Accepted Manuscript Title: Microstructure, rheological and wound healing properties of collagen-based gel from cuttlefish skin Author: Mourad Jridi Sana Bardaa Dorsaf Moalla Tarak Rebaii Nabil Souissi Zouheir Sahnoun Moncef Nasri PII: DOI: Reference:
S0141-8130(15)00172-5 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.03.020 BIOMAC 4954
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
4-2-2015 6-3-2015 10-3-2015
Please cite this article as: M. Jridi, S. Bardaa, D. Moalla, T. Rebaii, N. Souissi, Z. Sahnoun, M. Nasri, Microstructure, rheological and wound healing properties of collagen-based gel from cuttlefish skin, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.03.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2
Microstructure, rheological and wound healing properties of collagen-based gel from cuttlefish skin
3
Mourad Jridi*1, Sana Bardaa2, Dorsaf Moalla2, Tarak Rebaii3, Nabil Souissi4, Zouheir
5
Sahnoun2, and Moncef Nasri1
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1. Laboratoire de Génie Enzymatique et de Microbiologie, Université de Sfax, Ecole Nationale d’Ingénieurs de Sfax, B.P. 1173-3038 Sfax, Tunisie. 2. Laboratoire de Pharmacologie, Faculté de Médecine Sfax, Avenue Majida Boulila, 3028 Sfax, Tunisie. 3. Laboratoire d’Histologie Embryologie, Faculté de Médecine Sfax, Avenue Majida Boulila, 3028 Sfax, Tunisie. 4. Laboratoire de Biodiversité et de Biotechnologie Marine, Institut National des Sciences et Technologies de la Mer, Centre de Sfax. B.P. 1037-3018 Sfax, Tunisie.
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* Corresponding author. Tel.: +216 28142818; fax: +216 74 275 595.
18
Mourad Jridi: Laboratoire de Génie Enzymatique et de Microbiologie, Université de Sfax,
19
Ecole Nationale d’Ingénieurs de Sfax, B.P. 1173-3038 Sfax, Tunisia.
20
E-mail address: [email protected]
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Abstract
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Collagen-based biomaterials are of the utmost importance for tissue engineering and
38
regenerative medicine. The aims of the present investigation were to evaluate structural and
39
rheological properties of collagen-based gel obtained from cuttlefish skin, and to investigate
40
its ability to enhance wound healing. Scanning electron microscopy of resulted gel showed a
41
dense fibrillar microstructure with high interconnection network with a smaller pore size. In
42
addition, the rheological characterization of collagen gel showed an excellent reversibility,
43
when subjected to a temperature variation. Moreover, in the wound-healing study, topical
44
application of collagen based gel increased significantly the percentage of wound closure over
45
a period of 12 days, when compared to the untreated and CICAFLORA®-treated groups.
46
Wound-healing activity of collagen gel was confirmed by histopathology study. Thus,
47
cuttlefish collagen based gel might be useful as a wound healing agent.
48
Keywords: Collagen based-gel; Microstructure; Wound-healing activity.
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1. Introduction Collagen is commonly used in medical and pharmaceutical industries as carrier
58
molecules for drugs, proteins and genes [1]. Especially, microfibrous collagen sheets are used
59
as promising drug carrier for the treatment of cancer [2]. In fact, collagen qualified as an
60
excellent material for wound healing due to its biodegradable and biocompatible properties.
61
Hence, implanted collagen will be degraded through native enzymatic pathways without any
62
toxic response. In order to produce collagen biomaterials, different methods were developed
63
to extract collagen from bio-sources. Modern extraction approaches are based on three basic
64
principles of solubilisation: acidic solution [3], neutral salt solution [4] and proteolytic
65
solution [5].
an
us
cr
ip t
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The utilization of fish skin collagen and gelatin is expected to attract the interest of the
67
industry as an alternative source. This may be due to comparative unpopularity of porcine
68
skin collagen and gelatin in relation to some religious reasons. At the same time, use of
69
bovine derived collagen and gelatin are also in active discussion due to the mad cow disease,
70
bovine spongiform encephalopathy (BSE) and the risk they pose in human. In contrast, fish
71
collagen and gelatin have relatively a low risk of possessing unknown pathogens such as BSE.
