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Radiation Physics and Chemistry 76 (2007) 17671770

Development of honey hydrogel dressing for enhanced wound healing

Norimah Yusofa,, A.H. Ainul Hafizaa, Rozaini M. Zohdia, Md Zuki A. Bakarb

aMalaysian Institute for Nuclear Technology Research (MINT), Bangi, 43000 Kajang, Selangor, MalaysiabFaculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

Received 26 December 2006; accepted 13 February 2007

Abstract

Radiation at 25 and 50 kGy showed no effect on the acidic pH of the local honey, Gelam, and its antimicrobial property againstStaphylococcus aureusbut significantly reduced the viscosity. Honey stored up to 2 years at room temperature retained all the properties

studied. Radiation sterilized Gelam honey significantly stimulated the rate of burn wound healing in Sprague-Dawley rats as

demonstrated by the increased rate of wound contraction and gross appearance. Gelam honey attenuates wound inflammation; and

re-epithelialization was well advanced compared to the treatment using silver sulphadiazine (SSD) cream. To enhance further the use of

honey in wound treatment and for easy handling, Gelam honey was incorporated into our hydrogel dressing formulation, which was then

cross-linked and sterilized using electron beam at 25 kGy. Hydrogel with 6% of honey was selected based on the physical appearance.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Honey; Radiation sterilization; Cross-linking; Hydrogel

1. Introduction

The healing properties of honey have been known since

time immemorial and have recently gained recognition

from the scientific community (Molan, 2000). Honey

accelerates wound healing whether applied topically or

administered systemically (Suguna et al., 1992). Therapeu-

tic effects of honey have been found in burn treatment, by

helping the rapid healing of wounds with less scarring

(Subrahmanyam, 1991). Investigations have shown that

honey acted more effective than other natural and synthetic

products in the management of burn wound. Although it is

generally thought that honey is sterile, microorganisms

such as Clostridium botulinum are still able to survive in

honey (Snowdon and Cliver, 1996) and there may be a riskof wound botulism when it is applied on the wound. Thus,

gamma irradiation that is used to sterile heat-sensitive

medical items such as surgical gloves and polymeric

dressing was suggested as a possible alternative to sterilize

honey for medical use (Molan and Allen, 1996). The

present study reports the effects of gamma sterilization on

the physicochemical parameters, microbial quality and

antibacterial activity of a Malaysian honey namely Gelam

honey over a 24-month storage period. This paper alsoreports the wound healing potential of Gelam honey in the

management of burn wounds. Attempts were also made to

incorporate Gelam honey into a hydrogel system to produce

a functional wound dressing using electron beam processing.

2. Materials and methods

2.1. Honey sample

Gelam honey, purchased from the Agricultural Depart-

ment of Malaysia, was sterilized by gamma irradiation at

25 and 50 kGy at MINTec-Sinagama (Model JS 8900,Cobalt 60, dose rate 2 kGy/h). Ceric/cerous sulphate

solution dosimeter analyzed using potentiometric method

was used. Samples were analyzed over 24 months during

storage at room temperature for the physicochemical

properties: pH value was measured using the Mettler

Toledo pH meter (model 320), viscosity was measured

directly using the Brookfield Viscometer (model DV II)

and water content was determined using oven drying

method at 60 C f o r 2 4 h (Ainul Hafiza, 2005). Honey

samples were also screened for their microbiological

ARTICLE IN PRESS

www.elsevier.com/locate/radphyschem

0969-806X/$ - see front matterr 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.radphyschem.2007.02.107

Corresponding author. Fax: +603 8928 2956.

E-mail address: norimah@nuclearmalaysia.gov.my (N. Yusof).

http://www.elsevier.com/locate/radphyschemhttp://localhost/var/www/apps/conversion/tmp/scratch_2/dx.doi.org/10.1016/j.radphyschem.2007.02.107mailto:norimah@nuclearmalaysia.gov.mymailto:norimah@nuclearmalaysia.gov.myhttp://localhost/var/www/apps/conversion/tmp/scratch_2/dx.doi.org/10.1016/j.radphyschem.2007.02.107http://www.elsevier.com/locate/radphyschem

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quality using filtration method for both aerobic and

anaerobic microorganisms. Antibacterial activity against

Staphyloccocus aureus (ATCC 25923) was studied using

assay plates (Corning 43111124524518 mm) contain-

ing TSA (Oxoid) seeded with log phase cultured bacteria.

