Sea cucumber (Stichopus hermanii) based hydrogel to treat burn

wounds in rats

Rozaini Mohd Zohdi,1 Zuki Abu Bakar Zakaria,2 Norimah Yusof,3

Noordin Mohamed Mustapha,4 Muhammad Nazrul Hakim Abdullah5
1
Department of Life Sciences, Faculty of Pharmacy, Universiti Teknologi MARA, 42300 Puncak Alam, Selangor, Malaysia
2
Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang,
Selangor, Malaysia
3
Division of Agrotechnology and Biosciences, Malaysian Nuclear Agency, Bangi, 43000 Kajang, Selangor, Malaysia
4
Department of Veterinary Microbiology and Pathology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400
Serdang, Selangor, Malaysia
5
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang,
Selangor, Malaysia

Received 3 June 2010; revised 21 November 2010; accepted 25 November 2010

Published online 18 April 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.31828

Abstract: Malaysian sea cucumber was incorporated into
hydrogel formulation by using electron beam irradiation tech-
nique and was introduced as novel cross-linked Gamat
Hydrogel dressing. This study investigated whether Gamat
Hydrogel enhanced repair of deep partial skin thickness burn
wound in rats and its possible mechanism. Wounds were
treated with either Gamat Hydrogel, control hydrogel,
OpSiteV film dressing or left untreated. Skin samples were
R
taken at 7, 14, 21, and 28 days post burn for histological and
molecular evaluations. Gamat Hydrogel markedly enhanced
healing time. The level of proinflammatory cytokines; IL-1a,
IL-1b, and IL-6, were significantly reduced in Gamat Hydrogel
treated wounds compared with other groups as assessed by
reverse transcription-polymerase chain reaction (RT-PCR).
In summary, our results showed that Gamat Hydrogel
promoted burn wound repair via a complex mechanism
involving stimulation of tissue regeneration and regulation of
pro-inflammatory cytokines. The resultant wound healing
effects were attributed to the synergistic effect of the
hydrogel matrix and incorporated sea cucumber. V C 2011 Wiley

wound contraction and improved histological reorganization Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 98B:
of the regenerating tissue. Furthermore, the dressing modu- 30–37, 2011.
lated the inflammatory responses, stimulated the activation
and proliferation of fibroblasts, and enhanced rapid produc- Key Words: burn, cytokines, hydrogel, wound healing,
tion of collagen fiber network with a consequently shorter wound dressing

INTRODUCTION inflammatory,5 antibacterial7 and antioxidant activities2

Marine natural products have recently attracted the atten-
tion of researchers worldwide due to their potential phar-
macological activities.1 Sea cucumber, or holothurian, is one
of the potential marine organisms that exert beneficial
effects on human health.2 This benthic organism is well
known in many parts of the globe for its medicinal benefits
and culinary delicacies especially in Asian countries.3 There
are several studies reporting on the biological activities of
sea cucumber including anticancer, antifungal, antioxidant,
and anti-inflammatory effects.2,4,5 In Malaysia, sea cucumber
or locally known as ‘‘gamat’’ has long been used as a tradi-
tional remedy for healing of various internal and external
wounds.6 Its extracts and active metabolites have been char-
acterized to induce tissue repair and wound healing.6
According to Ridzwan,7 one particular species called Sticho-
pus hermanii or known locally as ‘‘Gamat Emas’’ has been
rated for its medicinal and healing properties. Several stud-
ies have shown that sea cucumber possesses strong anti-
which are imperative to accelerate the process of wound
healing. The compounds isolated from sea cucumbers have
shown to act as serine protease inhibitors which play a
major role in the direct inactivation of the inflammatory
mediators.8 The presence of elevated levels of serine prote-
ase in nonhealing wounds has been associated with the deg-
radation of important growth factors and fibronectin neces-
sary for wound healing.9 Meanwhile, the antioxidant
activities of sea cucumber are attributed to its phenolic con-
stituent particularly flavonoids which act against oxidative
reactions, notably the ones initiated by peroxyl radicals
resulting in a decreased risk of oxidative damage to tissue.2
The potential clinical application of this marine orga-
nism has lead to an extended interest in the use of it with
hydrogel wound dressing. Hydrogel dressings were origi-
nally invented to function merely as burn wound covering.10
As well as providing a moist milieu for wound healing,
hydrogels are nonparticulate, nontoxic, and nonadherent.11

