Gellan gumclay hydrogels for tissue engineering application Mechanical, thermal behavior, cell viability, and antibacterial properties Journal of Bioactive and Compatible Polymers 1 –19 © The Author(.
643106 research-article2016 JBC0010.1177/0883911516643106Journal of Bioactive and Compatible PolymersMohd et al JOURNAL OF Bioactive and Compatible Polymers Original Article Gellan gum/clay hydrogels for tissue engineering application: Mechanical, thermal behavior, cell viability, and antibacterial properties Journal of Bioactive and Compatible Polymers 1–19 © The Author(s) 2016 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0883911516643106 jbc.sagepub.com Saffawati Syazwani Mohd1, Mohd Aidil Adhha Abdullah1 and Khairul Anuar Mat Amin1,2 Abstract In this study, sodium montmorillonite (Na-MMT) was successfully modified by using n-hexadecyl trimethyl ammonium bromide (CTAB) via cationic exchange to obtain an organophilicmontmorillonite (CTAB-MMT) The Na-MMT, CTAB-MMT, and a commercial montmorillonite, that is, Cloisite15A were incorporated into gellan gum (GG) hydrogel and their mechanical, physical, thermal properties, biocompatibility, and antibacterial activities were investigated The mechanical performance results show that the GG hydrogels containing Cloisite15A required smallest volume to achieve optimum compression stress, modulus, and compression strain at 5% (w/w) compared to both Na-MMT and CTAB-MMT at 10% (w/w) Swelling ratio of GG hydrogels increased upon addition of MMT, and water vapor transmission rate (WVTR) values of all hydrogels were in the range of 1106–1890 g m−2 d−1, which were comparable to WVTR values of commercial wound dressings Thermal behavior shows that the inclusion of Cloisite15A in GG hydrogel improved the thermal stability than its counterparts Cell studies exhibit that the GG incorporated with Na-MMT is non-cytotoxic to human skin fibroblast cells (CRL2522), and in contrast, the GG hydrogels incorporated CTAB-MMT and Cloisite15A revealed that the cells were dying and the cell growth depleted after being cultured for 72 h Qualitative antibacterial study revealed that GG hydrogel containing CTAB-MMT only in the sample exhibits inhibition against the Gram-positive bacteria, that is, Staphylococcus aureus and Bacillus cereus, while there was no inhibition exhibited against Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae) 1School of Fundamental Science, Universiti Malaysia Terengganu, Kuala Terengganu, Malaysia of Marine Biotechnology, Universiti Malaysia Terengganu, Kuala Terengganu, Malaysia 2Institute Corresponding author: Khairul Anuar Mat Amin, School of Fundamental Science, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia Email: kerol@umt.edu.my Downloaded from jbc.sagepub.com by guest on May 17, 2016 Journal of Bioactive and Compatible Polymers Keywords Gellan gum, hydrogel, montmorillonite, Cloisite15A, compression, biocompatibility Introduction Every year, there are approximately 165 million cases worldwide requiring wound treatments across different types of wounds.1 Surgical wounds occupy the vast majority of injuries (103 million), followed by lacerations (20 million), diabetic ulcers (11 million), and burn wounds (10 million) Although there are more than thousand wound products available in the market, the efforts in finding new materials or methods to improve the healing process are continuing At present, biopolymers are receiving greater attention than synthetic petrochemical-based polymers due to environmental concerns A variety of renewable biopolymers such as polysaccharides, for example, chitosan (CH) and gellan gum (GG) derived from chitin and Pseudomonas elodea, respectively, have been studied in the development of wound dressing materials Studies regarding modified-montmorillonite (MMT) cross-linked with bio-polymers hydrogel have been scarcely reported As far as our knowledge goes, a study has been reported using a biopolymer, that is, CH in producing hydrogel-incorporated modified-MMT (Cloisite 15A) focusing on drug release.2 A few studies utilized the CH hydrogel by using pure-MMT in understanding the physical properties.3,4 However, there are more studies that have been using synthetic polymer materials such as poly(vinyl alcohol), poly-(N-isopropylacrylamide), polyacrylamide, and many more to produce hydrogel-incorporated MMT to focus on their mechanical and physical properties, their antibacterial properties,5 biocompatibility,6 drug discovery7,8 nuclear waste storage,9 and surface modification of clays/polymer as an adsorbent.10,11 A study also approved the amount of hydrophilic and hydrophobic montmorillonite as the filler highly controlled the swelling behavior of copolymer (poly(NIPAAm-co-AAm), poly(NIPAAm-co-AAc) and poly(AAm-co-AAc)) gels.12 In term of biocompatibility of modified-MMT by using n-hexadecyl trimethyl ammonium bromide (CTAB), the PVA-hydrogel shows no cytotoxic effects against the erythroleukemia cell line (K562, ATCC).13 In our review, no study has utilized GG in the formation of clay composites or hydrogel GG is selected as a base hydrogel material due to its biocompatibility and is approved by the United States Food and Drug Administration (US FDA) and the European Union (EU) for use in the food industry It is also used as a scaffold material for tissue engineering application.14,15 GG has been reported to have good biocompatibility on mouse fibroblast cells (L929),16 human skin fibroblast cells (CRL2522),17 and human fetal osteoblasts (hFOBs 1.19).18 In general, hydrogel can be classified as a three dimensional polymer network which has received more attention in past decade due to its ability to absorb and retain high amount of water This characteristic enables the hydrogel to be used in various application, including as a carrier in drug discovery, soft