Original Article
Evaluation of the Antibacterial Activity and
Toxicity of 2 New Hydrogels: A Pilot Study
Katia Vandenbulcke,* Lada-Ivana Laenen Horvat,** Martine De Mil,†
Guido Slegers,* and Hilde Beele†
*
Ghent University, Department of Radiopharmacy, Ghent, Belgium; **Virco-Tibotec, Mechelen,
Belgium (formerly Research and Development Flen Pharma, Edegem, Belgium); and
†
Ghent University Hospital, Tissue Bank and Department of Dermatology, Ghent, Belgium
Wound bed preparation remains a very important issue in
wound healing. To promote the production of granulation
tissue, it is necessary to remove necrotic tissue and to con-
trol infection. Necrotic tissue may be removed using
a hydrogel preparation. Flaminal® and Flaminal® Hydro
(Flen Pharma, Belgium) are 2 new hydroactive colloid gel
dressings with state antibacterial properties. These prop-
erties are attributed to an enzymatic complex in their
formulation. In the study described in this report, the
W ound repair involves a complex and temporal
integration of cytokines, various blood compo-
nents, extracellular matrix, and cells. The normal
healing process can be impeded at any step along its
path by a variety of factors.1,2 To promote the formu-
lation of granulation tissue and, in turn, epitheliza-
tion, a satisfactory wound bed has to be available.
Wound bed preparation includes the removal of
necrotic tissue and the control of bacterial overload.3,4
A wide variety of agents are available for treatment
of wounds, including ointments and dressings.5,6
Selection of agents suited to an optimal treatment
process for contaminated or infected wounds continues
to be controversial.7
Topical antimicrobial agents are essential in wound
care management. However, their use has also been
Correspondence should be sent to: Professor Hilde Beele, MD, PhD,
Tissue Bank and Department of Dermatology, Ghent University
Hospital, De Pintelaan 185, B-9000 Ghent, Belgium; e-mail:
Hilde.Beele@Ugent.be.
Conflict of interest: None declared.
DOI: 10.1177/1534734606289507
© 2006 Sage Publications
LOWER EXTREMITY WOUNDS 5(2);2006 pp. 109-114
antibacterial effects of Flaminal and Flaminal Hydro
were confirmed in an in vitro as well as an in vivo setting.
It was also demonstrated that Flaminal and Flaminal
Hydro are not toxic to keratinocytes in vitro using an MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide] test.
Key words: alginates, antibacterial, hydrogel, leg ulcers,
toxicity, wound healing
associated with cytotoxicity, delayed healing, the
emergence of bacterial strains resistant to common
antimicrobial agents, and the appearance of contact
dermatitis.3,8 Systemic antibiotic therapy should be
used judiciously; there is a lack of evidence in favor
of giving systemic antibiotics to patients with chronic
wounds. Given the clinical significance of contami-
nation and infection, there is a need to explore alter-
native management approaches for contaminated or
infected wounds.
The aim of this study was to compare the antibac-
terial activities and toxicity profiles of 2 new hydro-
gel formulations with other products commonly
used in the preparation of the wound bed. Intrasite®
(Smith and Nephew, UK) and Purilon® (Coloplast,
Denmark) and DuoDERM® hydrogel (ConvaTec, UK)
are hydrogels often used to remove necrotic tissue from
the wound bed. Isobetadine® (Viatris, Switzerland)
and Flammazine® (Solvay, France) are commonly used
in wounds because of their antimicrobial activity.3,9
Flaminal® and Flaminal® Hydro (Flen Pharma,
Belgium) are 2 new hydroactive colloid gel dressings
that contain acidic chemical polymers based on acry-
lates. Flaminal has a high concentration of alginates
and is used on exudating wounds, whereas Flaminal
109
 VANDENBULCKE ET AL

Hydro contains a lower amount of alginate and is Table 1. Patients Characteristics