Correspondence to: Z. Abu B. Zakaria; e-mail: zuki@vet.upm.edu.my

30 V
C 2011 WILEY PERIODICALS, INC.

Currently, there are numerous studies describing the use of
hydrogels in controlled release formulations and to immobi-
lize biological active species in hydrogel matrices for wound
healing applications.12,13 Many biomaterials have been used
to prepare active wound dressings, including chitin,14 chito-
san,15 alginate,16 and collagen.17
In this study, the composite matrix derived from sea
cucumber and hydrogel dressing were introduced as novel
cross-linked Gamat Hydrogel dressings having reservoir
capacities for the delivery of ‘‘gamat,’’ which were prepared
by irradiation technique. The development of a sustained
release system would substantially increase the utility of
incorporated sea cucumber in tissue repair and effectively
interact with the burn wounds, thus facilitate healing. Gamat
Hydrogel dressing is expected to have synergic beneficial
aspects of both sources. Therefore, in order to study the ef-
ficacy of Gamat Hydrogel in improving the outcome of burn
wound, a rat model with deep partial thickness burn was
utilized. We sought to determine the morphological and mo-
lecular changes of the burn wound tissue healing after
treatment with Gamat Hydrogel. This study clearly demon-
strated the effects of Gamat Hydrogel on the various cellular
elements and proinflammatory cytokines involved in the
healing process and its promoting effect may be due to the
stimulation of tissue regeneration and modulation of inflam-
matory response.
MATERIALS AND METHODS
Sea cucumber
Water-soluble extracts of local sea cucumber species, Sticho-
pus hermanii, was provided by International Islamic Univer-
sity Malaysia (IIUM). The extract was spray dried (Niro
Model 2000A, Denmark) and the powder was packed,
sealed and irradiated with 25 kGy gamma irradiation using
radioactive source Cobalt 60 (Model JS 8900) with the dose
rate of 2 kGy per hour for sterilization purposes at MINTec-
Sinagama, Malaysian Nuclear Agency. Dosimetry was per-
formed with ceric/cerous sulphate solution using potentio-
metric method of analysis.
Gamat Hydrogel dressing
Polyvinyl pyrolidone (PVP) with molecular weight of 650
kDa (Kollidon 90) and Polyethylene glycol (PEG) with mo-
lecular weight of 400 kDa were obtained from BASF, Lud-
wigshafen, Germany. Technical grade agar was supplied by
Oxoid. The mixture of 15% (w/v) PVP (Kollidon 90), 1%
(w/v) Protein Free Agar solution, and 1% (v/v) PEG were
added with 5% (w/v) gamat powder. A control hydrogel
was prepared similarly without further addition of gamat
powder. The mixture was poured into plastic molds (5 cm
in diameter; 3–4 mm in thickness) left to set at room tem-
perature before being covered with polyethylene sheet and
individually packed. The gels were cross-linked and steri-
lized by electron beam at 25 kGy at Alutron Irradiation Fa-
cility, Malaysian Nuclear Agency (Model EPS-3000, conveyer
speed of 4.4 m/min, beam current of 10 mA and energy of
3 MeV).
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JUL 2011 VOL 98B, ISSUE 1
ORIGINAL RESEARCH REPORT