72
A wound is a break in the normal tissue continuum, resulting in a variety of cellular and
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molecular sequels. The wound may be created by physical, chemical, thermal, microbial, or
74
immunological, abuse of the tissue [6]. Wound healing is a complex multifactorial process
75
involving inflammation, proliferation, remodeling, which behave in a harmonious way in
76
order to guarantee tissue reparation. The process of wound healing may be hampered by the
77
presence of free radicals, which can damage wound surrounding cells [7]. Many biomaterials
78
used as skin substitutes represented natural components existing in the wound that will be
79
activated during the healing process. Recently, gelatin forming gel has also been investigated
3 Page 3 of 26
80
successfully for its use in wound healing alone [8]. Due to its intrinsic biological properties
81
and biocompatibility, this material is advantageous. Results of previous study investigating the production of gelatin from the skin of
83
cuttlefish proved that this protein can serve as a potential biomaterial [9]. In addition,
84
advanced researches are carrying out to find novel source of collagen, with a potential tissue
85
repairing ability [10,11,12,13].
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The aims of this paper were to evaluate structural and rheological properties of collagen
87
based gel, obtained from cuttlefish skin, and to investigate its ability to enhance wound
88
healing.
89
2. Materials and Methods
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2.1. Cuttlefish skin preparation
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Cuttlefish by-products were obtained from marine processing industry “IMPEX”
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located in Sfax City, Tunisia. The samples were packed in polyethylene bags, placed in ice
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with a sample/ice ratio of approximately 1:3 (w/w). They were washed twice with water to
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eliminate the dark ink. Finally, cuttlefish outer skin was collected and then stored in sealed
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plastic bags at -20 °C until used for gelatin extraction and analysis.
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2.2. Collagen extraction
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In order to remove non-collagenous proteins, washed skins were first soaked in 0.05 M
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NaOH with a skin/solution ratio of 1/10 (w/v) for 2 h at 4 °C and the solution was changed
99
every 30 min. The alkaline treated skins were then washed with cold tap water until neutral
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pH wash water was obtained. The alkaline-treated skins cuttlefish were soaked in 0.1 M acetic
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acid with a solid/solvent ratio of 1:10 (w/v), as described by Jridi et al. [9]. The mixture was
4 Page 4 of 26
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stirred for 48 h at 4 °C. Finally, the pH of the mixture was raised to 5 using 10 M NaOH and
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stirred gently for 1 h at 4 °C. The extracted solution was filtered with a double layer of gauze and the filtrate was
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centrifuged at 10000 x g for 30 min. The filtrate was then freeze dried (Bioblock Scientific
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Christ ALPHA 1-2, IllKrich-Cedex, France) and the resulting extract was used for further
107
investigations.
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2.3. Viscoelastic properties
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Dynamic studies were performed on an AR1000 Rheometer (Physica MCR-301, Anton
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Paar, Germany) using a cone-plate geometry (cone angle 2°). Collagen solution at 6.67%
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(w/v) was prepared by dissolving in distilled water under continuous stirring at 45 °C for 20
112
min. The viscosity measurement was performed at a scan rate of 1 °C /min, frequency 1 Hz,
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oscillating applied stress of 3.0 Pa and gap 0.15 mm. During the testing, the gel was heated
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from 5 to 50 °C, cooled from 50 to 5 °C and then kept at 5 °C for 10 min. The tan δ was
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calculated from the ratio of G” and G’ obtained from frequency sweep tests and result was
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represented as a function of temperature.
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2.4. Scanning electron microscopy
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Collagen based gel microstructure was visualized using a scanning electron microscope
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(Cambridge Scan-360 microscope) at an accelerating voltage of 3.0 kV. The sample was
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frozen under liquid nitrogen. Prior to visualisation, sample was mounted on brass stub and
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sputtered with gold in order to make the sample conductive. Samples were photographed with
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an angle of 90° to the surface to allow observation of the films cross section.
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2.5. In vivo wound healing study
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2.5.1. Animals Healthy young female Wistar rats weighing between 150 and 200 g were housed in
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individual clean polyethylene cages under controlled conditions (12 h high-dark cycle at 22-
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25 °C and 60-70% relative humidity). Animals were left for 2 weeks at room conditions for
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acclimatization. They were maintained on standard pellet diet and water throughout the
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experiment. Procedures and animals comfort were controlled by the International Guidelines
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for Animal Care.
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2.5.2. Excision wound healing model
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After total anesthesia with ketamine (100 mg/kg body weight) by intramuscular
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injection, a circular area of approximately 150 mm2 wound was made on depilated thoracic
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region of rats. The wounding day was considered as day 0. The animals were housed
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individually and periodically weighted before and after the experiment. The ointment was
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topically applied every two days till the complete epithelization.
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2.5.3. Wound healing activity
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Eighteen rats in all were used in the study. They were divided into three groups
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consisting of six animals each. Group I was untreated and served as the control (just cleaning
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the wounds with a physiologic serum). Group II was treated with «CICAFLORA®» and
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served as a reference standard (positive control). Group III was treated with cuttlefish skin
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collagen gel (6.67%).