used for the treatment of slightly exudating wounds.
Location
The products induce lysis of necrosis and crusts Wound Patient Underlying of the
due to hydration of necrotic tissue and autolytic Number Number Age Gender Disease Wound
processes.8 These hydrogels also contain an enzy-
matic complex, a combination of glucoseoxidase and 1 1 72 M Diabetes Heel
lactoperoxidase that could protect against microbial 2 1 72 M Diabetes Big toe
3 2 72 F Venous Lower leg
contamination, and ultimately against infection.8 It is
insufficiency
speculated that alginate polymers with their strong and diabetes
absorption capacity absorb microorganisms into the 4 2 72 F Venous Ankle
gel and that a catalytic conversion process leads to an insufficiency
oxidative antimicrobial effect. and diabetes
5 3 85 F Venous Ankle
MATERIALS AND METHODS insufficiency
6 4 71 F Rheumatoid Lower leg
arthritis
Wound-Healing Products 7 4 71 F Rheumatoid Lower leg
arthritis
The topical wound-healing products used in
these experiments were Isobetadine, Flaminal and
Flaminal Hydro, Intrasite, Purilon, DuoDERM hydro-
gel, and Flammazine.
Belgium. Prior informed consent was obtained from
Antimicrobial Activity In Vitro all patients whose wounds were sampled in this study.
The wounds were caused by different etiologies:
The antimicrobial activities of Flaminal and Flaminal venous disease, diabetes, and rheumatic disease.
Hydro were tested in vitro, using such pure cul- Patient characteristics are presented in tabular form
tures as Escherichia coli (ATCC10536), Pseudomonas in Table 1. All ulcers showed clinical signs of major
aeruginosa (ATCC10145), Proteus vulgaris (ATCC contamination or colonization, such as increased
33420), Staphylococcus aureus (ATCC25923), and exudation, foul odor, friable granulation tissue, dis-
Candida albicans (ATCC10231). The bacteria were coloration, or augmentation of slough. The wounds
incubated on Trypticase Soy Agar and the yeasts on differed in size, redness of the skin around the ulcer,
Sabouraud Agar. The standardized plates were over- degree of edema, and pain. There were no signs of
laid with 3 g of Flaminal or Flaminal Hydro and systemic infection. The duration of the ulcers prior
incubated for at least 48 hours at 37°C, for bacteria, to first sampling was at least 4 weeks.
and at 28°C, for yeast and molds. To test the stability Specimens were taken using sterile swabs prior
of the antibacterial activity at higher temperature, to treatment. The wound bed was first cleaned with
plates were also incubated at 50°C for at least 48 hours. sterile saline solution, and superficial slough was
The numbers of CFU/mL (colony forming units), for debrided using tweezers and scissors. Premoistening
each detected species, were calculated. A 3 g overlay the swab with sterile saline was considered when the
of Flammazine, and a 3 g overlay of DuoDERM surface of the wound was dry. The tip of the swab was
hydrogel were used as positive and negative controls, rolled on its side in a zigzag pattern for at least 1 full
respectively. rotation. The swab was taken in the granulation tissue
with the most obvious signs of bacterial presence (eg,
Antimicrobial Activity In Vivo a zone with friable granulation tissue).
The swabs were put in transport medium and were
In a clinical pilot study, we studied the in sent to the laboratory, where they were processed
vivo antimicrobial activity on 7 chronic wounds on immediately. Standard methods for isolation and iden-
4 patients. We evaluated, in vivo, the antimicrobial tification of aerobic and anaerobic bacteria were used.
properties of Flaminal or Flaminal Hydro. The Flaminal (n = 3) or Flaminal Hydro (n = 4)
patients with chronic lower extremity wounds had was chosen, according to the type of ulcer, based on
attended the outpatient facility of the Department the degree of exudation. A 3 to 5 mm layer of gel
of Dermatology, Ghent University Hospital, Ghent, was applied to the wound and sterile gauze and

110 LOWER EXTREMITY WOUNDS 5(2);2006

 HYDROGELS AND ANTIBACTERIAL ACTIVITY

Table 2. Recovery of Microorganisms In Vitro After Treatment With Flammazine®,

DuoDERM®, Flaminal®, and Flaminal® Hydro

CFU/mL

After 48 Hours
® ®
Species Day 0 Flammazine DuoDERM Flaminal® Flaminal® Hydro

Gram– bacilli
E coli 1.15 × 107 0 1.0 × 107 0 0
P vulgaris 1.62 × 108 0 1.42 × 107 0 0
E aerogenes 6.58 × 107 0 5.91 × 107 0 0
P aeruginosa 1.95 × 107 0 1.6 × 107 0 0
Gram+ cocci
S epidermidis 1.50 × 108 0 1.50 × 108 0 0
S aureus 2.29 × 108 0 1.8 × 108 0 0
S pyrogenes 1.23 × 108 0 1.4 × 108 0 0
Yeasts
C albicans 8.73 × 107 0 8.21 × 107 0 0