Animals and experimental designs
A total of 96 male Sprague-Dawley rats (weight 200–300
g) were used in this study and randomly divided into four
experimental groups of six rats each. The experimental
protocol was approved by the Animal Care and Use Com-
mittee (ACUC) of Faculty of Veterinary Medicine, Universiti
Putra Malaysia (UPM) (Reference No: 08R36/July 08–Jun
09). Animals were acclimatized to the laboratory condi-
tions for one week prior to onset of experiment. The rats
were individually caged and given commercial pellet and
water ad libitum throughout the study. Animal death
occurred at 7, 14, 21, and 28 days post burn by an over-
dose of halothane inhalation and skin samples were either
taken for histopathological examination or snap frozen in
liquid nitrogen and stored at
80 C until RNA isolation
was performed.
Skin preparation and burn lesion
Rats were anesthetized with an intramascular (IM) injec-
tion of ketamine (50 mg/kg) and xylazine (5 mg/kg).
Under anesthesia, the back and flank of both sides of the
body were shaved. Following this procedure, rats were
returned to their cages for 24 h to allow any edema
caused by the shaving procedure to recede. A method
described by Kaufman et al.18 was used with modification.
Cylindrical aluminum templates (2.5 cm diameter  3 cm
length, a handle measuring 24 cm, and total weight 400 g)
were heated in a water bath at a constant temperature of
85 C for 3 h prior to inflicting burn areas on the skin of
the rats. Five templates were heated simultaneously and
used alternately and then were returned to the water bath
to ensure maintenance of the desired temperature of the
template surface.
Approximately 5 min elapsed between each use of a
template. Rats were again anesthetized with an IM injec-
tion of ketamine (50 mg/kg) and xylazine (5 mg/kg). The
anesthetized rat was positioned on sternal recumbency,
restrained and stretched on a metal stage. The location of
the burn was marked between the last ribs and the hori-
zontal line of the sacroiliac joints. Deep partial thickness
burn was inflicted on the dorsal part of the rat between
the last thoracic vertebra and the first sacrum by placing
the heated and moistened template at right angles perpen-
dicular to the dorsum of the rat on the premarked location
for 5 s using an analogue stopwatch. Minimal and constant
pressure was applied to ensure a perfect contact between
the template surface and the skin. The shaved skin was
smooth to ensure sufficient contact and uniform pressure
over the entire lesion.
Treatment protocol
All wounds in the treatment groups were dressed with
control hydrogel or Gamat Hydrogel followed by OpSiteV
R
film dressing (Smith and Nephew, Hull, England) as sec-
ondary dressing. The dressings were held in place with
sterile gauze by suturing the gauze on rat’s skin with
coated vicryl 4/0 (Ethicon, Johnson&Johnson, Belgium) in a
simple interrupted fashion. The covering of gauze gave
31
TABLE I. Sequences of Primers and the Size of Product Produced Based on Previously Published Work19
Primer Direction Sequences (50 to 30 ) Product size (bp)