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After rinsing wounds with physiologic serum, the collagen gel and the standard drug
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(CICAFLORA®) were applied, in a fine layer covering the surface of the wound, every two
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days till the wound was completely healed. On the last day, all the rats were anaesthetized
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with ether, sacrificed and the granulation tissues were excised from the sacrificed animals. A
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part of wet tissue was preserved for hydroxyproline estimation and another one was fixed in
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formalin 10% (v/v), embedded in paraffin and processed for histological observation.
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2.6. Wound healing evaluation parameters
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2.6.1. Chromatic study
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This study consisted on attributing a chromatic code to each wound rate as the
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following: bright red = blood covering the wound; dark red coagulation of blood in the
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epidermis, red = granulation tissue and pink epithelialization step [14].
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2.6.2. Wound contraction and epithelialization time
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An excision wound margin was traced after wound creation by using transparent paper
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and area was measured [15]. Wound contraction was measured every 2 days interval, until
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complete wound healing and expressed in percentage of healed wound area. The percentage
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of wound closure was calculated using the following expression:
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d
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Wound closure (%) = (A0 – Ad)/A0 x 100
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where A0 and Ad are the initial wound area (day 0) and the area of wound on day (d),
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respectively. The period of epithelization was calculated as the number of days required for
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falling of the dead tissue without any residual raw wound.
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2.6.3. Hydroxyproline estimation
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Hydroxyproline content of tissue samples was estimated according to the method of Lee
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and Tong [16]. Wound tissues were dried in a hot air oven at 60-70 °C to a constant weight
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and then hydrolyzed in 6 N HCl for 4 h at 130 °C in sealed glass tubes. The hydrolysates were 7 Page 7 of 26
neutralized to pH 7.0 and then were subjected to Chloramine-T oxidation for 20 min. The
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reactions were terminated by addition of 0.4 M perchloric acid and the color, developed with
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the help of Ehrlich reagent at 60 °C, was measured at 650 nm using a spectrophotometer.
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Hydroxyproline concentrations were calculated from the linear standard curve and presented
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as mg/100 mg of dry tissue weight.
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2.6.4. Histological study
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Wound skin samples were fixed in 10% neutral-buffered formalin and then embedded in
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paraffin. Sections of 5 μm in thickness were stained with hematoxylin and eosin (H&E) and
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studied by a routine light microscope. The criteria that were studied in histopathological
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sections consisted of reepithelialization, cornification of the epithelium, fibroblast content,
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revascularizations, fibroblast and inflammatory cells.
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2.7. Statistical analysis
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Statistical analyses were performed with SPSS ver. 17.0, professional edition using
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ANOVA analysis. Differences were considered significant at p < 0.05. All tests were carried
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out in triplicate.
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3. Results and discussion
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3.1. Scanning electron microscopy analysis
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The potential fibrous structure of cuttlefish skin collagen-based gel was evaluated by
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scanning electron microscopy imaging. The image of cuttlefish collagen gel, presented in Fig.
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1, showed that the morphology of cuttlefish collagen gel material is a typical collagen
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structure, with its triple helical structure (Fig 1.a). Cuttlefish collagen based gel contains
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dense network of short and thicker fibers organized in sheets. In addition, the morphology of 8 Page 8 of 26
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the collagen gel shows a low porosity microstructure with visibly very small pores, compared
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to collagen from other sources such as commercial acid-soluble collagen skin [17], rat-tail
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tendons [18] and horse mackerel bones [19]. Fig.1
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Generally, hydrogel with such structure is useful for a large variety of applications,
194
especially as medical biomaterials in tissue engineering. Collagen hydrogel with small pore
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size leads to absorb a large amount of water and serve as texturing, gelling, stabilizing and
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emulsifying agent. Furthermore, the micro-structure indicates the potential of collagen gel to
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act as a scaffold to support cells, allow transport of nutrients and metabolic wastes and thus
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promote tissue development [20].
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The results obtained are in agreement with those of Wang et al. [21] who reported that
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collagen gel extracted from Amur sturgeon skin, showed a dense fibrillar microstructure with
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high interconnection network and small pore size. In addition, Annabi et al. [22] suggested
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that the lower pore size of hydrogel was suitable for in vivo investigations.
