653145 research-article2016
JOB0010.1177/0883911516653145Journal of Bioactive and Compatible PolymersYamdej et al
JOURNAL OF
Original Article
Superior physicochemical and biological properties of poly(vinyl alcohol)/sericin hydrogels fabricated by a non-toxic gamma-irradiation technique
Bioactive and Compatible Polymers Journal of Bioactive and Compatible Polymers 2017, Vol. 32(1) 32–44 © The Author(s) 2016 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0883911516653145 jbc.sagepub.com
Rungnapa Yamdej1, Karnwalee Pangza2, Teerapol Srichana3 and Pornanong Aramwit1
Abstract Gamma irradiation was used to fabricate crosslinked poly(vinyl alcohol)/sericin hydrogels with different sericin concentrations, and the physicochemical and biological properties of the gammairradiated poly(vinyl alcohol)/sericin hydrogels were characterized. Following gamma irradiation, the hydrogels had a high gel fraction (80%–95%), implying a high degree of crosslinking. Fourier transform infrared spectra confirmed the crosslinking bonds within the hydrogels, as seen by the characteristic shift in the peak. Furthermore, a low tensile modulus together with a high elongation percentage indicated that the hydrogels were easy to handle. We also showed that all hydrogels released sericin simultaneously. The poly(vinyl alcohol)/sericin hydrogels with high sericin content released more sericin, possibly due to less crosslinking of the hydrogels. When L929 cells were cultured with the hydrogel extracts, the cells were viable and could proliferate, particularly for the cells cultured with the hydrogels containing a high sericin content, which released more sericin. Migration assays also demonstrated that the cells migrated toward the medium extract of hydrogels containing high sericin. We suggest that sterile gamma-irradiated
1Bioactive
Resources for Innovative Clinical Applications Research Unit and Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand 2Gems Irradiation Center, Thailand Institute of Nuclear Technology (Public Organization), Nakhon Nayok, Thailand 3Department of Pharmaceutical Technology and Drug Delivery System Excellence Center, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla, Thailand Corresponding author: Pornanong Aramwit, Bioactive Resources for Innovative Clinical Applications Research Unit and Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Chulalongkorn University, PhayaThai Road, Pathumwan, Bangkok 10330, Thailand. Email:
[email protected]
Yamdej et al.
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poly(vinyl alcohol)/sericin hydrogels could be used as a wound dressing for the treatment of dry and low-exudate wounds. Keywords Sericin, poly(vinyl alcohol), gamma irradiation, hydrogel, crosslinking
Introduction Natural wound-healing processes typically consist of hemostasis, inflammation, proliferation, and remodeling phases in which a number of complex and orchestrated biochemical signals and cells play crucial roles in each phase.1 However, in partial- and full-thickness wounds, the natural healing is limited, resulting in delayed healing and scar formation. To solve this issue, a wound dressing is applied to the wound to facilitate the healing process. The ideal properties of a wound dressing are the biocompatibility, controlled wound environment (moisture, pH, etc.), absorption of wound exudate, and non-adhesion to the wound surface, as well as promoting the re-epithelialization and collagen formation of the regenerated tissue.2 The biomaterials used to fabricate the wound dressing are one of the key factors that determine these properties. Either natural or synthetic polymers have been introduced as the main components of wound dressings. The advantages of natural polymers, such as proteins and polysaccharides, are the biocompatibility, biodegradability, low immunological responses, and wound-healing acceleration due to their biologically active components. Nevertheless, most natural polymers show poor mechanical properties and cannot form a mechanically stable wound dressing. On the other hand, synthetic polymers, such as poly(vinyl alcohol) (PVA) and poly(lactide-co-glycolide) (PLGA), usually have superior mechanical properties compared with the natural polymers, but they rarely contain active components to promote biological activities and they sometimes provoke an immunological reaction due to the toxicity of their degradation products. Silk fiber produced by Bombyx mori silkworms consists of two types of protein: fibroin (~75%) and sericin (~25%). Fibroin is the core, while the sericin is a glue-like protein coating on the fibroin fibers. Recently, various biological activities of sericin, such as ultraviolet