The Effect of Various Wound Dressings on the Activity of Debriding

ORIGINAL INVESTIGATION
The Effect of Various Wound Dressings on the
Activity of Debriding Enzymes
Lei Shi, PhD; Ryan Ermis, BS; Brett Kiedaisch, MS; and Dennis Carson, PhD, DABT
exudate and bioburden management, debridement, and tissue
regeneration. Overall, the authors’ testing found that collagenase
was observed to be more tolerant when used with the dressings
tested than papain. These findings merit further exploration in
clinical wounds to confirm clinical validity.
ABSTRACT
OBJECTIVE: To understand the compatibility between the
debriding enzymes collagenase and papain, and various wound
dressings.
DESIGN: The extracts from a silver dressing (Acticoat; Smith &
Nephew, St Petersburg, Florida), iodine dressings (Iodoflex and
Iodosorb; Smith & Nephew), a pigment-complexed polyvinyl
alcohol (PVA) dressing (Hydrofera Blue; Healthpoint, Ltd, Fort
Worth, Texas), and collagen dressings (Hydrofera Blue and
FibraCol Plus; Systagenix Wound Management, Quincy,
Massachusetts) were examined in vitro with collagenase and
papain (papain was used in papain-urea debriding agents,
no longer available on today’s US market).
SETTING: All testing was in vitro and performed at Healthpoint, Ltd.
PATIENTS: Testing was not performed using human or animal
subjects. All in vitro testing was conducted in the lab using
artificial wound eschar substrate and other lab equipment.
MAIN OUTCOME MEASURES: The main outcome measure was
percent collagenase and papain activity lost when combined with
each type of dressing tested.
MAIN RESULTS: The results demonstrated that the
pigment-complexed polyvinyl alcohol dressing and the collagen
dressing were compatible with collagenase, whereas the
iodine dressings inhibit the activity of collagenase. The
nanocrystal silver dressing (Acticoat) caused more than a 50%
loss in activity when combined with collagenase. Papain displayed
varying levels of inhibition with all dressings tested with the
enzyme. The iodine dressings significantly inhibit papain activity,
whereas the other dressings exhibited inhibitory activity ranging
from 10% to 30%.
CONCLUSION: Antimicrobial dressings are widely used for
management of wound bioburden. Frequently, they are used in
combination with other topical therapeutic drugs, such as
enzymatic debriding agents for the removal of wound necrotic
tissues. Such combined applications may have greater potential to
achieve multiple healing activities simultaneously, including
INTRODUCTION
Wound healing can be delayed if necrotic tissues are left in
the wound site.1 Therefore, wound debridement is necessary
to remove this necrotic tissue, cellular waste, and harmful
exudate. This has become an essential step in wound bed
preparation.2 Among various debridement methods, enzymatic
wound debridement uses proteolytic enzymes to hydrolyze the
denatured proteins in wound eschar tissue. This highly selective
debriding method has been widely used to remove necrotic/
devitalized tissues,3 – 7 particularly in patient populations not
amenable to surgical debridement.8,9 Over the years, enzymatic
debridement has been shown to be a clinically effective, safe, and
inexpensive method of removing necrotic tissue. There
is sufficient evidence in clinical practice and in the clinical
literature that enzymatic debridement is a well-established
practice with skin and wound care professionals.7,10,11
Successful topical treatment of chronic wounds requires
not only adequate debridement, but also control of bioburden
and moisture balance. Wound dressings are commonly used for
controlling wound bioburden and managing wound exudate.
Frequently, enzymatic debriding agents are applied in combination with other topical therapeutic drugs, such as antimicrobial and moisture control dressings. Bolton and Fattu10
discussed in their review of the literature that enzymatic debriding agents are typically used in conjunction with moist
wound dressings and serve as adjuncts to the autolytic debridement process. Clearly, such combinations may allow
several treatment objectives to be addressed simultaneously,
including exudate and bioburden management, debridement,
Lei Shi, PhD, is Scientific Advisor; Ryan Ermis, BS, is Scientist; Brett Kiedaisch, MS, is Senior Scientist; and Dennis Carson, PhD, DABT, is Senior Director, all in the Department of
Research and Development, Healthpoint, Ltd, Fort Worth, Texas. Acknowledgments: The authors thank Renée Carstens for medical writing contributions. Healthpoint, Ltd, financially
supported this research.