b-Actin Forward GTG GGC CGC TCT AGG CAC CAA 540
Reverse CTT TAG CAC GCA CTG TAG TTT CTC
IL-1a Forward ATG GCC AAA GTT CCT GAC TTG TTT 625
Reverse C TGG TCG GGC AC A ACG ACT TCC
IL-1b Forward ATG GCA ACT GTT CCT GAA CTC ACC T 563
Reverse TT TCC TTT CTT AG A TAT GGACAG GAC
IL-6 Forward ATG AAG TTC CTC TCT GCA AGA GAC T 638
Reverse CTC TAG ATG AGC CGT TTG GAT CAC

mechanical protection of the dressings while OpsiteV R film analyzer (Analysis LS Research) attached to a light micro-
dressing was used to prevent hydrogels from being scope (Olympus BX51, Japan). The wounds were evaluated
absorbed by the gauze thus permitting full utilization of for the extent of re-epithelialization, granulation tissue for-
Gamat Hydrogel healing properties on the wound. Sterile mation, architecture, cellularity, and inflammation.
technique was utilized when changing the dressings every
seven days to minimize introduction of pathogens to the
RNA extraction and reverse transcription polymerase
wound site.
chain reaction
The hydrogels were easily applied to the wound with
Total RNA was extracted from wound tissues using RNeasyV R
proper adherence and can be peeled off easily from the
Fibrous Tissue Mini Kit (QIAGEN, Hilden, Germany) accord-
wound when the dressing needs changing without irritating
ing to the manufacturer’s instructions. Single-stranded cDNA
the wound surface. Change of dressings were done every
was synthesized from total RNA using MMLV reverse tran-
seven days based on a pilot study done on the release pro-
scriptase (Promega, Madison, WI) and oligo (dT)15 as
file of gamat (data not shown). The release of gamat from
primer. Polymerase chain reaction (PCR) was performed
hydrogel matrix displayed a sustained release profile up to
using cDNA product with the sequence-specific primer pairs
seven days. OpSiteV R film dressing, a commercially available
listed in Table I.19 The housekeeping b-actin gene was used
wound dressing preparation was selected as the positive
as reference. The thermal cycling condition set consisted of
control group for comparison. The use of OpSiteVR as a posi-
denaturation for 30 s at 95 C, annealing for 30 s at 60 C
tive control was imperative in this study to ascertain that
and elongation for 45 s at 45 C with a final extension for
healing effects of the tested Gamat Hydrogel dressing were
7 min at 72 C. A total of 30 PCR amplification cycles
due to the composition of the hydrogels and not as a result
are performed using a Mastercycler thermal cycler machine
of secondary dressings. The negative control group received
(Eppendorf, Germany). The PCR products were electro-
identical burn and environmental exposure but no further
phoresed through a 1% agarose gel and visualized by
treatment were given.
0.5 lg/mL ethidium bromide staining and UV transillumi-
nation using GelDocTM 2000 (Bio-Rad, USA). The band
Wound contraction determination
intensities were measured and quantitated by image analy-
All wounds were digitally photographed in the presence of
sis by comparing PCR products with b-actin.
a standard reference ruler. The wounds area was measured
immediately by placing a transparent tracing paper over the
wound and tracing it out. The tracing paper was placed on
a 1 mm2 graph sheet, and traced out. The squares were
counted and the area recorded. The wound size measure-
ments taken at the time of burn infliction and at the time of
healing evaluation were used to calculate the percentage of
wound contraction, using the following equation: % Wound
contraction ¼ (A0
At)/A0  100, where A0 is the original
wound area, At is the area of the wound at predetermined
time points (7, 14, 21, and 28 post burn day). The wound
area was assessed by the same blinded observer.

Histopathological analysis FIGURE 1. Effect of Gamat Hydrogel on wound contraction rate in

Skin samples were fixed in 10% formalin solution and em-
bedded in paraffin. Tissue sections of 4–5 lm thickness
were cut, stained with hematoxylin and eosin (H&E), and
examined under light microscope. Digital photomicrographs
were captured at representative locations using an image
burn wound model in rats. Deep partial thickness burn wounds were
V
R
dressed with Gamat Hydrogel, control hydrogel, OpSite film dress-
ing or left untreated. Analysis of the contraction rate was performed
on days 7, 14, 21, and 28 post burn. Values are expressed as mean 6
S.E.M. (n ¼ 6). [Color figure can be viewed in the online issue, which
is available at wileyonlinelibrary.com.]

32 ZOHDI ET AL. Stichopus hermanii BASED HYDROGEL TO TREAT BURN WOUNDS IN RATS
 ORIGINAL RESEARCH REPORT

FIGURE 2. a–d: Representative photographs of wounds from animals treated with (A) Gamat Hydrogel (B) control hydrogel (C) OpSite (D) con-
trol group on days 7, 14, 21, and 28 post burn respectively. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]

Statistical analysis and time. The analyses were performed using the SPSSV
R

Data were expressed as means and standard error (S.E). Statistical package (SPSS, Version 15.0, Chicago, IL).
Collected data were analyzed using the two-way blocked p values of less than 0.05 were considered to be
time effects ANOVA to determine the effects of treatment significant.

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JUL 2011 VOL 98B, ISSUE 1 33
 FIGURE 2. (Continued) [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

RESULTS nificant differences (p > 0.05) observed in the measurement

Effect of Gamat Hydrogel on wound contraction and
appearance
Topical application of Gamat Hydrogel accelerated the rate
of wound healing as demonstrated by increased rate of
wound contraction (Figure 1) and better gross appearances
(Figure 2). At days 7 and 14 post burn, there were no sig-
of wound contraction between all experimental groups
(Figure 1). However, the rate of wound contractions meas-
ured at days 21 and 28, revealed that Gamat Hydrogel sig-
nificantly increased wound contraction rates (p < 0.05)
compared with other treatments at later stage of repair. In
particular, at day 21 post burn, wounds treated with Gamat