resistance, anti-tyrosinase, anti-coagulation, anti-oxidation, anti-bacteria, and anti-cancer activities, have been recognized.3–5 In wound-dressing applications, sericin induces only a minimal inflammatory response and its hydrophilic properties mean that it can maintain a moist environment and absorb wound exudate.2,6,7 Furthermore, sericin promotes attachment and proliferation of skin cells, for example, fibroblasts and keratinocytes8,9 and induces collagen formation.10 Our group has developed various types of sericin-releasing materials to be applied as wound dressings, most of which were fabricated by freeze-drying, salt-leaching, or solvent precipitation techniques.11,12 Usually, dry scaffolds with high absorption ability were obtained. As an alternative to these techniques, gamma irradiation is a new technique to fabricate the sericin-releasing hydrogels because it has many advantages, including that it is a fast and easy process and produces high-water-content hydrogels with unique properties. The radiation can induce chemical reactions that alter the structure of polymers in either solid or liquid states13,14 and are reproducible because the crosslinking can be easily adjusted by controlling the radiation dose.15 Further sterilization of hydrogels is not required because gamma irradiation also sterilizes the materials,16 and the hydrogels do not contain any residual toxic substances because there is no addition of any chemicals during the process. PVA was blended with sericin to improve the mechanical properties of the wound dressing.17 Among synthetic polymers, PVA and its degradation products lead to a low immunological response.18 Due to a number of hydroxyl groups, PVA can be easily crosslinked by either physical
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or chemical techniques.19 Gamma irradiation has also been used to fabricate crosslinked PVAbased hydrogels,20–22 and thus, we investigated whether the gamma irradiation could be used to form PVA/sericin-blended hydrogels and how it affects the properties of hydrogels obtained. In this study, the PVA/sericin hydrogels at various sericin concentrations were fabricated using gamma-irradiation technique. The physicochemical and biological properties of the PVA/sericin hydrogels obtained were systematically characterized. We propose that the high-water-content hydrogels that are non-toxic and release sericin would be an effective and safe alternative for the treatment of dry wounds.
Materials and methods Materials White-shell Bombyx mori silk cocoons were kindly supplied by Chul Thai Silk Co., Ltd (Phetchabun Province, Thailand). The cocoons were cut into small pieces and the sericin was extracted using a high-temperature and high-pressure degumming technique (1 g cocoon/15 g water), as reported previously.10,23 A silk sericin solution of approximately 0.1% w/v, as measured using the BCA Protein Assay Reagent (Pierce, Rockford, IL, USA), was obtained. The sericin solution was concentrated to 10% w/v by a solvent evaporation technique using heat treatment. PVA (molecular weight 77,000–82,000) was purchased from Ajax Finechem (New South Wales, Australia). Glycerin and other chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA) and used without further purification.
Preparation of PVA/sericin hydrogels using gamma irradiation PVA was dissolved in deionized (DI) water at 80°C for 4 h to prepare a PVA solution (6% w/v). The final concentration of PVA was fixed at 6% w/v, while the different concentrations of sericin (0%, 10%, 15%, 20%, and 25% w/v) were mixed with the PVA solution to prepare 6P0S, 6P10S, 6P15S, 6P20S, and 6P25S hydrogels, respectively. The final sericin concentrations in 6P0S, 6P10S, 6P15S, 6P20S, and 6P25S hydrogels were 0%, 0.6%, 0.9%, 1.2%, and 1.5% w/v, respectively. The mixed solution (20 mL) was poured into a 10-cm Petri dish and irradiated by the gamma source (Co-60) at 75 kGy (Paul Stephens Consultancy Ltd, Swindon, UK) at the Thailand Institute of Nuclear Technology. After irradiation, gamma-irradiated PVA/sericin hydrogels were obtained (Figure 1).
Measurement of water content The PVA/sericin hydrogels (2 × 2 × 0.3 cm3) obtained after gamma irradiation were weighed (Ww). Then, the hydrogel was frozen at −20°C and lyophilized for 72 h. The dry weight of the hydrogel was measured (Wd), and the percentage of water content of the hydrogel was calculated according to the following equation % water content =
Ww − Wd × 100 Wd
where Ww and Wd represent the weights of the hydrogel before and after freeze-drying, respectively (n = 3).
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Figure 1. Macroscopic images of the gamma-irradiated PVA/sericin hydrogel.