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ADVANCES IN SKIN & WOUND CARE & OCTOBER 2011
ORIGINAL INVESTIGATION
the substrate to collagenase, a collagen dressing (FibraCol Plus;
Systagenix Wound Management, Quincy, Massachusetts) was
tested only with CSO for investigating the potential interaction
between them. The second experiment was designed to examine
collagenase used in CSO and papain used in Accuzyme (ACC;
Healthpoint, Ltd) for their ability to degrade artificial eschar
materials containing the major wound proteins, collagen, fibrin,
and elastin, when used in conjunction with HB. The results from
these studies may provide clinicians with a better understanding
of how the currently approved enzymatic debriding agent can be
used most effectively in combination with various wound
dressings.
and tissue regeneration. To allow maximum debriding efficacy, a good delivery system, a sustained period of enzyme
activity, and an optimal wound environment are required. In
conjunction with other wound dressings, such as antimicrobial
or moisturizing, the compatibility between the debriding enzyme and dressings may account for the potential loss of efficacy of the debriding enzyme. Enzymes are proteins, and any
factors that could cause a conformational change to a protein
could potentially affect enzyme activity. In addition, molecules
that can bind to the active site of an enzyme may block substrate binding to the enzyme and inhibit enzyme activity.
The objective of this study supported by Healthpoint, Ltd
(Fort Worth, Texas), was to evaluate the compatibility between
2 debriding enzymes, Clostridium collagenase and papain, with
various wound dressings. Both collagenase and papain have
been extensively used for wound debridement7,12 – 16 and have
a long history of good efficacy and safety. Recently, the US Food
and Drug Administration required the manufacturing of papaincontaining topical products to cease, effective November 2008
in the United States.17 Although these papain products are
no longer on the market in the United States today, the work
with papain is included in this article as a comparator to demonstrate the diversity of enzymatic debriding agents. This information may prove helpful to those transitioning from a
papain debrider to a collagenase debrider. In addition, it will
also benefit the users outside the United States. In this article,
2 activity-based experiments were designed. The first was to
test the activity of the 2 enzymes, with and without incubation,
in the extracted solution from each dressing. Both enzymatic debriding agents were treated with the extracts from 4 commonly
used wound dressings including a silver dressing (Acticoat; Smith
& Nephew, St Petersburg, Florida), iodine dressings (Iodosorb
and Iodoflex; Smith & Nephew), and a polyvinyl alcohol (PVA)
foam dressing (Hydrofera Blue [HB]; Healthpoint, Ltd). The 2
types of antimicrobial dressing, silver and iodine, were chosen
because they have been frequently used for wound disinfection.
Although the product insert for the collagenase ointment product, Collagenase Santyl Ointment (CSO; Healthpoint, Ltd),
contains a precaution advising users to avoid use with heavy
metals, such as mercury or silver, which may inactivate the enzyme, the authors believed their study should include this type of
dressing for comparison purposes, despite the known inhibitory
effect on collagenase. The article also includes the other protease,
papain. The test results of silver with both enzymes will provide some insights between the 2 enzymes. Considering the
application of moist dressings concurrently used with debridement agents, a PVA foam dressing, HB, was selected because it
could provide a highly moisturizing wound environment and is
friendly for use with the enzyme ointments. Because collagen is
ADVANCES IN SKIN & WOUND CARE & VOL. 23 NO. 10
MATERIALS AND METHODS
Unless otherwise indicated, all chemicals and substrates were purchased from Sigma Aldrich Chemical Company (St Louis, Missouri).