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3.2. Viscoelastic properties
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The result of phase angle evolution during heating and cooling between 5 °C and 50 °C
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of collagen solution (6.67% w/v) are presented in Fig. 2. Melting and gelling temperatures of
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collagen gel were about 21.6 °C and 16.5 °C, respectively. During the cooling of collagen
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solution, the triple helix interactions improve the gel formation and the gelling point presents
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the point from which the viscosity began to increase sharply with decreasing temperature.
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During the collagen gel formation, Djabourov et al. [23] reported that there is a lag phase
210
where the primary aggregates (dimers and trimers of collagen molecules) are nucleated. Then,
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microfibrillar aggregation starts with the lateral aggregation of sub-units until equilibrium
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reaching. These results suggested the excellent reversibility of collagen-based gel, when subjected
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to a temperature variation, which reflects its potential use as a gelling skin care product.
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Moreover, the ability of collagen gel to return to its initial viscosity represents an important
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property for a topical cosmetic product, to remain on the skin and not flow off after
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application [24].
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Fig.2
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3.3. Wound healing studies
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3.3.1. General characteristic of animals
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The body weight evolutions of the different groups before and after treatments are
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presented in Table 1. The comparison between body weights averages of the different groups
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of rats did not reveal any statistical significant difference before and after treatment.
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Therefore, the different batches are consistent and comparable. Moreover, a slight increase in
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weight of the rats of different groups after treatment was noted suggesting their normal
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growth.
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Table 1
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3.3.2. Wound closure
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The size of the wound area and the percentage of contraction rates were monitored
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during the 12-days experimental period to assess the wound healing potential of cuttlefish
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collagen-based gel. Results of all groups in the excision wound model are shown in Table 2
10 Page 10 of 26
and Fig. 3. As shown in Fig. 3, the use of collagen hydrogel as ointment exhibited higher
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wound healing ability (Table 2).
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Table 2
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As expected, the untreated group showed the lower wound cloture than the treated one
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(pc; p < 0.05).
Different letters in the same column indicate significant differences
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Groups
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Table 3: Hydroxyproline content in wounds at the end of the experiment.
Group 1 Group 2 Group 3
Hydroxyproline (mg/100 mg of tissue) 20.97±0.1 c 24.68±0.8 b 32.95±0.35 a
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Data are expressed as mean±SEM (n=6). Group 1 was untreated and served as the negative
451
control (physiologic serum); Group 2 and 3 were treated with «CICAFLORA®» and collagen
452
gel, respectively.
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(a>b>c; p < 0.05).
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Different letters in the same column indicate significant differences
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Figure captions:
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Fig. 1. Scanning electron microscopy micrograph of collagen gel from cuttlefish skin taken at 1000.
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Fig. 2. Evolution of the phase angle during cooling from 50 °C to 5 °C and heating from 5 °C to 50 °C (B) of collagen gel from cuttlefish skin.
490 491 492
Fig.3. Representative photographs of visual appearance of wound excised (1.5 cm×1 cm) on rat on day 0, 7 and 13 of untreated group and served as control (1); CICAFLORA® treated group (2) and collagen gel treated group (3).
493 494 495 496 497
Fig. 4. Representative photomicrographs of the effect of physiological serum (1); «CICAFLORA®» (2) and collagen gel (3) on wound healing. Treatment of rats revealed epidermal and dermal architecture of wounds on the 13th day. HE-stained histological sections are 5 mm thick and photomicrographs are taken at 100 (A) and 400 (B) magnifications. e: epidermis ; d: dermis; : inflammatory cells ; : vessels.
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Fig. 1. 400nm
1µm
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530 531 532 533 534 535 536
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Fig. 2.
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100 90
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Phase angle
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80 70 60 50
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40 30
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10 0 5
10
15
20
25 30 Temperature (°C)
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570 571 572 573 574
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d
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an
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Heating
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Cooling
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Fig. 3.
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Group 1
Group 2
Group 3
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Day 0
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Day 6
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Day 12
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597 598 599 600 601 602 603
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Fig. 4.