34 ZOHDI ET AL. Stichopus hermanii BASED HYDROGEL TO TREAT BURN WOUNDS IN RATS
 ORIGINAL RESEARCH REPORT

FIGURE 3. Representative micrographs of histological sections at seven days post burn stained with H&E. Note the advanced epidermal regener-
ation in wound treated with (A) Gamat Hydrogel with increased blood vessel formation. Marked inflammatory response with necrosis were still
persists in (B) control hydrogel (C) OpSiteV
R (D) control group. (E, epidermis: g, granulation tissue; arrow, blood vessels; arrowhead, necrosis;

40 mag). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Hydrogel showed 82.31% contraction compared to control
hydrogel (58.94%), OpSiteV R (60.06%) and untreated wounds
(56.72%). At day 28 post burn, the contraction increased to
94.43% (Gamat Hydrogel), 72.75% (control hydrogel),
80.54% (OpSiteV R ), and 70.86% (untreated wounds). There
were prominent visible differences in wound appearances by
day 28 post burn (Figure 2). The superficial aspect of the
wound treated with Gamat Hydrogel was at the same level
with the surface of the surrounding skin, while in the control
hydrogel groups this superficial aspect was raised slightly
above it. Small crusts were still observed in the OpSiteV R Effect of Gamat Hydrogel on proinflammatory cytokines
treated group and untreated control group.
Effect of Gamat Hydrogel on histological changes of
burn wound tissue
Microscopical observation at day seven revealed that the
wound surfaces of all experimental groups were covered by
necrotic tissue which consisted of a mixture of tissue debris
and inflammatory cells (Figure 3). However, compared with
was thickened at its cut edges as a result of mitotic activity
of basal cells. Signs of healing were noted when neo-vascu-
larization and granulation tissue developed in the lower
margin of the wounds in all groups. In the dermal layer of
Gamat Hydrogel treated animals, there were increased blood
vessel-like structures with prominent vasodilatations and
less inflammation. However, residual necrosis with marked
infiltration of inflammatory cells can still be observed in the
other experimental groups.
in burn wound tissue
The levels of IL-1a, IL-1b, and IL-6 mRNA in wounds
treated with Gamat Hydrogel and Honey Hydrogel were
maintained at statistically significant lower levels (p < 0.05)
throughout the healing process as compared to other exper-
imental groups (Figure 4). The mRNA levels for these proin-
flammatory cytokines in wounds treated with control hydro-
gel, OpSiteV
R film dressing, and untreated control group

the other groups, burn wounds treated with Gamat Hydro-
gel showed evidence of advanced healing whereby early re-
epithelialization were observed with proliferating and
migrating keratinocytes projected toward the center of the
wound field immediately above the newly formed granula-
tion tissue and below the scab (Figure 3). The epidermis
remained higher and generally decreased only after 14 days
post burn.
DISCUSSION
The results presented in this study illustrated that burn
wound healing was best obtained when treated with Gamat

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JUL 2011 VOL 98B, ISSUE 1 35

