Accepted Manuscript Title: Bio-inspired aloe vera sponges for biomedical applications Author: S.S. Silva M.B. Oliveira J.F. Mano R.L. Reis PII: DOI: Reference:
S0144-8617(14)00512-8 http://dx.doi.org/doi:10.1016/j.carbpol.2014.05.042 CARP 8909
To appear in: Received date: Revised date: Accepted date:
27-11-2013 6-5-2014 14-5-2014
Please cite this article as: Silva, S. S., Oliveira, M. B., Mano, J. F., & Reis, R. L.,Bioinspired aloe vera sponges for biomedical applications, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.05.042 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.
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Bio-inspired aloe vera sponges for biomedical applications
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S S Silva a,b, M B Oliveira a,b, JF Mano a,b, RL Reis a,b
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a
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University of Minho, Headquarters of the European Institute of Excellence on
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Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas,
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Guimarães, Portugal
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b
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Portugal
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3B’s Research Group - Biomaterials, Biodegradables and Biomimetics,
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ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães,
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Corresponding-Author:
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3B´s
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Department of Polymer Engineering, University of Minho, Headquarters of the
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European Institute of
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Medicine – AvePark, Zona Industrial da Gandra – Caldas das Taipas – 4806-
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909 Guimarães- Portugal – Fax: +351 253510909; ICVS/3B´s- PT Government
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Associate Laboratory, Braga/Guimarães, Portugal
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[email protected] te
Simone S. Silva
Research Group- Biomaterials,
Biodegradables and Biomimetics,
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Excellence on Tissue Engineering and Regenerative
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Highlights
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- Sponges showed heterogeneous pore formation and interconnected pores
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- Swelling behavior of the sponges was dependent on the pH 1 Page 1 of 26
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- The sponges provided the sustained release of BSA-FTIC over 3 weeks
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Abstract
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Chemical composition and biological properties of Aloe vera (AV), a tropical
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plant, explain its potential use for cosmetic, nutritional and biomedical
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applications.
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nutrients and polysaccharides. This work proposes using AV gel complex
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structure and chemical composition, associated with freeze-drying, to produce
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sponges. To increase the structures stability in aqueous media, a thin coating of
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gellan gum (GG), was applied onto AV gel. AV-based sponges showed a
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heterogeneous porous formation, interconnected pores and good porosity (72-
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77%). The coating with a GG layer onto AV influenced the stability, swelling
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behavior and mechanical properties of the resulting sponges. Moreover,
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sponges provided the sustained release of BSA-FTIC, used as a model protein,
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over 3 weeks. Also, in vitro cell culture studies evidenced that sponges are not
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cytotoxic for a mouse fibroblast-like cell line. Therefore, developed AV-based
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sponges have potential use in biomedical applications.
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AV gel present in AV leaves is rich in several compounds,
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Keywords: aloe vera gel; sponges; gellan gum; regenerative medicine
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1. Introduction
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Worldwide, there is a need for the search for new therapies and drugs due to
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the incidence of diseases such as cancer and diabetes. As such, the use of
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plants as a natural resource of bioactive phytochemical compounds may be
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useful for developing new formulations and strategies to enhance life quality. It
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is recognized that some plant extracts contain compounds and polysaccharides,
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which display anticancer, antioxidant, antibacterial and antiviral activities. Aloe
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vera (AV), a tropical plant belonging to the liliaceae family, is known to be the
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oldest medicinal plant in Nature (Gage, 1996). AV plant per se is an unique
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resource of the natural compounds, nutrients and polysaccharides with several
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bioactivities (Boudreau & Beland, 2006; Hamman, 2008; Reynolds & Dweck,
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1999). This chemical diversity has encouraged its utilization in cosmetic
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formulations, food supplements and medical devices (Cole & Heard, 2007;
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Hamman, 2008; Rodriguez Rodriguez, Darias Martin & Diaz Romero, 2010;
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Silva, Caridade, Mano & Reis, 2013; Silva et al., 2013). By its turn, the
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properties of AV gel, contained in the inner parts of the fresh AV leaves
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(Hamman, 2008) have anti-inflammatory, antioxidant, antiviral and antibacterial
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action. These properties are associated to its complex composition. AV gel has
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also been useful to design and create functional films, microspheres and
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sponges. Most of these biomaterials have been prepared by combining AV gel
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with bacterial cellulose, chitosan, alginate, and gelatin (Chen, Wang & Weng,
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2010; Pereira, Tojeira, Vaz, Mendes & Bartolo, 2011; Saibuatong &
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Phisalaphong, 2010; Silva, Caridade, Mano & Reis, 2013; Silva et al., 2013).