Fluorescein isothiocyanate (FITC)–labeled collagen and rhodaminelabeled elastin were obtained from Elastin Products Company
(Owensville, Missouri). A Tris-buffered saline (TBS) solution was
used consisting of 50 mM Tris, 100 mM NaCl, and 10 mM CaCl2
(pH 7.4).
Enzyme and Dressing Materials
& Clostridium collagenase is the active enzyme used in CSO.
& USP papain is the active enzyme used in the papain-urea
ointment, ACC.
Note: This product is no longer commercially available.
& Hydrofera Blue is a bacteriostatic foam dressing made of a
PVA sponge containing the antimicrobial dyes methylene blue
(0.025%) and gentian violet (0.025%). Prior to use, it must be
moistened with sterile saline or water, and then squeezed out
before being secured to the wound bed with an appropriate
secondary dressing.
& Acticoat is a silver dressing, in which nanocrystalline silver is
uniformly deposited across polyethylene layers.
& Iodoflex consists of individual applications of a cadexomer
iodine paste consisting of a macrogol ointment base incorporating sterile, yellow-brown microspheres or beads 0.1 to 0.3
mm in diameter.
& Iodosorb-medicated dressing: Each unit dose contains
cadexomer iodine (60% wt/wt) equivalent to 0.9% wt/wt
available iodine; with macrogol as the vehicle base.
& FibraCol Plus is a wound dressing consisting of 90% collagen
and 10% alginate.
Extraction
All dressing materials were cut into 2 2-in squares and
incubated in 8 mL TBS containing 50 mM Tris, 100 mM NaCl,
2
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ORIGINAL INVESTIGATION
Bovine collagen (type I), bovine fibrinogen, and elastin were
used to make an artificial wound eschar (AWE) substrate.
Collagen-FITC, elastin-rhodamine, and fibrin-coumarin were
the raw materials used for producing the AWE substrate.21
To prepare 1 g of AWE substrate, 650 mg collagen-FITC and
100 mg each of elastin-rhodamine and fibrin-coumarin were
weighed into a 50 mL-tube and homogenized in 10 mL of
TBS. In a separate tube, 10 mL of fibrinogen solution was prepared at 15 mg/mL with TBS. The 2 solutions were combined and thoroughly mixed. A thrombin solution (0.25 mL at
50 U/mL) was added and quickly mixed, and the solution
was poured into a Petri dish containing a 90-mm nonreactive
membrane filter. As a result of the thrombin-induced fibrinogen polymerization, the material began to form a soft
sheet on top of the membrane filter by clotting the dyed
proteins into a solid matrix. The clotted AWE substrate was
allowed to solidify for 30 minutes and then rinsed with water
for 15 minutes to remove the thrombin. The AWE substrate
was further dehydrated to 75% moisture content in preparation for use.
With the AWE substrate still attached to the membrane,
a 35-mm diameter piece was punched out using a hole
punch. The AWE substrate punch was placed on the top flat
face of a Franz Diffusion Cell System (Hanson Research,
Chatsworth, California), and a nonstick sample holder is
placed on top. Enzyme products were loaded in the center of
the sample holder, and any excess sample was removed by
scraping. HB and moist gauze were cut into small pieces,
wetted according to the manufacturer’s directions, and applied on top of the enzyme product layer. With the dressing
in place, the whole assembly was fastened with a screw clamp
to the receptor cell filled with TBS containing 1% penicillinstreptomycin, without any air bubbles, and surrounded by
a 35- C water bath. The solution was sampled from the
receptor cell in 1-mL increments at time 0 and 24 hours.
Once finished, the samples were analyzed by fluorescence
measurement.
CSO was tested for collagenolytic activity based on the degradation of collagen in AWE. Because papain has broader specificity, fibrinolytic activity of ACC based on the degradation
of fibrin in AWE was also examined, in addition to collagen digestion. However, ACC was not tested with the collagen dressing.
and 10 mM CaCl2 (pH 7.4) for 2 hours on a rotary shaker at
37- C. After incubation, the dressing materials were removed,
and the dressing extraction solutions were allowed to rest at
room temperature before further testing.