FIGURE 4. Modulation of proinflammatory cytokines by Gamat Hydro-
gel dressing: mRNA levels of (A) IL-1a (B) IL-1b (C) IL-6 at various
time points after burn injury, as determined by semiquantitative RT-
PCR. [Color figure can be viewed in the online issue, which is avail-
able at wileyonlinelibrary.com.]
Hydrogel. Topical application of Gamat Hydrogel signifi-
cantly stimulated the rate of wound healing as demon-
strated by the increase in the rate of wound contraction
and better cosmetic appearances. Wound contraction facili-
tates wound closure as a result of the centripetal movement
of the adjacent skin forming the wound margin which
appears to be mediated by myofibroblasts, a contractile
fibroblast phenotype presence in the granulation tissues.20
The accelerated onset of wound contraction in Gamat
Hydrogel treated wounds, judged by qualitative comparison
of photographic records as well as percentage of wound
contraction, suggested either that their granulation tissue
contained a more effective or greater number of contractile
myofibroblasts than other groups.
36 ZOHDI ET AL.
Histological findings of this study also indicated
advanced re-epithelialization in Gamat Hydrogel treated
wounds. Fredalina et al.21 reported that Malaysian sea
cucumber contains high composition of fatty acids. Fatty
acid was shown to affect cell metabolism and division, mod-
ulate expression of certain gene in dermal fibroblasts and
endothelial cells, stimulate early angiogenesis and promote
wound healing in vivo.22 Furthermore, the fatty acids in the
sea cucumber might act as an energy source for cellular di-
vision and epidermal regeneration.21,23
This study also demonstrated that Gamat Hydrogel sig-
nificantly modulated wound inflammatory response with
the reduction of proinflammatory cytokines. One of the
goals of wound therapy is to reduce excess inflammatory
responses as well as to prevent wound from becoming
infected.24 Early reparative activity of wound healing
observed in this study could be partly attributed to the
early attenuation of inflammatory changes. The anti-inflam-
matory and antioxidants activities of sea cucumber could be
ascribed as major components in controlling the inflamma-
tory reaction generated by burn injuries. A decreased in the
proinflammatory cytokines may result with fewer neutro-
phils and macrophages recruited to the wound and less
cytokines being released in the wound to provide a permis-
sive milieu for wound healing process to proceed.25 Exces-
sive production of proinflammatory cytokines ultimately can
lead to the development of further tissue injury.25
CONCLUSIONS
In summary, this study provides firm evidence to support
that Gamat Hydrogel represent a feasible and productive
approach to support dermal wound healing by regulation of
multiple events that are central to the process of healing.
Gamat Hydogel enhanced wound contraction and improved
histological reorganization of the regenerating tissue. It
modulated the inflammatory responses, by cytokine modula-
tion with a consequently shorter healing time. It is likely
that accelerated wound closure in our burn model is attrib-
uted in part due to sea cucumber being release from the
hydrogel matrix that act in synergy with the moist environ-
ment provided by the hydrogel system. Its use may provide
a new and effective alternative treatment for wound healing
in clinical practice.
ACKNOWLEDGMENTS
The authors wish to express their appreciation to Mrs. Asnah
Hassan for her conscientious technical assistance.
REFERENCES
1. Mayer AMS, Glaser KB, Cuevas C, Jacobs RS, Kem W, Little RD,
McIntosh JM, Newman DJ, Potts BC, Shuster DE. The odyssey of
marine pharmaceuticals: A current pipeline perspective. Trends
Pharmacol Sci 2010;31:255–265.
2. Mamelona J, Pelletier E, Girard-Lalancette K, Leagult J, Karboune
S, Kermasha S. Quantification of phenolic contents and antioxi-
dant capacity of Atlantic sea cucumber, Cucumaria frondosa.
Food Chem 2007;104:1040–1047.
3. Zhong Y, Khan A, Shahidi F. Compositional characteristics and
antioxidant properties of fresh and processed sea cucumber
(Cucumaria frondosa). J AgricFood Chem 2007;55:1188–1192.
Stichopus hermanii BASED HYDROGEL TO TREAT BURN WOUNDS IN RATS