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A
Group 1
B
e
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617
Group 2
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Group 3
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Collagen gel showed a dense fibrillar microstructure and smaller pore size
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Ability of collagen to enhance wound healing
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Collagen gel from cuttlefish increased wound contraction
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Histological analysis revealed that collagen enhanced wound epithelialization
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S0141-8130(15)00172-5 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.03.020 BIOMAC 4954
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
4-2-2015 6-3-2015 10-3-2015
Please cite this article as: M. Jridi, S. Bardaa, D. Moalla, T. Rebaii, N. Souissi, Z. Sahnoun, M. Nasri, Microstructure, rheological and wound healing properties of collagen-based gel from cuttlefish skin, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.03.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2
Microstructure, rheological and wound healing properties of collagen-based gel from cuttlefish skin
3
Mourad Jridi*1, Sana Bardaa2, Dorsaf Moalla2, Tarak Rebaii3, Nabil Souissi4, Zouheir
5
Sahnoun2, and Moncef Nasri1
ip t
4
6
1. Laboratoire de Génie Enzymatique et de Microbiologie, Université de Sfax, Ecole Nationale d’Ingénieurs de Sfax, B.P. 1173-3038 Sfax, Tunisie. 2. Laboratoire de Pharmacologie, Faculté de Médecine Sfax, Avenue Majida Boulila, 3028 Sfax, Tunisie. 3. Laboratoire d’Histologie Embryologie, Faculté de Médecine Sfax, Avenue Majida Boulila, 3028 Sfax, Tunisie. 4. Laboratoire de Biodiversité et de Biotechnologie Marine, Institut National des Sciences et Technologies de la Mer, Centre de Sfax. B.P. 1037-3018 Sfax, Tunisie.
17
* Corresponding author. Tel.: +216 28142818; fax: +216 74 275 595.
18
Mourad Jridi: Laboratoire de Génie Enzymatique et de Microbiologie, Université de Sfax,
19
Ecole Nationale d’Ingénieurs de Sfax, B.P. 1173-3038 Sfax, Tunisia.
20
E-mail address: [email protected]
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1 Page 1 of 26
Abstract
37
Collagen-based biomaterials are of the utmost importance for tissue engineering and
38
regenerative medicine. The aims of the present investigation were to evaluate structural and
39
rheological properties of collagen-based gel obtained from cuttlefish skin, and to investigate
40
its ability to enhance wound healing. Scanning electron microscopy of resulted gel showed a
41
dense fibrillar microstructure with high interconnection network with a smaller pore size. In
42
addition, the rheological characterization of collagen gel showed an excellent reversibility,
43
when subjected to a temperature variation. Moreover, in the wound-healing study, topical
44
application of collagen based gel increased significantly the percentage of wound closure over
45
a period of 12 days, when compared to the untreated and CICAFLORA®-treated groups.
46
Wound-healing activity of collagen gel was confirmed by histopathology study. Thus,
47
cuttlefish collagen based gel might be useful as a wound healing agent.
48
Keywords: Collagen based-gel; Microstructure; Wound-healing activity.
50
51
52
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M
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49
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54
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2 Page 2 of 26
56
1. Introduction Collagen is commonly used in medical and pharmaceutical industries as carrier
58
molecules for drugs, proteins and genes [1]. Especially, microfibrous collagen sheets are used
59
as promising drug carrier for the treatment of cancer [2]. In fact, collagen qualified as an
60
excellent material for wound healing due to its biodegradable and biocompatible properties.
61
Hence, implanted collagen will be degraded through native enzymatic pathways without any
62
toxic response. In order to produce collagen biomaterials, different methods were developed
63
to extract collagen from bio-sources. Modern extraction approaches are based on three basic
64
principles of solubilisation: acidic solution [3], neutral salt solution [4] and proteolytic
65
solution [5].
an
us
cr
ip t
57
The utilization of fish skin collagen and gelatin is expected to attract the interest of the
67
industry as an alternative source. This may be due to comparative unpopularity of porcine
68
skin collagen and gelatin in relation to some religious reasons. At the same time, use of
69
bovine derived collagen and gelatin are also in active discussion due to the mad cow disease,
70
bovine spongiform encephalopathy (BSE) and the risk they pose in human. In contrast, fish
71
collagen and gelatin have relatively a low risk of possessing unknown pathogens such as BSE.
72
A wound is a break in the normal tissue continuum, resulting in a variety of cellular and
Ac ce p
te
d
M
66
73
molecular sequels. The wound may be created by physical, chemical, thermal, microbial, or
74
immunological, abuse of the tissue [6]. Wound healing is a complex multifactorial process
75
involving inflammation, proliferation, remodeling, which behave in a harmonious way in
76
order to guarantee tissue reparation. The process of wound healing may be hampered by the
77
presence of free radicals, which can damage wound surrounding cells [7]. Many biomaterials
78
used as skin substitutes represented natural components existing in the wound that will be
79
activated during the healing process. Recently, gelatin forming gel has also been investigated
3 Page 3 of 26
80
successfully for its use in wound healing alone [8]. Due to its intrinsic biological properties
81
and biocompatibility, this material is advantageous. Results of previous study investigating the production of gelatin from the skin of
83
cuttlefish proved that this protein can serve as a potential biomaterial [9]. In addition,
84
advanced researches are carrying out to find novel source of collagen, with a potential tissue
85
repairing ability [10,11,12,13].
cr
ip t
82
The aims of this paper were to evaluate structural and rheological properties of collagen
87
based gel, obtained from cuttlefish skin, and to investigate its ability to enhance wound
88
healing.