Collagenase Activity Assay
Collagenase activity was determined using N-[3(2-Furyl)
Acryloyl]-Leu-Gly-Pro-Ala (FALGPA).18,19 A 500-Ag/mL enzyme solution was prepared in the same TBS as above. The
enzyme solution was mixed with each dressing extraction solution in a 1:1 ratio. The control was prepared with the same
enzyme solution mixed with the TBS. The mixture solutions were
allowed to incubate on the bench for 30 minutes. The 5-mM
FALGPA solution was prepared in the TBS and vortexed prior
to use to ensure no particles were left in the solution. Using a
96-well microplate, 100 AL of the enzyme solution was mixed
with 150 AL of the FALGPA solution. The kinetic reaction was
monitored for 30 minutes, and kinetic rates (for enzymatic activity) were determined by recording the absorbance change
at 345 nm. Rates were reported as milli-OD per minute.
Papain Activity Assay
Papain activity was determined using N-benzoyl-L-arginine-pnitroanilide (BAPNA).20 The inhibitory activity of papain was
tested under 2 different conditions, with activation and without
activation. Papain is a cysteine (Cys) protease, and in its native
state, inactivation of enzyme commonly occurs due to oxidation. To reactivate, thiol reagents such as Cys are used to recover the activity. In these experiments, 2 solutions were used to
prepare enzyme solutions. Solution A was a 10 mM dibasic
sodium phosphate buffer (pH 6.0) containing 38 mM Na2 EDTA
and 50 mM L-Cys as activating agents, and solution B contained 10 mM phosphate buffer (pH 6.0) minus EDTA or Cys.
Enzyme solutions were prepared at 100 Ag/mL and mixed with
the dressing extraction solutions at a 1:1 ratio. After mixing,
the solutions were allowed to rest for 30 minutes at room
temperature. Substrate BAPNA solution was prepared in solution
B, and the solution was checked visually prior to use to ensure
complete dissolution. In a 96-well microplate, 100 AL of the
enzyme mixture solution was mixed with 100 AL of the BAPNA
solution. The kinetic reaction was monitored at 37- C for 20 minutes, and kinetic rates (for enzymatic activity) were determined
by recording the absorbance change at 410 nm. The rates were
reported as milli-OD per minute.
Statistical Analysis
In Vitro Testing of PVA Foam Dressing With Enzymatic
Debriding Agents
Enzyme inhibition data were averaged from a group of 4 samples (n = 4). SD was calculated and displayed in the graphs. Data
between the enzyme itself and the dressing treated were statistically compared using a t test (a = .05). To determine statistical
An in vitro study was conducted to evaluate the compatibility of HB with CSO and papain-urea ointment (ACC).
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ADVANCES IN SKIN & WOUND CARE & OCTOBER 2011
ORIGINAL INVESTIGATION
solution containing extractions and those without extractions.
Figure 1 presents the inhibition percentages for each of the
dressings: FibraCol, HB, Acticoat, Iodoflex, and Iodosorb. No
inhibition was found for HB. FibraCol showed less than 5% inhibition, which can be considered insignificant inhibition. Acticoat, Iodoflex, and Iodosorb inhibited collagenase activity by
52%, 94%, and 87%, respectively.
Figure 1.
PERCENTAGE OF COLLAGENASE INHIBITION BY VARIOUS
DRESSINGS CALCULATED USING 100% SUBTRACTED BY
THE PERCENTAGE OF THE REMAINING ACTIVITY OVER
THE ORIGINAL ACTIVITY (*P < .05)
Inhibitory Effect on Papain
The inhibitory effect of the dressings on papain was tested
under 2 conditions: 1 with activator and 1 without (refer to
Figure 2). With activators, no inhibition was found for HB. Low
inhibition was found for Acticoat and Iodosorb (approximately
12% and 26%, respectively). Iodoflex was found to significantly
inhibit papain with greater than 70% inhibition. Without the
activators, papain became more sensitive to the dressings. HB
and Acticoat all showed some inhibition, 10% to 30%, whereas
Iodoflex and Iodosorb displayed greater than 90% inhibition.