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For instance, Silva et al (Silva et al., 2013) reported that a small portion of AV
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gel into chitosan provided blended membranes with adequate roughness,
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degradation rate, wettability and mechanical properties to be used as wound
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dressing materials. Due to the wide use of AV, a high number of possibilities are
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still open to investigation. Therefore, in the present work, the exploitation of AV
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gel, in the development of sponges using freeze-drying technique was
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evaluated. It is well known that freeze drying is one of the most popular
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technique for producing three dimensional (3D) porous structures based on
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natural or synthetic polymers (Reys et al., 2011; Silva et al., 2008). This
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technique is based on the formation of ice crystals that induce porosity through
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ice sublimation and desorption (Mano et al., 2007); that could be useful for
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increasing the shelf-life of Aloe components (Krokida, Pappa & Agalioti, 2011).
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To increase the stability of the structures in aqueous media, a thin coating layer
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of gellan gum (GG), was applied onto AV gel. GG is a well-known water soluble,
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biocompatible polysaccharide that could be used as an adhesive to apply
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seasoning to the surfaces of foods (Oliveira et al., 2010b). In addition, GG has
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been proposed for tissue engineering applications (Oliveira et al., 2010a;
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Oliveira et al., 2010b; Silva-Correia et al., 2013). We hypothesized that the
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developed AV-based sponges could represent a natural therapeutic strategy,
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where the natural biological activity of AV may act in different stages of the
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tissue regeneration. To evaluate the potential use of AV-based sponges as a
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carrier for therapeutic molecules, the sponges were labelled with a model
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protein, namely bovine serum albumin labelled with fluorescein isothiocyanate
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(BSA-FTIC). Furthermore, the studies presented in this article focus on
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understanding the morphological features, mechanical properties and cellular
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response of the AV-based sponges.
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2. Materials and methods
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2.1. Materials
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Fresh whole aloe vera (Aloe barbadensis Miller) leaves, obtained from a
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Portuguese botanic shop, were used as the raw material in all experiments. The
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studied leaves, between 30 and 40 cm of length, corresponded to 4-year old
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plants. Low-acyl gellan gum (GG, GelzanTM) was purchased from Sigma-Aldrich
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Co., USA. All other reagents were obtained from Sigma Aldrich Co. (USA),
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unless otherwise indicated.
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2.2. Methods
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2.2.1. Preparation of the aloe vera-based sponges
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All whole aloe vera leaves were washed with distilled water to remove dirt from
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the surface. The skin was separated from the parenchyma using a scalpel-
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shaped knife. The AV filets were extensively washed with distilled water to
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remove the exudates from their surfaces. AV fillets were cut in small cubes
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(diameter 0.8 cm). Gellan gum (GG) powder was mixed with distilled water
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under constant stirring at room temperature to obtain a concentration of 1%
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(w/v). The solution was heated to 90C during 20-30 minutes. Afterwards, the
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temperature was progressively decreased to 50C (Oliveira et al., 2010a).
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Further, the molded AV gels were immersed into GG solution during 30
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seconds, in order to create a thin layer on the material. After that, AVG matrices
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were rinsed with phosphate buffer solution (PBS). Then, both matrices (AV and
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AVG) were stabilized at 65C for 15-20 minutes, and at 4C for 5 minutes,
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followed by freezing
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sponges were also prepared without any GG coating. The identification of the
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AV-based sponges was AVG and AV, for those prepared with and without GG
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coating, respectively.
