digestion slough staph

Two in vitro studies on the micro-organism
absorption capacity and influence on fibroblasts
of TenderWet active
Summary of two publications:
C. Courderot-Masuyer, S. Robin, X. Bertrand, P. Plesiat, N. Tholon, P. Humbert.
Étude du comportement des fibroblastes sains et d’ulcère veineux après infection par Pseudomonas
aeruginosa et en présence du pansement HYDROCLEAN active. Journal des Plaies et Cicatrisations
2005; 51: 3-7
R.Bruggisser. Bacterial and fungal absorption properties of a hydrogel dressing with a superabsorbent
polymer core. Journal of Wound Care 2005; 9: 438-442
Background: Chronic wounds covered with slough and necrotic tissue require effective
debridement and a moist wound environment for healing. Removing slough and necrotic tissue
reduces the bacterial bioburden, and in a physiological moist wound environment the wound
can exert its own debriding capacity.
Objective: The efficiency of the wound dressing TenderWet active, marketed under the brand
name Hydroclean in France, to absorb and retain bacteria and the influence of the dressing on
fibroblasts were investigated in two in vitro studies.
Methods: Quantity of S. aureus, S. epidermidis, P. aeruginosa and C. albicans and the
behaviour of fibroblasts – the key cells in granulation tissue formation and wound contraction
– were investigated in the presence and absence of TenderWet active.
Results: TenderWet active significantly reduced the quantity of all micro-organisms tested.
At the same time, the dressing exerted a positive effect on fibroblast behaviour.
Conclusion: TenderWet active reliably removes und retains pathogenic bacteria from the
wound, recovers the biological behaviour of fibroblast and supports the healing process
in chronic ulcers. Clinical studies and experiences confirm these in vitro results and underline
the fast cleansing and debriding effect of TenderWet active in patients with chronic wounds
of different aetiologies.
Introduction
Chronic wounds, particularly those covered
with slough and necrotic tissue, provide a
favourable environment for microbial growth
(1). The most frequently detected bacteria in
chronic ulcers are Pseudomonas aeruginosa
and Staphylococcus aureus (2). Colonisation
alone without any clinical signs of infection
does not impair wound healing. But if a
critical colonisation or infection develops and
clinical signs of infection can be diagnosed,
the exudative phase is prolonged and wound
healing is further delayed. Especially multi-
morbid and immunocompromised patients
are at risk, suffering increased pain, and
it may take months or sometimes even years
until the lesion has healed (3).
Chronic wounds with slough and necrotic tissue require effective debridement and a
moist wound environment which supports the
wound healing process. Debriding slough
and necrotic tissue reduces the bacterial bioburden (4). The physiological moist wound
environment enables the wound to exert its
own debriding capacity (5). During this
autolytic debridement, cells in the wound
area release endogenous protelolytic enzymes
and activate phagocytes. Thus, necrotic
tissue and slough are digested and separated
from healthy tissue, promoting the formation
of granulation and epithelial tissue (6).
Fibroblasts and myofibroblasts are key cells in
the formation of granulation tissue and
wound contraction (7). During the process of
cutaneous repair, the dermal fibroblasts
migrate to the area of the lesion where they
proliferate, synthesise the constituents of
the extracellular matrix and differentiate into
myofibroblasts. Myofibroblasts, the fibroblasts of granulation tissue, play an active role
in the process of wound contraction and
are characterised by the presence of bundles
of actin-filaments in the cytoplasm (8).
The moisture-activated polyacrylate dressing
pad TenderWet active supports the autolytic
debridement of the wound itself (see page 5).
The dressing pad attracts and permanently
removes proteins from necrotic tissue, attracts
and retains toxins and bacteria and provides
a moist and physiological environment for
enzymatic autolysis (9).
Two in vitro studies have now investigated
the bacterial absorption and retention
properties of TenderWet and the resulting
effects on fibroblasts.
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Material and Methods
Behaviour of fibroblasts in contact with
TenderWet active
The French study of Courderot-Masuyer et al.
analysed the in vitro behaviour of two
different kinds of fibroblasts in the presence
of Pseudomonas aeruginosa and TenderWet
active. Normal fibroblasts and fibroblasts
obtained from incipient venous ulcers in a
75-year-old female patient were used. The in
vitro model investigated in the study was
a stretched skin equivalent known as tense
collagen lattice (10). This model can be likened to granulation tissue which is involved
in cutaneous scar formation. In this situation,
cell growth is increased and the extracellular
matrix is non-retractable and under tension
(11). In a collagen matrix, fibroblasts exhibit
behaviour resembling their in vivo behaviour
(12). Courderot-Masuyer et al. therefore used
this in vitro stretched skin equivalent model
to evaluate the effect of TenderWet active
on the behaviour of healthy and venous ulcer
fibroblasts.