bandages were secured. The gel was applied every
2 days. Remnants of Flaminal or Flaminal Hydro
were wiped away from the ulcer site with gauze
soaked in a sterile saline solution. On day 8, the
ulcer area was sampled again. Antiseptics were not
used on the wound prior to sampling. Sampling was
done as described above.
In Vitro Toxicity
The culture media, fetal calf serum, and other
additives used were purchased from Invitrogen
(Merelbeke, Belgium) or Sigma (Bornem, Belgium).
Human keratinocytes were isolated from skin biop-
sies obtained from healthy young donors undergoing
elective plastic surgery (eg, breast reduction) with
prior written informed consent. The use of human
keratinocytes was approved by the Ethics Committee
of the Ghent University Hospital.
The resulting keratinocyte suspension was seeded
on a feeder layer made of irradiated 3T3 mouse
fibroblasts and cultured at 37°C in a humidified
atmosphere containing 10% CO2 as described by
Rheinwald and Green.10 At confluency, cells were
trypsinized and the resulting cell suspension was cry-
opreserved in a DMSO (10%) containing medium.
For this study, secondary keratinocyte cultures were
set up, using thawed cells, which were seeded on a
feeder layer as described by Rheinwald and Green.10
When these secondary cultures were subconfluent,
the keratinocytes were used in subsequent experi-
ments. The different products to be tested were
diluted 10% (weight/volume) in culture media and
heated, if necessary, in a water bath at 37°C for max-
imum 1 hour to obtain solubilization. The solutions
were filter sterilized (pressure filtration with a 0.2
µm diameter pore filter, Nalgene, Rochester, NY) and
were then used as culture media for the subconfluent
keratinocytes. After 24 and 48 hours of exposure to
the different products, MTT [3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide] tests were
carried out as recommended by the manufacturer’s
instructions (Promega, Madison, WI). MTT tests are
often used to evaluate the toxicity of topically applied
skin products as described by Poon and Burd11 who
studied the toxicity of topical antibacterial products
using this technique.
In the in vitro tests, cell cultures of 2 different donors
were evaluated. After trypsinization of the cultures,
the cell suspensions were diluted to 1 × 106 cells/mL.
Cell respiration was assessed by the mitochondrial-
dependent reduction of MTT (25 µL, 5 mg/mL). Blue
formazan crystals were measured by spectrometry at
570 nm after dissolution of the crystals with stop/
solubilization buffer (100 µL added). Each experi-
ment was repeated thrice. On each occasion, 12 to
24 samples were analyzed for each condition of the
experiment.

LOWER EXTREMITY WOUNDS 5(2);2006 111

 VANDENBULCKE ET AL

DATA ANALYSIS Table 3. Recovery of Microorganisms

in Leg Ulcers Before and After Treatment With
The Wilcoxon signed-rank test was done using Flaminal® or Flaminal® Hydro
an SPSS 10 software package to compare the number
of isolated species before and after treatment of the Number of Different Number of Different
Microorganisms Microorganisms
lower extremity wounds. This test was also used to Identified on Day 0 Identified on Day 8
compare the treated samples in the toxicity aspect of Wound
the experiment. P < .05 was accepted as the thresh- Number Number Description Number Description
old of statistical significance.
1 6 S aureus 2 S aureus
E coli S epidermidis
RESULTS P aeruginosa
E aerogenes
Antimicrobial Activity In Vitro S epidermidis
P vulgaris
2 6 S aureus 2 S aureus
Flaminal and Flaminal Hydro were found to be C albicans E cloacae
fully active against all microorganisms tested, irre- P aeruginosa
spective of the incubation temperature. After incuba- E coli
tion with the antimicrobial agent, silver sulfadiazine E cloacae
(Flammazine), no colony growth was observed. The E aerogenes
plate overlaid by DuoDERM hydrogel did not show 3 7 S aureus 1 S aureus
any effects of the gel on microbial population, and E coli
P aeruginosa
confluent growth of the bacteria and yeasts was E aerogenes
observed. These data are shown in Table 2. S epidermidis
E cloacae
Antimicrobial Activity In Vivo C albicans
4 6 S aureus 2 S aureus
E coli E coli
On day 0, a large number of different types of E aerogenes
bacteria were detected in all lower extremity wounds. S epidermidis
All wounds harbored more than one species. S aureus C albicans
and P aeruginosa were isolated from almost all S pyogenes
wounds. P vulgaris was less often isolated. The pre- 5 4 S aureus 0
dominant flora, in addition to those mentioned E coli
P aeruginosa
above, were aerobes or facultative anaerobes such as E cloacae
Staphylococcus epidermidis and E coli. Candida 6 5 S aureus 1 S epidermidis
albicans was found in more than half of the chronic E coli
wounds. C albicans
Eight days after the start of the treatment with S epidermidis
Flaminal or Flaminal Hydro, wounds had become P aeruginosa
7 4 S epidermidis 1 S epidermidis
negative for several bacterial species and C albicans.
S agalactiae
In the case of S aureus and S epidermidis, the erad- S pyogenes
icating effect of Flaminal and Flaminal Hydro was P aeruginosa
not complete. These results are presented in Table 3.
The number of different types of isolated species
decreased significantly (P = .018) after the use of
Flaminal or Flaminal Hydro. Isobetadine showed a significant reduction of the opti-
cal density (P < .01). The reduction of the metabolic
In Vitro Toxicity activity in the case of Intrasite was modest (approxi-
mately 5% reduction in optical density). With
Results are presented in Figure 1 as mean values Isobetadine, however, the optical density was reduced
with standard deviation and level of significance. by approximately 60%. The Flammazine-treated
After 24 hours incubation of the keratinocytes with samples could not be evaluated with the MTT test
the above-mentioned products, only Intrasite and because the solution was too opaque to count the cells.