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2.2.2. Bovine serum albumin (BSA) incorporation
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The AV-based sponges were incubated into bovine serum albumin labeled with
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fluorescein isothiocyanate (BSA-FTIC) at a concentration of 2 mg/ml overnight
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at 4C to avoid the gel oxidation. Afterwards, the samples were frozen at -80 C
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and submitted to freeze-drying for 3 days.
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3. Characterization
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3.1. Scanning electron microscopy (SEM)
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Samples of the freeze-dried hydrogels were observed by a Leica Cambridge
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S360 Scanning Electron Microscope. The materials were fixed by mutual
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conductive adhesive tape on aluminum stubs and covered with gold palladium
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using a sputter coater.
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at -80C overnight and freeze-drying for 3 days. AV
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3.2. Micro-computed tomography (µ-CT)
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The microstructure of the sponges was evaluated using a high-resolution μ-CT
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Skyscan 1072 scanner (Skyscan,Kontich, Belgium) with a resolution pixel size
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of 6.59 μm and integration time of 1.9 s. The x-ray source was set at 32 keV
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and 143 μA. Approximately 500 projections were acquired over a rotation range
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of 180◦with a rotation step of 0.45◦. Data sets were reconstructed using
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standardized cone-beam reconstruction software (NRecon v1.4.3, SkyScan).
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The output format for each sample was 500 serial 1024 ×1024 bitmap images.
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A representative data set of 500 slices was segmented into binary images with
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a dynamic threshold of 57–255 (grey values). The same representative volume
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of interest (VOI) was analysed for all the samples. These data sets were used
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for morphometric analysis (CT Analyser, v1.5.1.5, SkyScan) and to build the 3D
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models (ANT 3D creator, v2.4, SkyScan).
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3.3. Swelling behavior
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Swelling tests were performed by immersing the sponges in different buffer
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solutions (pH 5, 7.4 and 10) up to 360 minutes. The swollen sample weights
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were measured after removing excess surface water by gently tapping the
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surface with filter paper. The percentage of water uptake was calculated using
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equation 1, where ws is the weight of the swollen sample and wd is the weight of
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the dry sample. Each experiment was repeated three times, and the average
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value was considered to be the water uptake value.
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Equation 1
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3.4. Stability
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The AVG matrices (6.5 mm diameter, 4.5 mm height) were immersed in
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phosphate buffer solution (PBS) at 37 C for up to 30 days. Samples were
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collected at different time points, washed gently with distilled water, dried at
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40C and weighed. Percentage loss in weight at respective time points was
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calculated.
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3.5. In vitro BSA release
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BSA-FTIC-loaded sponges were weighted and suspended in 3 ml of PBS. The
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samples were immersed in a thermostatic water bath at 37C and 60 rpm.
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Aliquots of 300 µl were withdrawn at predetermined time intervals, and the
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same volume of fresh buffer was added to the suspension. The samples were
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analyzed by UV-spectroscopy at 280 nm and 495 nm to eliminate the
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contribution of the FITC absorption at 280 nm in a microplate reader (Synergy
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HT, Bio-Tek Instruments, USA). The results presented are the average of three
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measurements. Calculations on the amount of drug released took into account
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the replacement of aliquots with fresh medium. The concentration of BSA-FTIC
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was calculated using a standard curve (concentrations ranging from 0.0 to 1
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mg/mL), relating the amount of BSA-FTIC with the intensity of light absorbance.
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3.6. Dynamical mechanical analysis (DMA)
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The viscoelastic measurements of the AV sponges before and after GG coating
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were performed using a TRITEC2000B DMA from Triton Technology (UK),
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equipped with a compression mode. The matrices were always analyzed in
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hydrated conditions through immersion of the samples in a Teflon reservoir
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containing PBS, used during all analysis period. After equilibration at 37C, the
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DMA spectra were obtained during a frequency scan between 0.1 and 10 Hz.