89
2. Materials and Methods
90
2.1. Cuttlefish skin preparation
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Cuttlefish by-products were obtained from marine processing industry “IMPEX”
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located in Sfax City, Tunisia. The samples were packed in polyethylene bags, placed in ice
93
with a sample/ice ratio of approximately 1:3 (w/w). They were washed twice with water to
94
eliminate the dark ink. Finally, cuttlefish outer skin was collected and then stored in sealed
95
plastic bags at -20 °C until used for gelatin extraction and analysis.
96
2.2. Collagen extraction
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In order to remove non-collagenous proteins, washed skins were first soaked in 0.05 M
98
NaOH with a skin/solution ratio of 1/10 (w/v) for 2 h at 4 °C and the solution was changed
99
every 30 min. The alkaline treated skins were then washed with cold tap water until neutral
100
pH wash water was obtained. The alkaline-treated skins cuttlefish were soaked in 0.1 M acetic
101
acid with a solid/solvent ratio of 1:10 (w/v), as described by Jridi et al. [9]. The mixture was
4 Page 4 of 26
102
stirred for 48 h at 4 °C. Finally, the pH of the mixture was raised to 5 using 10 M NaOH and
103
stirred gently for 1 h at 4 °C. The extracted solution was filtered with a double layer of gauze and the filtrate was
105
centrifuged at 10000 x g for 30 min. The filtrate was then freeze dried (Bioblock Scientific
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Christ ALPHA 1-2, IllKrich-Cedex, France) and the resulting extract was used for further
107
investigations.
108
2.3. Viscoelastic properties
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Dynamic studies were performed on an AR1000 Rheometer (Physica MCR-301, Anton
110
Paar, Germany) using a cone-plate geometry (cone angle 2°). Collagen solution at 6.67%
111
(w/v) was prepared by dissolving in distilled water under continuous stirring at 45 °C for 20
112
min. The viscosity measurement was performed at a scan rate of 1 °C /min, frequency 1 Hz,
113
oscillating applied stress of 3.0 Pa and gap 0.15 mm. During the testing, the gel was heated
114
from 5 to 50 °C, cooled from 50 to 5 °C and then kept at 5 °C for 10 min. The tan δ was
115
calculated from the ratio of G” and G’ obtained from frequency sweep tests and result was
116
represented as a function of temperature.
117
2.4. Scanning electron microscopy
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Collagen based gel microstructure was visualized using a scanning electron microscope
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(Cambridge Scan-360 microscope) at an accelerating voltage of 3.0 kV. The sample was
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frozen under liquid nitrogen. Prior to visualisation, sample was mounted on brass stub and
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sputtered with gold in order to make the sample conductive. Samples were photographed with
122
an angle of 90° to the surface to allow observation of the films cross section.
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2.5. In vivo wound healing study
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2.5.1. Animals Healthy young female Wistar rats weighing between 150 and 200 g were housed in
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individual clean polyethylene cages under controlled conditions (12 h high-dark cycle at 22-
127
25 °C and 60-70% relative humidity). Animals were left for 2 weeks at room conditions for
128
acclimatization. They were maintained on standard pellet diet and water throughout the
129
experiment. Procedures and animals comfort were controlled by the International Guidelines
130
for Animal Care.
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2.5.2. Excision wound healing model
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After total anesthesia with ketamine (100 mg/kg body weight) by intramuscular
133
injection, a circular area of approximately 150 mm2 wound was made on depilated thoracic
134
region of rats. The wounding day was considered as day 0. The animals were housed
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individually and periodically weighted before and after the experiment. The ointment was
136
topically applied every two days till the complete epithelization.
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2.5.3. Wound healing activity
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Eighteen rats in all were used in the study. They were divided into three groups
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consisting of six animals each. Group I was untreated and served as the control (just cleaning
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the wounds with a physiologic serum). Group II was treated with «CICAFLORA®» and
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served as a reference standard (positive control). Group III was treated with cuttlefish skin
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collagen gel (6.67%).
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After rinsing wounds with physiologic serum, the collagen gel and the standard drug
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(CICAFLORA®) were applied, in a fine layer covering the surface of the wound, every two
145
days till the wound was completely healed. On the last day, all the rats were anaesthetized
6 Page 6 of 26
with ether, sacrificed and the granulation tissues were excised from the sacrificed animals. A
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part of wet tissue was preserved for hydroxyproline estimation and another one was fixed in
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formalin 10% (v/v), embedded in paraffin and processed for histological observation.