The tense collagen lattices were cultured for
4 days, and on the 5th day TenderWet active
dressing was placed on the surface of the
tense collagen lattice in the presence or absence of P. aeruginosa.
Bruggisser assessed the growth of microorganisms in a suspension before and
after placing the dressing in the cell culture
flask. To visualise the absorption properties,
TenderWet contaminated with S. aureus
was photographed using a scanning electron
microscope. Additionally, the behaviour of
the micro-organisms under the dressing was
studied. For that purpose, samples of the
microbial film on the agar were collected and
the number of micro-organisms in the border
Results
Behaviour of fibroblasts in contact with
TenderWet active
As Courderot-Masuyer et al. report in their
publication, a significant decrease in the
number of P. aeruginosa could be detected in
the presence of TenderWet active in the culture medium of normal fibroblasts and fibro-
Colony forming units of P. aeruginosa
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infected with P. aeruginosa
infected with P. aeruginosa plus TenderWet
10
8
6
4
2
*
*
0
Fibroblasts (normal)
Viability of fibroblasts was measured on:
– normal fibroblasts or fibroblasts from venous ulcer not infected by P. aeruginosa;
– normal fibroblasts or fibroblasts from
venous ulcer infected with P. aeruginosa;
– normal fibroblasts or fibroblasts from
venous ulcer not infected but on which
TenderWet active was applied;
– normal fibroblasts or fibroblasts from
venous ulcer infected with P. aeruginosa
and on which TenderWet active was
applied.
area and in the area below the dressing was
counted.
Fibroblasts (from venous ulcer)
Figure 1 Number of colony forming units of P. aeruginosa detected in the culture media of normal fibroblasts and
fibroblasts from venous ulcer in the absence and presence of TenderWet (* p<0.01 versus groups: fibroblasts infected
with P. aeruginosa)
Viability of fibroblasts in %
100
*
*+
80
60
Additionally, the myofibroblast differentiation
was investigated by immunohistochemical
analysis of alpha-smooth muscle actin (alphaSM-actin, specific for myofibroblast) and
F-actin (specific for the cytoskeleton of fibroblasts and myofibroblasts).
Micro-organism absorption properties
The in vitro study of Bruggisser demonstrated
the bacterial and fungal absorption and
retention capacity of TenderWet. Overall, four
organisms were analysed:
– Staphylococcus aureus,
– Staphylococcus epidermidis,
– Pseudomonas aeruginosa and
– Candida albicans.
40
*
20
0
Fibroblasts (normal)
control
plus TenderWet
Fibroblasts (from venous ulcer)
plus P. aeruginosa
plus P. aeruginosa and TenderWet
Figure 2 Study of the viability of fibroblasts after 24 hours of culturing in tense collagen lattices in the absence and
presence of TenderWet and P. aeruginosa (JPC 2005; 51: 3-7). There is no statistically significant difference between
the viability of the fibroblasts before and after application of TenderWet active. The application of TenderWet does not
adversely affect the viability of the fibroblasts (+p < 0.05 versus control; *p < 0.05 versus Fibroblasts +
P. aeruginosa)
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Number of viable germs in suspension with or without TenderWet
CFU/ml
Staphylococcus aureus
10 10
10
**
**
9
10 8
10 7
10 6
10 5
10 4
10 3
10 2
10 1
control (0h)
CFU/ml
10
control (24h)
control with
Ringer (24h)
TenderWet (24h)
Staphylococcus epidermidis
**
10
gauze (24h)
**
Figure 5 Electron microscopic images of superabsorbent
polyacrylate within TenderWet without (a) and with (b)
Staphylococcus aureus (J Wound Care 2005; 9: 438-442)
10 9
10 8
10 7
duced the viability of the fibroblasts. The use
of TenderWet in the presence of P. aeruginosa
was associated with a considerable number
of viable fibroblasts (Figure 2).