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The experiments were performed under constant strain amplitude (30 µm). The
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average value of four tests was reported for each sample.
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3.7. Cytotoxicity tests
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Before testing, the samples were sterilized under an ethylene oxide
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atmosphere. To assess the possible cytotoxicity of the samples, extracts
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(leachables) of both sponges (AV and AVG) were evaluated following MEM
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extraction tests (72 h), according to ISO-10993-5 guidelines (ISO/10993, 1992).
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In MEM extraction tests, both materials and the control (latex as positive
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control) were extracted by incubation in culture medium (the same used for cell
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culture) for 24 h at 37C and 60 rpm (in thermostatic bath) (Gomes, Reis,
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Cunha, Blitterswiijk & de Bruijn, 2001). After the defined period of incubation,
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the resulting solution was removed and filtrated, obtaining the extraction fluid.
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For these tests a mouse fibroblast-like cell line, L929 cells, (European
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Collection of Cell Cultures-ECACC, UK) at passage 16 was used. L929 cells
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were cultured in basic medium: Dulbecco’s Modified Eagle’s Medium (DMEM,
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Sigma-Aldrich, USA) with phenol red and supplemented with 10% foetal bovine
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serum, FBS (Gibco, UK) and 1% antibiotic/antimycotic (A/B, Gibco, UK) solution
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and incubated at 37◦C in an atmosphere containing 5% CO2 until achieving
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90% confluence. Then, a cell suspension was prepared with a concentration of
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1×10
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h. The metabolic activity of L929 cells in contact with the extracts of the
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sponges was assessed using an AlamarBlue assay. For this assay, an Alamar
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Blue solution (AlamarBlueCell Viability Assay Protocol, Life Technologies)
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was prepared, added to the medium and incubated for 4 h at 37C. The optical
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cells ml–1 and seeded on extracts in 24-well plates and incubated for 72
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density (OD) was read at 570 and 600 nm in a multiwell microplate reader
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(Synergy HT, BioTek Instruments). The L929 cell viability (%) was determined
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for each extract and compared to tissue culture polystyrene (TCPS) wells, used
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as a negative control of cell death. Latex extracts were considered as a positive
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control of cellular death.
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3.8 Statistical analysis
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All quantitative experiments were run in triplicate and results were expressed
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as mean ± standard deviation for n=3. Statistical analysis of the data was
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conducted using two-way ANOVA with Bonferroni´s posthoc comparisons by
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means of using the GraphPadPrism version 5.0 for Windows (GraphPad
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Software, San Diego, http://www.graphpad.com). Differences between the
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groups with p0.05 were considered to be statistically significant.
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4. Results and discussion
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4.1. Morphological features
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For construction of AV–based sponges (AV and AVG), the AV gel was molded
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in cylindrical shapes (0.8 cm diameter, Figure 1a). Even though the matrices
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showed good mechanical stability, the stabilization of AV gel should be
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considered before use. Then, the stabilization of AV-based structures (AV and
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AVG) was performed by applying high temperature (65C) for a short period of
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time (15-20 min). This process will preserve the biological activity of the AV gel.
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GG is a linear anionic polysaccharide that forms thermoreversible gels, and it
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has demonstrated non-cytotoxicity in different studies (Oliveira et al., 2010b). In
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our work, a thin layer of GG solution (1 wt%) was applied on molded AV gel.
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Since the gelation of GG solutions is influenced by the presence of cations
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(Oliveira et al., 2010a), the matrices were rinsed with PBS which allowed the
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formation of a three dimensional network on the AV gels, increasing their
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stability and mechanical properties.
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Both developed AV an AVG sponges showed a heterogeneous porous
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formation distributed in the matrix (Figure 1b-1d). Moreover, the freeze-drying
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process allowed retaining properly the biological activity of the gel and
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preserving the cell wall polysaccharides network of AV. It is also increased the
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shelf-life of the produced sponges (Krokida, Pappa & Agalioti, 2011).