Under this condition, which was similar to the condition used
for collagenase, greater inhibitory effects were found for papain
with HB, Iodoflex, and Iodosorb, with only Acticoat exhibiting
less inhibitory effect on papain, when compared with collagenase. Thus, collagenase was found to be more stable, therefore
less sensitive to these dressings, with the exception of Acticoat.
significance, P G .05 was the decision level. In the in vitro debridement experiment, a 1-way analysis-of-variance F test was
performed. Statistical decisions were made at P = .05.
RESULTS
Inhibitory Effect on Collagenase
The activity of collagenase in the presence of the various dressing
extraction solutions was compared with the enzyme solution
without dressing extractions. The inhibition percentages were
calculated using the percentage of the activity for each enzyme
Inhibitory Effect on Formulated Enzyme Debriding Agents
An in vitro study using AWE was conducted to evaluate the
application of HB with enzymatic debriding products, CSO
Figure 2.
PERCENTAGE OF PAPAIN INHIBITION BY VARIOUS DRESSINGS WITH AND WITHOUT CYSTEINE (CYS) AND EDTA,
CALCULATED USING 100% SUBTRACTED BY THE PERCENTAGE OF THE REMAINING ACTIVITY OVER THE ORIGINAL
ACTIVITY (*P < .05)
ADVANCES IN SKIN & WOUND CARE & VOL. 23 NO. 10
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ORIGINAL INVESTIGATION
Figure 3.
COLLAGENOLYTIC ACTIVITY OF CSO ALONE OR WITH HB OR MOIST GAUZE TESTED ON AWE FOR 24 HOURS. ONE-WAY
ANALYSIS-OF-VARIANCE TEST (SIGNIFICANT DECISION LEVEL AT .05) SHOWED NO SIGNIFICANT DIFFERENCE
BETWEEN THE TEST ARTICLES
and without HB. HB, when used as the dressing on top of
ACC, did not change the papain enzymatic activities significantly, indicating that HB was compatible with ACC. Data also
showed collagenolytic activity of ACC to be much lower than
its fibrinolytic activity.
and ACC. In the experiment with CSO, moist gauze was included as a control. Figure 3 illustrates the collagenolytic activity of 24-hour treatment. CSO in combination with HB or
moist gauze displayed slightly higher collagenase digestive
power on collagen than CSO alone. However, statistical analysis indicated no significant difference between them, suggesting that HB was very compatible with collagenase, confirming
the results obtained from the enzyme/dressing extraction
study discussed.
ACC was also tested with HB to determine compatibility
between the dressing and the papain enzyme. Figure 4 demonstrates results of both the collagenolysis and fibrinolysis with
DISCUSSION
The successful topical treatment of chronic wounds requires
adequate debridement, management of bioburden, and moisture balance. Enzymatic debridement has been widely used to
remove necrotic tissue, including hard eschar in burn wounds
and soft slough in chronic wounds. This method of wound
Figure 4.
COLLAGENOLYTIC AND FIBRINOLYTIC ACTIVITIES OF ACC WITH AND WITHOUT HB TESTED ON AWE FOR 24 HOURS.
NO SIGNIFICANT DIFFERENCE WAS FOUND BETWEEN TESTS WITH AND WITHOUT HB USING A t TEST (*P < .05)
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ADVANCES IN SKIN & WOUND CARE & OCTOBER 2011
ORIGINAL INVESTIGATION
where the native zinc resides. This could lead to the possible
replacement of zinc ions or interference with the orientation of
the zinc ions, both effects capable of changing the enzyme activity. Similar effects could happen to papain. Because papain
is a Cys protease, silver ions could directly interact with the
sulfhydryl group at the active site to block the enzyme’s activity.
However, the results from this study did not show significant
inhibition of papain, with or without EDTA. Papain appeared
more tolerant of nanocrystal silver.
