clinicians will have to rely on
“Best-Practice Principals”.
6. Acknowledgement
Bo Jørgensen is acknowledged for sampling; Vibeke Rudkjøbing, Masumeh Chavoshi,
Martin Aasholm, Yijuan Xu and Per Halkjær Nielsen are acknowledged for scientific work
and input.
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