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Figure 1. Photographs of the AV gel (A), AVG sponge (B) and representative
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SEM image of the surface of the AV (C) and AVG sponges (D).
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Micro-CT analysis was used to obtain quantitative information of the 3D
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architecture of the aloe-based sponges. The 3D micro-CT images, obtained
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from digital geometry processing from a series of two-dimensional x-ray images,
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are illustrated in Figure 2. From the 3D AV image we observed a close pore
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structure as compared to AVG. The values of interconnectivity (Table 1)
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indicated that all structures have interconnected pores, which may help
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homogeneous cell distribution and transferring of nutrients effectively, as well as
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to facilitate cell growth within the 3D porous structures (Madihally & Matthew,
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1999). The pore size distribution of the samples (Figures 3) presented values
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ranging between 50 and 300 µm. The AV sponges have also a wide pore size
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distribution, while AVG sponges have a narrow pore size distribution. In one
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hand, AV gel is a nature made sponge, possessing high values of porosity (76.9
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± 4.6 %) and interconnectivity (79.3 ± 10.1%) that could be adequate for
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regenerative medicine approaches. On the other hand, the morphological
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features of AV sponges were affected by the GG coating, where the AVG
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sponges have values of porosity and interconnectivity lower than AV (Table 1).
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Figure 2. 3D image reconstructions of the sponges (AV and AVG) obtained by
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µ-CT seen on a lateral and top views.
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Table 1: Values of porosity and interconnectivity for AV and AVG sponges.
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Sample
AV
AVG
Porosity (%)
76.9 ± 4.6
72.3 ± 9.4
Interconnectivity (%)
79.3 ± 10.1
74.6 ± 4.9
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Figure 3. Porosity distribution of AV and AVG.
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Swelling and stability behavior
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Depending on the site of implantation, the biomaterials are subjected to different
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pH environments, which could affect their degradation, mechanical and swelling
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properties. To determine how the pH affects the swelling behavior, AVG
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sponges were immersed in different buffer solutions at pH 5, 7.4 and 10. AVG
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samples immersed at pH 10 and pH 7.4 showed an appreciable swelling
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compared to the ones immersed at pH 5. Statistical differences (p< 0.05) were 14 Page 14 of 26
observed for pH 5 in relation to pH 10 and pH 7.4 along the swelling time. The
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AV gel is rich in hydroscopic polysaccharides such as acemannan (Hamman,
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2008), which together with the acid resistance of the coating layer of GG at low
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pH (Picone & Cunha, 2011), could explain the greater swelling in high pH and
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its decreasing at pH 5. GG is able to form gels through an ionotropic gelation
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mechanism (Oliveira et al., 2010b); where an increase in pH leads to the
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formation of a reduced number of junction regions (Picone & Cunha, 2011).
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Other studies on aloe vera/alginate films demonstrated that an increase in AV
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content implied an increase in the water absorption ability at pH 7.4 (Pereira,
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Tojeira, Vaz, Mendes & Bartolo, 2011), as result of the hydrophilicity of AV.
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Figure 4: Swelling profiles of AVG sponges in different buffer pH´s (A) and
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weight loss (B) in function of immersion time on PBS at 37C. Data represent
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the mean ± standard deviation (p< 0.05, two-way ANOVA).
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In tissue engineering approaches, the 3D structures are expected to degrade
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during tissue regeneration. Thus, it is important to characterize the structural
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stability of AV-based sponges to understand their potential for application in 15 Page 15 of 26
tissue engineering strategies. The structural stability of the AV and AVG
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sponges was investigated by weight loss after their incubation in PBS up to 30
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days in order to simulate physiological conditions. During the assay, the AV
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sponges gradually dissolved, leading to the formation of gels and loss of their
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integrity after a few days (data not shown). Compared with AV, AVG sponges
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showed a delayed disintegration and increased structural stability. This result
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could be attributed to the presence of the GG coating on AV. In the presence of
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salt ions, contained in PBS, the number of junction zones in GG is enhanced
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(Picone & Cunha, 2011). This leads to an increase in the gel rigidity, increasing
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its stability to aqueous medium. Furthermore, the AVG sponges showed a
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weight loss of AVG was about 30 wt% along the degradation period (Figure 4B)
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that can be related with the soluble compounds.