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2.6. Wound healing evaluation parameters
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2.6.1. Chromatic study
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This study consisted on attributing a chromatic code to each wound rate as the
152
following: bright red = blood covering the wound; dark red coagulation of blood in the
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epidermis, red = granulation tissue and pink epithelialization step [14].
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2.6.2. Wound contraction and epithelialization time
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An excision wound margin was traced after wound creation by using transparent paper
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and area was measured [15]. Wound contraction was measured every 2 days interval, until
157
complete wound healing and expressed in percentage of healed wound area. The percentage
158
of wound closure was calculated using the following expression:
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d
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Wound closure (%) = (A0 – Ad)/A0 x 100
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where A0 and Ad are the initial wound area (day 0) and the area of wound on day (d),
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respectively. The period of epithelization was calculated as the number of days required for
162
falling of the dead tissue without any residual raw wound.
163
2.6.3. Hydroxyproline estimation
164
Hydroxyproline content of tissue samples was estimated according to the method of Lee
165
and Tong [16]. Wound tissues were dried in a hot air oven at 60-70 °C to a constant weight
166
and then hydrolyzed in 6 N HCl for 4 h at 130 °C in sealed glass tubes. The hydrolysates were 7 Page 7 of 26
neutralized to pH 7.0 and then were subjected to Chloramine-T oxidation for 20 min. The
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reactions were terminated by addition of 0.4 M perchloric acid and the color, developed with
169
the help of Ehrlich reagent at 60 °C, was measured at 650 nm using a spectrophotometer.
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Hydroxyproline concentrations were calculated from the linear standard curve and presented
171
as mg/100 mg of dry tissue weight.
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2.6.4. Histological study
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Wound skin samples were fixed in 10% neutral-buffered formalin and then embedded in
174
paraffin. Sections of 5 μm in thickness were stained with hematoxylin and eosin (H&E) and
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studied by a routine light microscope. The criteria that were studied in histopathological
176
sections consisted of reepithelialization, cornification of the epithelium, fibroblast content,
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revascularizations, fibroblast and inflammatory cells.
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2.7. Statistical analysis
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Statistical analyses were performed with SPSS ver. 17.0, professional edition using
180
ANOVA analysis. Differences were considered significant at p < 0.05. All tests were carried
181
out in triplicate.
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3. Results and discussion
183
3.1. Scanning electron microscopy analysis
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The potential fibrous structure of cuttlefish skin collagen-based gel was evaluated by
185
scanning electron microscopy imaging. The image of cuttlefish collagen gel, presented in Fig.
186
1, showed that the morphology of cuttlefish collagen gel material is a typical collagen
187
structure, with its triple helical structure (Fig 1.a). Cuttlefish collagen based gel contains
188
dense network of short and thicker fibers organized in sheets. In addition, the morphology of 8 Page 8 of 26
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the collagen gel shows a low porosity microstructure with visibly very small pores, compared
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to collagen from other sources such as commercial acid-soluble collagen skin [17], rat-tail
191
tendons [18] and horse mackerel bones [19]. Fig.1
193
Generally, hydrogel with such structure is useful for a large variety of applications,
194
especially as medical biomaterials in tissue engineering. Collagen hydrogel with small pore
195
size leads to absorb a large amount of water and serve as texturing, gelling, stabilizing and
196
emulsifying agent. Furthermore, the micro-structure indicates the potential of collagen gel to
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act as a scaffold to support cells, allow transport of nutrients and metabolic wastes and thus
198
promote tissue development [20].
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The results obtained are in agreement with those of Wang et al. [21] who reported that
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collagen gel extracted from Amur sturgeon skin, showed a dense fibrillar microstructure with
201
high interconnection network and small pore size. In addition, Annabi et al. [22] suggested
202
that the lower pore size of hydrogel was suitable for in vivo investigations.
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3.2. Viscoelastic properties
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The result of phase angle evolution during heating and cooling between 5 °C and 50 °C
205
of collagen solution (6.67% w/v) are presented in Fig. 2. Melting and gelling temperatures of
206
collagen gel were about 21.6 °C and 16.5 °C, respectively. During the cooling of collagen
207
solution, the triple helix interactions improve the gel formation and the gelling point presents
208
the point from which the viscosity began to increase sharply with decreasing temperature.