Wells cut into the agar were filled up by 0:1ml honey

sample (50% v/v) in quadruplicate. The plates were left topre-diffuse at 4 C for 1 h before incubated at 37 C for

57 h until growth inhibition zone could be seen.

2.2. Wound healing assessment

In vivo healing assessment was conducted using 45 male

Sprague-Dawley rats (weight between 200 and 300 g). The

three experimental groups namely untreated control, Gelam

honey and SSD, comprised of 15 rats each. Deep partial skin

thickness burn was inflicted on the dorsum of rats body

using a cylindrical stainless steel template heated in a water

bath at a constant temperature of 85

C, using a methoddescribed byKaufman et al. (1990)with slight modification.

The rate of wound contraction and microscopic observations

were assessed at 3, 7, 14, 21 and 28 days post burned as

discussed previously (Rozaini et al., 2005).

2.3. Honey hydrogel dressing

Gelam honey (6%, 8%, 10% and 15%) was added into

the mixture of 15% polyvinyl pyrrolidone (PVP) (Kollidon

90), 1% protein free agar (Oxoid) solution, and 1%

polyethylene glycol (PEG). The mixture was poured into

a 10cm10 cm plastic mold with 34 mm thickness, left to

set at room temperature before covered with polyethylene

sheet and individually packed. The gel was cross-linked as

well as sterilized by electron beam at 25 kGy at Alutron

Irradiation Facility, MINT (Model EPS-3000, conveyer

speed of 4.4 m/min, beam current of 10 mA and energy of3 MeV).

3. Results and discussion

No significant changes p40:05 was observed in thephysicochemical properties of the irradiated Gelam honey,

except for the viscosity value that reduced almost 50%

after irradiated at 25 and 50kGy (Table 1). On the

contrary,Sabato (2004)could not detect any changes when

honey irradiated at lower doses of 5 and 10 kGy. The pH of

Gelam honey (3.553.57) conformed to the EU Standard

(Council Directive 74/409/EEC, 1974) i.e. must be less than5; whilst water content (1721%) conformed to the Codex

Standard 1996, i.e. must be o21% (Ainul Hafiza, 2005).

The microbial count of Gelam honey was o5cfu=mlmostly comprised of aerobic microorganisms. The low

microbial counts indicated that the honey was of high

quality. No growth was detected in the irradiated samples

and sterility was maintained throughout the 24 months of

storage. The findings also suggested that the irradiated

honey properly stored in cool dark place up to 2 years at

room temperature retained its antimicrobial properties

ARTICLE IN PRESS

Table 1

The physicochemical and biological properties of Gelam honey over 24 months of storage

Dose (kGy) p H Viscosity (cps) Water content (%) Zone diameter (cm) Microbial quality (cfu/ml)

0 mont hs 24 month s 0 month s 24 mon ths 0 mont hs 24 month s 0 month s 24 mon ths 0 mont hs 24 month s

0 3:57a 3:58a 512:40a 513:40a 20a 20a 1:84 0:20a 1:840:16a 4:4a 4:9a

25 3:57a 3:55a 225:40b 223:20b 20a 21a 1:90 0:10a 1:960:24a

50 3:59a 3:55a 233:60b 258:40b 17a 20a 1:94 0:08a 1:860:17a

: not detected.

Means in a column followed by same letters are not significantly different.

Table 2

Measurements of wound contraction at different days as percentage of original wound size

Days of injury Percentage of wound contraction (%) (meanSD)

Transverse measurement Longitudinal measurement

Control Gelam SSD Control Gelam SSD

7 9:50 7:36ab;w 8:58 4:60ab;w 3:43 3:04a;w 1:30 2:01a;w 5:60 3:65a;w 3:41 5:34a;w

14 24:26 17:29ab;wx 42:90 30:83b;x 32:20 7:46ab;x 6:75 5:20a;wx

33:10 33:97ab;x 18:904:70ab;wx

21 38:10 19:97a;xy 60:65 3:04a;y 55:13 23:15a;y 19:41 9:92a;x 56:10 9:50a;y 42:0029:92a;xy

28 50:21 27:35a;y 87:50 15:36b;z 69:60 16:31ab;y 46:48 27:10a;y

83:86 18:44b;y 63:0019:28ab;y

Means with different superscripts within a row were significantly different at po0:05 due to treatment.