Evaluation of gel fraction The gel fraction of the PVA/sericin hydrogels was evaluated following the method reported by Kamoun et al.24 Briefly, the hydrogel (2 × 2 × 0.3 cm3) obtained after gamma irradiation was dried in a vacuum oven at 50°C for 24 h. The dry hydrogel was weighed (Wo) and immersed in DI water for 24 h. The non-crosslinked part of the hydrogel was solubilized in this step. Then, the swollen hydrogel was dried in a vacuum oven again. The dry weight of the hydrogel was measured (WG). The gel fraction percentage of the hydrogels was calculated according to the following equation % gel fraction =
WG × 100 Wo
where Wo and WG represent the dry weights of the hydrogel before and after being immersed in DI water, respectively (n = 3). The amount of sericin solubilized into the immersed water was measured using the BCA protein assay kit. The absorbance of the solution was measured at 562 nm, and the amount of sericin was determined from a standard curve prepared from different concentrations of sericin solution (n = 3). The percentages of sericin and PVA loss (non-crosslinked fraction) were calculated.
Fourier transform infrared spectroscopy The functional groups in the PVA/sericin hydrogels were examined using Fourier transform infrared spectroscopy (FT-IR) (PerkinElmer, Waltham, MA, USA). The information on structural contributions was collected in the FT-IR analysis using PerkinElmer Spectrum GX (FT-IR system). The FT-IR analysis was based on the identification of absorption bands attributed to the vibrations of functional groups present in the samples. All spectra were recorded in the wave number range from 4000 to 400 cm−1 at a resolution of 4 cm−1.
Mechanical test A tensile test was performed on the PVA/sericin hydrogels (W × L × H = 10 × 60 × 2 mm3) at room temperature using a universal testing machine (Hounsfield H10KM, UK) equipped with a 10-kN load cell at a constant rate of 30 mm/min. The curves of force as a function of deformation (mm)
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were automatically recorded by the software. The tensile strength (N/mm2) and percentage of elongation at break were calculated according to the ASTM D638-01 method (n = 6).
In vitro enzymatic biodegradation test The known dry-weight PVA/sericin hydrogel (Wo) was incubated in 1.6 µg/mL lysozyme enzyme solution (hen egg white, 92,717 U/mg, lot no. 1316456; BioChemika, Fluka Sigma–Aldrich, Buchs, Switzerland) at pH 7.4 and 37°C. At each time interval, the remaining hydrogel was removed from the solution, rinsed repeatedly with DI water, and freeze-dried. The dried hydrogel was weighed (Wt), and the percentage of weight remaining was calculated according to the following equation % remaining weight =
Wt × 100 Wo
where Wo and Wt represent the dry weights of the hydrogel before and after being immersed lysozyme solution, respectively (n = 3).
In vitro release of sericin from the hydrogels PVA/sericin hydrogels were placed into 10 mL of phosphate-buffered saline solution (PBS, pH 7.4) at 37°C with continuous stirring at 100 r/min in a closed container. Aliquots of the PBS solution (1.5 mL) were collected at the predetermined times, and the amount of sericin released into the solution was measured using a BCA protein assay kit as described previously (n = 3).
In vitro cytotoxicity of the hydrogels The cytotoxicity the PVA/sericin hydrogels was evaluated using an indirect method (extract method) on L929 mouse fibroblast cells according to ISO 10993—Part 5. The hydrogels were incubated in Dulbecco’s modified eagle medium (DMEM) without fetal bovine serum at 37°C/5% CO2 for 24 h in order to obtain the hydrogel extracts. L929 cells were seeded at a density of 105 cells/well in 48-well plates and incubated at 37°C/5% CO2 for 24 h. Next, the cell culture medium was changed to the hydrogel extract medium. After 1, 2, and 3 days of culture, the viability of cells was measured using a MTT assay.25
In vitro migration assay L929 mouse fibroblast cells at a concentration of 1 × 105 cell/well (1.5 mL) were seeded in the upper compartment, and fresh medium (2.5 mL) was added to the lower compartment of six-well plates (Transwell; Corning, New York, USA). Then, the hydrogels were put into the lower compartment, followed by incubation at 37°C/5% CO2 for 24 h. A lower compartment that contained medium without the hydrogel was used as a negative control. The number of cells migrated into the lower compartment was counted by microscopy (20×) and calculated as the migration percentage compared with the negative control.
Statistical analysis All results were statistically analyzed using the unpaired Student’s t test, and p