4. Dang NH, Thanh NV, Kiem PV, Huong LM, Minh CV, Kim YH.
Two new triterpene glycosides from the Vietnamese sea cucum-
ber Holothuria scabra. Arch Pharm Res 2007;30:1387–1391.
5. Himaya SWA, Ryu B, Qian ZJ, Kim SK. Sea cucumber. Stichopus
japonicus ethyl acetate fraction modulates the lipopolysaccharide
induced iNOS and COX-2 via MAPK signaling pathway in murine
macrophages. Environ Toxicol Pharmacol 2010;30:68–75.
6. Ridzwan BH. 2007. Sea Cucumber: The Malaysian Heritage, 1st ed.
Kuala Lumpur: International Islamic University Malaysia; 176 p.
7. Farouk AEA, Ghouse FAH, Ridzwan BH. New bacterial species iso-
lated from Malaysian sea cucumbers with optimized secreted
antibacterial activity. Am J Biochem Biotechnol 2007;3:60–65.
8. Collin PD. Inhibition of angiogenesis by sea cucumber fractions.
United State Patent. US005985330. Coastside Bio-Resources, Sto-
nington, Maine, 1999.
9. Schultz GS, Mast BA. Molecular analysis of the environments of
healing and chronic wounds: Cytokines, proteases and growth
factors. Primary Intent 1999;7:7–14.
10. Rosiak JM, Ulanski P, Pajewski A, Yoshii F, Makuuchi K. Radiation
formation of hydrogels for biomedical purposes: Some remarks
and comments. Radiat Phys Chem 1995;46:161–168.
11. Hoffman AS. Hydrogels for biomedical applications. Adv Drug
Deliv Rev 2003;43:3–12.
12. Costache MC, Qu H, Ducheyne P, Devore DI. Polymer-xerogel
composites for controlled release wound dressings. Biomaterials
2010;31:6336–6343.
13. Balakrishnan B, Mohanty M, Fernandez AC, Mohanan PV, Jayak-
rishnan A. Evaluation of the effect of incorporation of dibutyryl
cyclic adenosine monophosphate in an in situ-forming hydrogel
wound dressing based on oxidized alginate and gelatine. Bioma-
terials 2006;27:1355–1361.
14. Jayakumar R, Prabaharan M, Nair SV, Tamura H. Novel chitin and
chitosan nanofibers in biomedical applications. Biotechnol Adv
2010;28:142–150.
15. Yang JM, Su WY, Leu TL, Yang MC. Evaluation of chitosan/PVA
blended hydrogel membranes. J Membr Sci 2004;236:39–51.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JUL 2011 VOL 98B, ISSUE 1
ORIGINAL RESEARCH REPORT
16. Cho WJ, Heang OS, Ho LJ. Alginate film as novel post-surgical
tissue adhesion barrier. J Biomater Sci Polym Ed 2010;21:
701–713.
17. Purna SK, Babu M. Collagen based dressing—A review. Burns
2000;26:56–62.
18. Kaufman T, Lusthaus SN, Sagher U, Wexler MR. Deep partial skin
thickness burns: A reproducible animal model to study burn
wound healing. Burns 1990;16:13–16.
19. Bryan D, Walker KB, Ferguson M, Thorpe R. Cytokine gene
expression in a murine wound healing model. Cytokine 2005;31:
429–438.
20. Dyson M, Steven Y, Pendle L, Webster DF, Lang SM. Comparison
of the effects of moist and dry conditions on dermal repair. J
Invest Dermatol 1988;91:434–439.
21. Fredalina BD, Ridzwan BH, Zainal-Abidin AA, Kaswandi MA, Zai-
ton H, Zali I, Kittakoop P, Jais AM. Fatty acid composition in local
sea cucumber, Stichopus chloronotus, for wound healing. Gen-
eral Pharmacology 1999;33:337–340.
22. Shingel KI, Faure MP, Azoulay L, Roberge C, Deckelbaum RJ.
Solid emulsion gel as a vehicle for delivery of polyunsaturated
fatty acids: implications for tissue repair, dermal angiogenesis
and wound healing. J Tissue Eng Regen Med 2008;2:383–393.
23. Kusakari Y, Ogawa E, Owada Y, Kitanaka N, Watanabe H, Kimura
M, Tagami H, Kondo H, Aiba S, Okuyamo R. Decreased kerati-
nocyte motility in skin wound on mice lacking the epidermal
fatty \acid binding protein gene. Mol Cell Biochem 2006;284:
183–188.
24. Cho MK, Sung MA, Kin DS, Park HG, Jew SS, Kim SG. 2-Oxo-
3,23-isopropylidene-asiatate (AS2006A), a wound-healing-asiatate
derivative, exerts anti-inflammatory effect by apoptosis of macro-
phages. Int Immunopharmacol 2003;3:1429–1437.
25. Zhang M, Caragine T, Wang H, Cohen PS, Botchkina G, Soda K,
Bianchi M, Ulrich P, Cerami A, Sherry B, Tracey KJ. Spermine
inhibits proinflammatory cytokines synthesis in human mononu-
clear cells: A counterregulatory mechanism that restrains the
immune response. J Exp Med 1997;185:1759–1768.
37