10 6
10 5
10 4
10 3
10 2
10 1
control (0h)
CFU/ml
control (24h)
control with
Ringer (24h)
TenderWet (24h)
Pseudomonas aeruginosa
**
10 10
gauze (24h)
**
10 9
10 8
10 7
10 6
10 5
10 4
10 3
10 2
10 1
control (0h)
control (24h)
control with
Ringer (24h)
TenderWet (24h)
gauze (24h)
Figure 3 Number of viable germs (CFU) in suspension with or without TenderWet compared to gauze (*p<0.001,
Student’s-test, n =18). The results for C. albicans are not shown, but look similar (J Wound Care 2005; 9: 438-442)
blasts from venous ulcer (Figure 1). The use of
TenderWet did not cause a significant decrease in the viability of the normal fibro-
TenderWet did not affect the organisation of cytoskeleton of fibroblasts
The presence of P. aeruginosa in the culture
medium resulted in the death of the fibroblasts. The use of TenderWet active in the
presence of P. aeruginosa allowed the fibroblasts to survive and the organisation of the
cytoskeleton (F-actin) to be preserved. Additionally, the use of TenderWet did not impair
myofibroblast differentiation (detection of
alpha-SM-actin fibres) of fibroblasts. The
use of TenderWet active in the presence of
P. aeruginosa allowed the fibroblasts to survive and to differentiate into myofibroblasts.
The fibroblasts cultured in the presence of
P. aeruginosa and TenderWet active exhibited
normal morphological and functional
(presence of alpha-SM-actin) characteristics
of myofibroblast differentiation.
blast or fibroblasts from venous ulcer cultured
in tense collagen lattices. In contrast,
the presence of P. aeruginosa significantly re-
Micro-organism absorption properties
Bruggisser underlines in her publication that
for all investigated strains, the number of
micro-organisms in the suspension without
a wound pad or with gauze as control was
significantly higher than in the suspension
containing TenderWet active. Figure 3 illustrates the results for S. aureus, S. epidermidis
and P. aeruginosa. In the electron microscopy
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pictures, the adherence of bacteria to the
superabsorber polymer core of the dressing
was obvious (Figure 4).
Number of colony forming units of selected micro-organisms under TenderWet
CFU
Pseudomonas aeruginosa
108
Border area
Area below TenderWet
107
106
105
104
Figure 5 depicts the number of different
micro-organisms under the wound dressing
and on the non-covered border area. Compared with the border area, a one to three
log10 reduction in the number of viable germs
was assessed under TenderWet. As the
author points out, the number of microorganisms in the non-covered border area
remained constant over the investigated
period of time.
103
0
20
40
60
80
Incubation time (h)
CFU
Candida albicans
10
6
Border area
Area below TenderWet
10
5
104
103
102
0
20
40
60
80
Incubation time (h)
CFU
Border area
Area below TenderWet
Staphylococcus aureus
107
106
105
0
20
40
60
Discussion
Both in vitro investigations clearly demonstrate that TenderWet active was capable
of absorbing and retaining bacteria such as
S. aureus, S. epidermidis, and P. aeruginosa
which often cause clinically relevant infections in chronic wounds and delay the healing process (2). Bruggisser emphasised
in her publication that a traditional wound
dressing such as gauze was not able to
achieve this significant reduction of microorganisms. Furthermore, TenderWet did
not re-contaminate the wound surface or the
nutrient broth within 24 hours, the clinically
recommended time interval for changing
the hydrogel dressing. The reduction of microorganisms by the wound dressing helps to
avoid or reduce excessive bacterial burden on
wounds, according to author’s statement.
80
Incubation time (h)
Figure 5 In vitro comparison of the number of colony forming units (CFU) of selected micro-organisms under TenderWet and in the margin region over a period of 72 hours. The results for S. epidermidis are not shown, but look similar
(J Wound Care 2005; 9: 438-442).
However, the effective absorption and retention capacity of TenderWet did not impair
the viability of fibroblasts, the key cells in the
wound healing process, and re-epithelialisation of the wound surface. Courderot-Masuyer and colleagues could not observe in
their study any disruption of the cytoskeleton
organisation (detection of F-actin) and myofibroblast differentiation (detection of alphaSM-actin) due to the action of TenderWet
active in the presence or absence of the bacteria. Even after P. aeruginosa had decreased
the viability of fibroblasts, TenderWet
removed the bacteria from fibroblasts and
retained them, resulting in a recovery
of the biological behaviour of fibroblasts.
The fact that the results of these in vitro assays can be transferred into clinical practice
had been shown previously in clinical studies,
in which TenderWet provided a fast cleansing and debriding effect and supported the
wound healing process in patients with
chronic wounds of different aetiologies (9,13).