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During the collagen gel formation, Djabourov et al. [23] reported that there is a lag phase
210
where the primary aggregates (dimers and trimers of collagen molecules) are nucleated. Then,
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211
microfibrillar aggregation starts with the lateral aggregation of sub-units until equilibrium
212
reaching. These results suggested the excellent reversibility of collagen-based gel, when subjected
214
to a temperature variation, which reflects its potential use as a gelling skin care product.
215
Moreover, the ability of collagen gel to return to its initial viscosity represents an important
216
property for a topical cosmetic product, to remain on the skin and not flow off after
217
application [24].
218
Fig.2
219
3.3. Wound healing studies
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3.3.1. General characteristic of animals
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The body weight evolutions of the different groups before and after treatments are
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presented in Table 1. The comparison between body weights averages of the different groups
223
of rats did not reveal any statistical significant difference before and after treatment.
224
Therefore, the different batches are consistent and comparable. Moreover, a slight increase in
225
weight of the rats of different groups after treatment was noted suggesting their normal
226
growth.
227
Table 1
228
3.3.2. Wound closure
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The size of the wound area and the percentage of contraction rates were monitored
230
during the 12-days experimental period to assess the wound healing potential of cuttlefish
231
collagen-based gel. Results of all groups in the excision wound model are shown in Table 2
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and Fig. 3. As shown in Fig. 3, the use of collagen hydrogel as ointment exhibited higher
233
wound healing ability (Table 2).
234
Table 2
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As expected, the untreated group showed the lower wound cloture than the treated one
236
(pc; p < 0.05).
Different letters in the same column indicate significant differences
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a,b,c
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Groups
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434 435 436 437 438 439
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Table 3: Hydroxyproline content in wounds at the end of the experiment.
Group 1 Group 2 Group 3
Hydroxyproline (mg/100 mg of tissue) 20.97±0.1 c 24.68±0.8 b 32.95±0.35 a
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445
450
Data are expressed as mean±SEM (n=6). Group 1 was untreated and served as the negative
451
control (physiologic serum); Group 2 and 3 were treated with «CICAFLORA®» and collagen
452
gel, respectively.
453
(a>b>c; p < 0.05).
cr
Different letters in the same column indicate significant differences
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a,b,c
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d te Ac ce p
457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484
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Figure captions:
486 487
Fig. 1. Scanning electron microscopy micrograph of collagen gel from cuttlefish skin taken at 1000.
488 489
Fig. 2. Evolution of the phase angle during cooling from 50 °C to 5 °C and heating from 5 °C to 50 °C (B) of collagen gel from cuttlefish skin.
490 491 492
Fig.3. Representative photographs of visual appearance of wound excised (1.5 cm×1 cm) on rat on day 0, 7 and 13 of untreated group and served as control (1); CICAFLORA® treated group (2) and collagen gel treated group (3).
493 494 495 496 497
Fig. 4. Representative photomicrographs of the effect of physiological serum (1); «CICAFLORA®» (2) and collagen gel (3) on wound healing. Treatment of rats revealed epidermal and dermal architecture of wounds on the 13th day. HE-stained histological sections are 5 mm thick and photomicrographs are taken at 100 (A) and 400 (B) magnifications. e: epidermis ; d: dermis; : inflammatory cells ; : vessels.
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an
498 499
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504 505 506 507 508
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d
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509 510 511 512
21 Page 21 of 26
513 514
Fig. 1. 400nm
1µm
515 516
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517 518 40nm
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519 520
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523 524
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530 531 532 533 534 535 536
te
529
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d
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537 538 539 540 541
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Fig. 2.
545
100 90
547
551 553
Phase angle
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80 70 60 50
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40 30
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10 0 5
10
15
20
25 30 Temperature (°C)
560
562
564
570 571 572 573 574
50
te Ac ce p
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45
d
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568
40
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567
35
an
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566
Heating
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Cooling
20
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575 576 577 578 579
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580
Fig. 3.
581 582
Group 1
Group 2
Group 3
583
Day 0
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584 585
Day 6
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586 587
Day 12
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588 589
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590 591
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592 593
597 598 599 600 601 602 603
te
596
Ac ce p
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d
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604 605 606 607 608
24 Page 24 of 26
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Fig. 4.
610 611
A
Group 1
B
e
612 613
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d
614
617
Group 2
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618
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619 620
622
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621
Group 3
626 627 628 629 630 631 632
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624
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623
633 634 635 636
25 Page 25 of 26
Collagen gel showed a dense fibrillar microstructure and smaller pore size
637
Ability of collagen to enhance wound healing
638
Collagen gel from cuttlefish increased wound contraction
639
Histological analysis revealed that collagen enhanced wound epithelialization
ip t
636
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26 Page 26 of 26