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Mechanical properties and in vitro release studies
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Almost all biological tissues show native viscoelastic properties (Sasaki, 2012).
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Those are conferred by constituents such as cells, extracellular matrix and
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proteins. These properties are of uttermost importance for the correct function
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of the tissue that must be able to stand the physiological load, as well as be
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capable of dissipating mechanical energy during cyclic solicitation. Upon the
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implantation of a biomaterial, it is important to make sure that the structure is
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compatible also with the tissue mechanical properties, so that it allows for
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correct biomaterial integration. Moreover, it was shown that the mechanical
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properties of the biomaterials influence the proliferation and even differentiation
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phenomena of cells (Engler, Sen, Sweeney & Discher, 2006). In order to
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investigate possible applications of the AVG sponges in the regenerative
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medicine field, we assessed the viscoelastic properties of the structures by
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dynamic mechanical analysis (DMA). Mechanical analysis of the AV sponge
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was not conducted as the sponge disintegrate along the tests. The AVG
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sponges developed in this work present a storage modulus (E’) – a measure of
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the material’s stiffness - varying from average values of 5 kPa to 10 kPa (Figure
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5A), over a biological relevant frequency range (0.1 Hz to 10 Hz, respectively).
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These E’ values are in the range of elastic modulus measured for several soft
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tissues present in the human body. For example, skeletal muscle was reported
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to have an elastic modulus of 7 kPa (Chino, Akagi, Dohi, Fukashiro &
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Takahashi, 2012) and subcutaneous fat tissue a modulus varied from 0.12 kPa
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to 30 kPa, according to the applied measuring method (Geerligs, van Breemen,
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Peters, Ackermans, Baaijens & Oomens, 2011; Hendriks, Brokken, Van
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Eemeren, Oomens, Baaijens & Horsten, 2003). Skin tissue in wet state was
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evaluated in 10 kPa–100 MPa (Derler & Gerhardt, 2012). Although in this case
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the E’ reported value is higher than the one of the biomaterial proposed herein,
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it was previously shown that the formation of extracellular matrix by cells
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seeded in biomaterials increase their stiffness (Popa, Caridade, Mano, Reis &
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Gomes, 2013; Silva-Correia et al., 2013), and the final tissue may show
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properties similar to the native tissue (Silva-Correia et al., 2013). Soft biological
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tissues play an important role in the mechanical integrity of the body. Indeed,
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these tissues transfer loads between bones to ligaments, or between muscles
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and bones to the tendons (Silva, Mano & Reis, 2010). They have an important
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viscous component in their behavior. The loss factor or damping (herein
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presented as tan ) is a measure of the viscoelasticity of materials, as it
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correlates the elastic and viscous components. Regarding the loss factor of the
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AVG sponges, the value 0.5 indicates that the material showed a marked
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viscoelastic behavior (Figure 5A). Moreover, this value is also close to the loss
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factors of native tissues, such as skin, which showed in in vivo testing a
358
damping of 0.6-0.8 (Sandford, Chen, Hunter, Hillebrand & Jones, 2013).
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Moreover, brain tissue showed loss factor values of 0.4-0.6 (Fallenstein, Hulce
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& Melvin, 1969). Considering the results obtained in the analysis of the
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viscoelastic properties of this new biomaterial, we conclude that it may have
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potential in tissue engineering of soft tissues, as its properties resemble those
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from the native tissues.
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Figure 5: Evaluation of the viscoelastic properties of AVG sponges by DMA.
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Frequency scans were performed in the range 0.1-10 Hz under wet conditions
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at 37C (A), and BSA-FTIC release profile of the samples (▲) AV and (*) AVG
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(B). Data represent the mean ± standard deviation (p< 0.05, two-way ANOVA.