Compared with silver, iodine displays more potent inhibition
of both enzymes. It has already been found that iodine could
effectively inhibit various proteases in wounds including metalloprotease and serine proteases.22 Iodine is strongly oxidative
and easily reacts with the amino, phenol, and thiol groups of
amino acids. For enzymes, this results in denaturation and inactivation. The observations in the authors’ experiments support
this possible mechanism. The authors’ data suggest that in a
clinical setting, combining an iodine-containing product with a
protein- or enzyme-based healing agent may result in decreased
activity from the protein or enzyme.
The experiments designed for this compatibility study focused
on the extracts from the dressings. Another possibility for interaction between an enzyme and dressing is simply that the
enzyme could bind to the dressing, thus changing its availability
and activity. Further research is necessary to fully understand
this potential interaction.
debridement is highly selective and uses naturally occurring
proteolytic enzymes specifically for eliminating devitalized tissue. Basically, this is a hydrolytic process of degrading proteinaceous material depending on the catalytic action of proteases
(debriding enzymes). Thus, a moist environment under physiological conditions is required for the enzymes to perform their
hydrolytic activity. The data from this study support the combined application of an enzymatic debriding agent with a moist
dressing. CSO used with moist gauze or HB dressing can increase the in vitro collagenolytic activity. Hydration would first
help solubilize the enzyme and accelerate the enzyme release,
which then provides conditions for the enzyme to perform the
hydrolytic action. Therefore, it is highly recommended to ensure
sufficient moisture in a wound when using enzymatic debriding
agents and keep the eschar moisturized during debridement.
To maintain wounds in a favorable healing environment, various antimicrobial wound dressings are used to control infection
and promote healing. When these dressings are used in conjunction with enzymatic debriding agents, compatibility between the enzyme and antimicrobial activity becomes a major
concern. In addition, extensive growth of bacteria can also
produce significant level of proteases, which may cause the
degradation of various proteins and enzymes, including the
exogenous proteins used for therapeutic purposes. In such
wounds, the half-life of the debriding enzymes used could be
significantly reduced. To generate an optimal environment for
clean wound beds, the combined application of debriding enzymes with an antimicrobial agent could not only remove the
devitalized tissues, but also control infection to minimize continuous tissue necrosis. The optimal combination should be such
that the dressing materials do not display any inhibitory activity
on the debriding enzymes, while the debriding enzymes do not
alter the therapeutic activity of the antimicrobial agent. In this
study, 3 types of antimicrobial dressings were investigated for
compatibility with the 2 debriding enzymes. A silver dressing
and iodine dressings displayed varying degrees of inhibition
to both collagenase and papain. The dye-based antimicrobial
dressing, HB, showed excellent compatibility with collagenase.
When tested with the enzyme in solution and CSO, HB showed
no inhibition of collagenase activity.
The silver dressing displayed significant inhibitory effects to
both debriding enzymes. Acticoat uses nanocrystalline silver as
the active agent. The nanocrystalline silver exists as both metallic
silver and ionic silver during solubilization, meaning it has a
higher potential to interact with enzymes in 2 possible ways: one
by binding to the surface of protein molecules through the side
chains of certain amino acids and the other by direct interaction
with the residues in the active site. For collagenase, because it is a
metalloprotease, silver has the potential to get into the active site
ADVANCES IN SKIN & WOUND CARE & VOL. 23 NO. 10
CONCLUSIONS
Two enzymatic debriding enzymes, collagenase and papain,
have been tested with various types of wound dressings, including antimicrobial dye, silver, iodine, and collagen. The dyebased and collagen-based wound dressings showed the greatest
compatibility with collagenase and papain. Extracts from Acticoat reduced enzyme activity by 50%. The silver dressing also
affected papain enzymatic activity at low levels. Both of the
iodine dressings, Iodoflex and Iodosorb, significantly inhibited
both collagenase and papain. An in vitro test with the CSO
demonstrated a greater activity with the HB dressing than the
enzyme alone. The in vitro debridement efficacy was enhanced
when moisturized dressings, such as HB and moist gauze, were
used. These findings merit further exploration in clinical wounds
to confirm clinical validity.
&
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