Means with different superscripts within a column were significantly different at po0:05 due to days post injury.

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(Table 1). The inhibition zone indicating antibacterial

activity against Staphyloccocus aureus was insignificantly

different for both control and irradiated honey, before and

after storage of 24 months. These results suggested that

gamma irradiation is suitable for honey sterilization, which

is in accordance with earlier suggestion by Molan and

Allen (1996).As presented in Table 2, Gelam honey significantly

stimulated the rate of burn wound healing as the rate of

wound contraction at day 7 (wound contraction: transverse

8.58% and longitudinal 5.6%) was greater compared to

those in SSD (3.43%, 3.41%) and untreated control groups

(9.5%, 1.3%). Similarly, Suguna et al. (1993) and Aljadi

et al. (2000), reported that honey hasten wound healing by

accelerating wound contractions. Microscopic evaluation

demonstrated that there was a significant acceleration of

dermal repair in wound treated with Gelam honey as

shown in Fig. 1. Histologically, early attenuation of

inflammatory reaction and early reparative activities were

observed in the honey treated wounds (Fig.1B) compared

to the control (Fig. 1A) and the SSD group (Fig. 1C),

whereby necrotic cells (n) and inflammatory cells (inf)

could still be observed. In addition, epithelial regeneration

(e) appeared to be significant in the Gelam honey treated

wound. These macroscopic and microscopic observations

under in vivo assessment suggested that the topical

application of Gelam honey might have favorable influence

on the various phases of burn wound healing hence

accelerating the healing process.

Hydrogel with 6% of honey seemed to be the most

acceptable, easily peeled from mold, transparent and

completely cross-linked compared to 8%, 10% and 15%after irradiation at 25 kGy (Table3). This observation was

further supported by our early data on the physical

properties. The elongation of the Gelam hydrogel

(337.04%) was higher than the hydrogel alone (211.11%)

even though the tensile strength was almost similar (Gelam

hydrogel 0.02647 mPa, hydrogel alone 0.02778 mPa). The

Gelam hydrogel with pH of 4.3 was slightly acidic

compared to the hydrogel alone (pH 5.3). Dressings with

a slightly acidic pH or similar to that of healthy skin (pH of

5.5) may be most comfortable to wear (Parson et al., 2005)

and the low pH creates an unfavorable environment for

bacterial growth (Molan, 2000). Therefore the Gelam

hydrogel has the potential as a wound dressing with healing

function.

4. Conclusion

Our study shows that gamma irradiation and

storage time do not incur any significant changes in the

physicochemical properties of Gelam honey. The honey

may play a positive role in modulating wound healing

when incorporated into hydrogel matrix. Hydrogel wound

dressing containing 6% of Gelam honey will not function

merely as coverage to provide clean moist environment for

healing but also directly contribute to enhanced cell

ARTICLE IN PRESS

Fig. 1. Photomicrographs of burn wound tissues at day seven post burned

stained with H&E: (A) untreated control wound; (B) Gelam honey treated

wound; (C) SSD-cream treated wound. Note the epidermal regeneration in

Gelam honey treated wound under scab formation. Necrotic debris still

present on the superficial layer of dermis in untreated control. Bars on the

photomicrograph represent 20mm: e, epidermis; g, granulation tissue; n,

necrotic debris; inf, inflammatory cells; s, scab.

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recovery. Further studies on physical properties of the

Gelam hydrogel, diffusion rate of the honey and healing

properties are on going.

Acknowledgment

We would like to thank Mrs. Asnah Hassan of MINT

for her excellent technical assistance. Our special gratitude

to Prof. J. Rosiak of Poland, who has continuouslyinspired us to work on hydrogel for wound healing.

References

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antibacterial agent M.Sc. Thesis, National University of Malaysia,

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inflicted wounds: an animal model. Med. J. Islamic Acad. Sci. 13 (3),

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Kaufman, T., Lusthaus, S.N., Sagher, U., Wexler, M.R., 1990. Deep

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ARTICLE IN PRESS

Table 3

Physical appearance of honey hydrogel with different concentrations of

Gelam honey

Honey (%) 6 8 10 15

Cross-link Yes Yes Yes No

Peeling Yes No No No

C olo r Gol den yell ow Gol den yel low Brow n Bro wn

Odor Hon ey sme ll Hon ey smell Ho ney smel l H one y smell

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