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In vitro release studies
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The characteristics of AV-based sponges could be interesting in the
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construction of a drug delivery system. In this sense, BSA-FTIC was used as a
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model to analyze their profile release from the AV-based sponges. The release
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profile of the samples was evaluated in PBS for a time period up to 3 weeks.
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We hypothesized that the presence of GG coating onto AV (AVG) will act as a
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barrier to BSA release. In Figure 5B the release profile of BSA from AV and
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AVG is shown. It seems that AVG sponges have a faster release than AV,
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suggesting that the protein was dispersed in the sponge. Although the
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encapsulated protein on AV could be easily released in the aqueous
380
environment, the findings suggested that the interactions between BSA-FTIC
381
and AV were stronger than AVG, controlling the protein release. Typically,
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conventional drug delivery systems provide a sharp increase in the
383
concentration of the drug, followed by a rapid decrease in subsequent time
384
periods, until new administration. In controlled delivery systems designed for
385
long term administration the concentration should increase slowly in the
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beginning of the process, followed by a steady release. The findings obtained
387
for both AV and AVG suggest that a controlled drug release system for
388
therapeutic molecules could be developed from them.
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Cytotoxicity assays
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Considering the potential use of these materials in regenerative medicine, a
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cytotoxicity assessment of both AV and AVG sponges was carried out as a
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preliminary approach to determine the toxicity potential the sponges. The cell
394
viability values found for AV, AVG extracts and tissue culture polystyrene were
19 Page 19 of 26
166.9 ± 3.9, 155.8 ± 6.4% and 151.4 ± 8.3%, respectively. The obtained data
396
indicates that both AV and AVG do not exert any cytotoxic effect on L929 cells,
397
which supports the concept that these materials could be used in contact with
398
the body. In fact, the complex composition of AV gel could enhance the wound
399
healing, influencing the wound vascularization and contraction due to the
400
presence of specific active compounds such as beta-sitosterol (Yadav, Kumar,
401
Basha, Deshmukh, Gujjula & Santhamma, 2012). For instance, AV gel extracts
402
have been shown to stimulate and speed up the production of hyaluronic acid
403
and dermatan sulphate (Chithra, Sajithlal & Chandrakasan, 1998). Therefore, it
404
is likely that the multiple active phytochemicals present in the AV gel may be
405
required to address different aspects of the wound healing. Further in vitro
406
direct cell contact assays will be performed to gain a better understanding of the
407
cellular behavior of AV-based sponges.
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5. Conclusions
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Versatile 3D sponges were successful developed from native aloe vera gel
411
using the freeze-drying technique. Both AV-based sponges (AV and AVG) have
412
a heterogeneous porous formation distribution in the matrix, with an
413
interconnected pore structure and good porosity. Moreover, the presence of a
414
coating layer of gellan gum onto aloe vera matrices improved the stability and
415
mechanical properties of AV sponges. The swelling of AVG was dependent on
416
the pH at which the sponges were prepared. Also, the sponges provided the
417
sustained release of BSA-FTIC, used as a release model for over 3 weeks.
418
Moreover, the low cytotoxicity levels of the sponge extracts are encouraging for
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further in vitro and in vivo studies. All findings suggest that the AV-based
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sponges can be promising candidates for biomedical applications, including in
421
the regenerative medicine field.
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Acknowledgments
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The authors acknowledge financial support from Portuguese Foundation for
425
Science
426
SFRH/BD/64601/2009),
427
Diferencial de Potencial Humano-POPH". This work was partially supported by
428
European Union Seventh Framework Programme (FP7/2007-2013) under grant
429
agreement no. KBBE-2010-266033 (project SPECIAL).
-FCT
(Grant
SFRH/BPD/45307/2008
and
us
Technology
“Fundo Social Europeu”-
FSE,
and “Programa
M
an
and
cr
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Graphical abstract
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