112 LOWER EXTREMITY WOUNDS 5(2);2006

 HYDROGELS AND ANTIBACTERIAL ACTIVITY

1.2 1.01 1.04

1.01 0.92 0.95 0.96
0.89 0.96 §
1 0.97 §
Optical Density 0.82 §
0.8

0.6
0.39 §
0.4 0.28 §

0.2

0
control Intrasite Purilon Flaminal Flaminal Hydro Isobetadine

24 h 48 h

Fig. 1 MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test: Optical density at 570 nm reflecting the metabolic
activity for cultures incubated with control medium, Intrasite®, Purilon®, Flaminal®, Flaminal® Hydro, and Isobetadine® for 24 hours and
48 hours. P values versus control at 24 hours and 48 hours, respectively: Instrasite®, P = .008 (s), P = .2 (ns); Purilon®, P = .08 (ns), P = .002
(s); Faminal®, P = .051 (ns), P = .004 (s); Flaminal® Hydro, P = .051 (ns), P = .13 (ns); Isobetadine®, P < .001 (s), P < .001 (s). P < .5 was
accepted as the threshold of statistical significance. Statistical significance of treated samples versus control samples is indicated by §.

After 48 hours of incubation of the keratinocytes
with the different products, 3 products exhibited a
significantly different optical density (P < .01) when
compared to the controls. For the cultures incubated
with Flaminal, a modest increase of the optical den-
sity was observed. A limited reduction in metabolic
activity was observed with Purilon. For Isobetadine,
an important decrease of the metabolic activity was
observed (approximately 70% decrease).
DISCUSSION
Chronic wounds are almost always contaminated
with microorganisms. In wound management, it is
very important to distinguish between wound conta-
mination, wound colonization, and wound infection.
In this context, the number and kind of microorgan-
isms present, their virulence, and host resistance are
important parameters.4,12,13 Small numbers of micro-
organisms present in wound exudate and on the sur-
face of chronic wounds do not necessarily delay
wound healing. In contrast with this, a count of 105 to
106 microbial cells per mm2 of wound surface or per
gram of tissue is reported to correlate with a critical bac-
terial colonization inside the wound. This may hinder
wound healing by local toxin release and by eliciting an
inflammatory reaction.9,13 It is therefore important to
keep the number of bacteria to minimal levels.
In this study, Flaminal and Flaminal Hydro
demonstrated antimicrobial effects, both in vitro and
in vivo. The effects are considered to be due to the
enzymatic complex present in these hydrogels.8 The
same antimicrobial effects were observed at higher
temperatures. The presence of organic material in
the in vivo situation does not seem to weaken the
antimicrobial effect.
Schultz et al described that organisms such as
Staphylococci or Streptococci are difficult to eradicate
without systemic antibiotics.14 The results from this
study showed that in 6 out of 7 patients those species
could not be removed with the Flaminal or Flaminal
Hydro treatments.
Furthermore, hydrogels with alginates such as
Flaminal and Flaminal Hydro permit the topical to
gellate and demonstrate autolytic debridement. In a
small clinical study, the use of these hydrogels resulted
in a surface and a volume reduction of wounds com-
pared with controls.8
In our study, an MTT test was used to evaluate the
toxicity profile of the new hydrogels. We were unable
to demonstrate major cytotoxicity in the keratinocytes,
treated with the hydrogels without antibacterial
activity. The results of the MTT test of keratinocytes
cultured with Flaminal and Flaminal Hydro were
comparable to the control samples, suggesting the
absence of cytotoxic effects. However, our findings
are consistent with reports of the cytotoxic effects
of iodine-containing products.3,15,16 We must hasten
to add that the observed in vitro cytotoxicity of
iodine-containing products does not necessarily
mean that these products also impede the wound-
healing process in vivo.16,17 On the contrary, the use of