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Conclusion
TenderWet active removes und retains pathogenic bacteria from the wound but also
exerts a positive effect on fibroblast and supports the healing process of chronic ulcers.
In both in vitro studies the wound dressing
exhibited a high absorbent and retention
capacity for all bacterial and fungal strains
tested. At the same time, TenderWet active
did not adversely affect the viability of the
fibroblasts and their myofibroblastic differentiation necessary for the process of wound
closure. Clinical studies and experiences underline this effective cleansing and debriding
effect of the wound dressing. Thus, TenderWet is suitable for all wounds requiring
a therapy that both provides a moist wound
environment and also absorbs and retains micro-organisms. The absorbed bacteria are
removed when the dressing is changed.
References
1. McGuckin M, Goldman R, Bolton L,
Salcido R. The clinical relevance of microbiology in acute and chronic wounds.
Adv Skin Wound Care 2003; 16: 12-25
2. Edwards R, Harding KG. Bacteria and
wound healing. Curr Opin Infect Dis
2004; 17: 91-96
3. Hess CT, Kirsner RS. Orchestrating wound
healing: Assessing and preparing the
wound bed. Adv Skin Wound Care 2003;
16: 246-259
4. Bowler PG, Duerden BI, Armstrong DG.
Wound microbiology and associated
approaches to wound management. Clin
Microbiol Rev 2001; 14: 244-269
5. Steed L. Debridement. Am J Surgery
2004; 187: 71S-74S
6. Dissemond J, Goos M. Options for
debridement in the therapy of chronic
wounds. JDDG 2004; 2: 743-751
7. Falanga V. Wound healing and its impairment in the diabetic foot. Lancet 2005;
366: 1736-1743
8. Clark RAF. Wound repair: Overview and
general considerations, Chapter 1. In:
Clark RAF (ed). The molecular and cellular biology of wound repair. New York,
London: Plenum Press, 1996: 3-35
9. Paustian C, Stegman MR. Preparing the
wound for healing: The effect of acti-
vated polyacrylate dressing on debridement. Ostomy Wound Manage 2003; 49:
34-42
10. Lucarz-Bietry A, Chapuis JF, Agache P,
Humbert PA. A histological and ultrastructural comparative study of retracted
and tense collagen lattis. Eur J Dermatol
1995; 5: 524-530
11. Grinnel F. Fibroblasts, myofibroblasts and
wound contraction. J Cell Biol 1994; 124:
401-404
12. Bell E, Ivarsson B, Merrill C. Production of
tissue-like structure by contraction of
collagen lattices by human fibroblasts of
different proliferative potential in vitro.
Proc Natl Acad Sci 1979; 76: 1274 -1278
13. König M, Vanscheidt W, Augustin M,
Kapp H. Enzymatic versus autolytic
debridement of chronic leg ulcers: a
prospective randomised trial. J Wound
Care 2005; 14: 320-323
Mode of action
TenderWet supports autolytic debridement
TenderWet active is a ready-to-use dressing
already pre-activated with Ringer’s solution;
it is available in two versions, one for flat
wounds, and the other for cavity wounds
(TenderWet active cavity). The dressing consists of three parts: a cover of non wound
adherent polypropylene knitted fabric,
a moisture repellent layer, and a superabsorbent polyacrylate pad pre-activated
with Ringer’s solution (a biocompatible sterile
isotonic solution based on sodium chloride,
potassium and calcium). Applied to the
wound, TenderWet continuously releases
Ringer’s solution up to over 24 hours and
supports autolytic debridement of the wound.
During this process, necrotic tissue and coatings are softened, loosened and rinsed out.
At the same time, the wound dressing pad
absorbs bacteria-laden wound exudate into
its absorbent core and binds it there. This
exchange - Ringer’s solution is released and
proteins are taken up – functions because
the superabsorber of the wound dressing pad
has a greater affinity for the protein-containing wound exudate than for the salt-containing Ringer’s solution. As the wound is being
cleansed of necrotic tissue and other slough,
an environment conducive to cellular
migration, angiogenesis and the development
of granulation tissue is provided.
A Necrotic tissue
colonised by bacteria and slough are
actively softened
and detached by
Ringer’s solution.
B At the same time
detached necrosis
and slough are absorbed (in the form
of protein exudate)
and bound into
the absorbent core.
C Further supply
of Ringer’s solution
promotes formation of granulation
tissue.
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