LOWER EXTREMITY WOUNDS 5(2);2006 113

 VANDENBULCKE ET AL
the same antiseptic products in a clinical setting has
proven to be beneficial for the wound-healing process.18
In conclusion, the results of this pilot study
demonstrate that Flaminal and Flaminal Hydro exert
antibacterial activity in vitro and in vivo, and using
the MTT test in cultured keratinocytes, toxicity was
not observed. These are preliminary observations
that raise the need for appropriately designed clini-
cal studies to demonstrate the lack of cytotoxicity in
vivo and to evaluate inherent properties desirable to
achieve wound healing.
ACKNOWLEDGMENTS
Professor Jan Philippé gave expertise for the pre-
liminary experiments with flow cytometry. Ir. Klaus
Bacher evaluated the results with the statistical
program. Financial funding was obtained from the
Fund of the Flemish Government for Scientific
Innovation IWT Ms/IS/48717.
REFERENCES
1. Machesney M, Tidman N, Waseem A, Kirby L, Leigh I.
Activated keratinocytes in the epidermis of hypertrophic scars.
Am J Pathol 1998;152:1133-41.
2. Jaffe AT, Heymann WR, Lawrence N. Epidermal maturation
arrest. Dermatol Surg 1999;25:900-3.
3. Reimer K, Vogt PM, Broegmann B, Hauser J, Rossbach O,
Kramer A, et al. An innovative topical drug formulation for
wound healing and infection treatment: in vitro and in vivo inves-
tigations of a povidone-iodine liposome hydrogel. Dermatology
2000;201:235-41.
4. Sibbald RG, Orsted H, Schultz GS, Coutts P, Keast D.
International Wound Bed Preparation Advisory Board; Canadian
Chronic Wound Advisory Board Preparing the wound bed 2003:
focus on infection and inflammation. Ostomy Wound Manage
2003;49:23-51.
114 LOWER EXTREMITY WOUNDS 5(2);2006
5. Moreno-Arias Ga, Izento-Menezes Cm, Carrasco Ma Camps-
Fresneda A. Second intention healing after Mohs micrographic
surgery. JEADV 2000;14:159-65.
6. Palmieri TL, Greenhalgh DG. Topical treatment of pediatric
patients with burns: a practical guide. Am J Clin Dermatol 2002;
3:529-34.
7. Harker J. The effect of bacteria on leg ulcer healing. Br J
Community Nurs 2001;6:126-34.
8. de la Brassinne M, Thirion l, Horvat L-IL. A novel method of
comparing the healing properties of two hydrogels in chronic leg
ulcers. JEADV 2006;20:131-5.
9. Bowler PG. Wound pathophysiology, infection and therapeutic
options. Ann Med 2002;34:419-27.
10. Rheinwald JG, Green H. Serial cultivation of strains of human
epidermal keratinocytes: the formation of colonies from single
cells. Cell 1975;6:331-44.
11. Poon VK, Burd A. In vitro cytotoxicity of silver: implication
for critical wound care. Burns 2004;30:140-7.
12. Dow G, Browne A, Sibbald RG. Infection in chronic wounds:
controversies in diagnosis and treatment. Ostomy Wound Manage
1999;45:23-40.
13. Ramelet AA, Perrenoud D. Bacteriology of leg ulcers. In:
Hafner J, Ramelet AA, Schmeller W, Brunner UV, editors.
Management of leg ulcers. Curr Probl Dermatol 1999;27:20-35.
14. Schultz GR, Sibbald RG, Falanga V, Ayello EA, Dowsett C,
Harding K, et al. Wound bed preparation: a systemic approach to
wound management. Wound Rep Reg 2003;11(Suppl):S1-28.
15. Balin AK, Pratt L. Dilute povidone-iodine solutions inhibit
human skin fibroblast growth. Dermatol Surg 2002;28:210-4.
16. Zhou LH, Nahm WK, Badiavas E, Yufit T, Falanga V. Slow
release iodine preparation and wound healing: in vitro effects
consistent with lack of in vivo toxicity in human chronic wounds.
Br J Dermatol 2002;146:365-74.
17. Falanga V. Iodine containing pharmaceuticals: a reappraisal.
Paper presented at Proceedings of the 6th European Conference on
Advances in Wound Management, October 1-4, 1996, Amsterdam.
New York: Macmillan; 1997. p. 191-4.
18. Fumal I, Braham C, Paquet P, Piérard-Franchimont C, Piérard
GE. The beneficial toxicity paradox of antimicrobials in leg ulcer
healing impaired by a polymicrobial flora: a proof-of-concept
study. Dermatology 2002;204(Suppl 1):70-4.