contact lens, corneal implants, cell carrier, and wound dressing.19–21 However, the insufficient mechanical and physical characteristics of hydrogel are a major drawback in hydrogel application, although it has great potentials This study highlights the use of GG hydrogels incorporated with MMT in understanding the physical characteristics, mechanical properties, thermal behaviors, biocompatibility, and antibacterial activities Three types of MMT with different basal-spacing values, that is, sodium-montmorillonite (Na-MMT, d≈12.72 Å), modified Na-MMT by using CTAB via cationic exchange reaction to obtain an organophilic CTAB-MMT (d≈19.20 Å), and a commercially modified MMT, that is, Cloisite15A (d≈28.04 Å), were mixed into GG hydrogels The difference in basal-spacing values used could contribute to better polymer chain intercalation, which could translate to increase in mechanical properties of the hydrogels The chemical interaction of GG hydrogels with different fillers (i.e Na-MMT, CTAB-MMT, and Cloisite15A) was confirmed by using attenuated total Downloaded from jbc.sagepub.com by guest on May 17, 2016 Mohd et al reflectance (ATR) and X-ray diffractometer (XRD), while the physical characteristics were carried out in investigating the mechanical performance, swelling, gel fraction, and water vapors transmission rates (WVTR) The thermal behavior of the GG hydrogels was examined by using thermogravimetric analyzer and differential scanning calorimetry (DSC) The cell viability and proliferation tests involved human fibroblast skin cell (CRL-2522, ATCC), while the antibacterial activities were assessed via in-vitro qualitative study against four bacterial strains, that is, Grampositive bacteria (Staphylococcus aureus and Bacillus cereus) and Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae) Materials and method Materials Low acyl GG (Gelzan™ CM, Mw ≈ 2–3 × 105 Da, product number-G1910, lot number SLBB0374V) was obtained from Sigma Aldrich (Malaysia) and CTAB (CTAB-product number-219374) from Merck (Malaysia) Sodium-montmorillonite (Na-MMT) was purchased from Kunimine Ind (Japan) with a cation capacity (CEC) of 1.19 cmol kg−1 Cloisite15A was obtained from Southern Clay (United States) According to the manufacturer’s specification, Cloisite15A (particles sizes ≈ 13 µm (90%), moisture < 2%) was modified by using quaternary ammonium salt with cation capacity of 1.25 cmol kg−1 All materials were used as initially received Synthesis of organo-montmorillonite Sodium montmorillonite (Na-MMT) was modified by cationic exchange method between Na+ in layered silicate galleries and CTAB cations in an aqueous solution using a mechanical mixer with constant stirring (≈70 rpm) at 80°C for 24 h The obtain mixture was filtered and washed several times to purify and remove the unreacted material The final product was then dried under vacuum to obtain organophilic-montmorillonite (CTAB-MMT) Preparation of hydrogel/MMT GG solutions were prepared by dissolving 1% (w/v) GG in deionized water (18.2 MΩ) at 80°C for 2 h Na-MMT, CTAB-MMT, and Cloisite15A at different concentrations (ranging from 2% to 20%, w/w) were dispersed in deionized water at 500 rpm, 90°C for 30 min Both solutions were then heated up to 80°C for 4 h followed by drop wise addition of 10 mM of CaCl2 aqueous solution The mixtures were then deposited onto petri dishes (90 mm × 15 mm) and dried at 25°C for at least 24 h The hydrogels were washed with deionized water to remove the non-cross-linked polymer fractions and pre-condition for next 24 h prior to any characterizations The GG hydrogels containing Na-MMT, CTAB-MMT, and Cloisite15A will be hereafter referred as GG/Na-MMT, GG/CTABMMT, and GG/Cloisite15A, respectively The clays loading into GG hydrogels were shown by the number at end of samples, for example, GG/Na-MMT10 containing 10% (w/w) of Na-MMT and same naming were applied to other samples XRD X-ray diffractometry was performed by using Rigaku Miniflex (II) XRD operating at a scanning rate of 2.00° min−1 The diffraction spectra were recorded at the diffraction angle, 2θ from 3° to 10° at room temperature Downloaded from jbc.sagepub.com by guest on May 17, 2016 Journal of Bioactive and Compatible Polymers Fourier transform infrared ATR-Fourier transform infrared (FTIR) spectra were collected using a Perkin Elmer Spectrum 100 FT-IR spectrophotometer with a PIKE Miracle ATR accessory (single-bounce beam path, 45° incident angle, 16 scans, 4 cm−1 resolution), and all spectra were corrected using the Perkin Elmer spectrum 100 software Elemental analysis The presence of carbon, hydrogen, nitrogen, and sulfur in Na-MMT, CTAB-MMT, and Cloisite15A was analyzed by using FLASHEA 1112 Series, CHNS-O analyzer It was carried out by placing the sample at 2–3 mg in a tin capsule at the high temperature with a constant helium flow Surface area analysis Nitrogen adsorption–desorption isotherms were measured using the ASAP 2020 volumetric adsorption analyzer The samples were degassed at 473 K for 1 h The specific surface area, SBET of the sample was calculated by the BET method, and the total pore volume, Vt was obtained at a relative pressure of 0.9746 Compression test Mechanical characterization of hydrogels was carried out by using Instron Universal Mechanical machine (model 3366) at the cross-speed set at 10 mm min−1 Hydrogels were cut into cubes (2 cm × 2 cm × 0.7 cm) for characterization Gel fraction The pieces of samples (2 cm × 2 cm) were dried for 6 h at 50°C and weighted (W1) Then, they were soaked in 10 mL deionized water for 24 h or to a constant reading and dried again for 6 h at 50°C (W2) The gel fraction was calculated by as follows Gel fraction ( % ) = ( W2 /W1 ) × 100 Swelling ratio Swelling ratio was determined by the weight ratio of absorbed water (Wwet) to dry (Wdry) hydrogel The hydrogels (2 cm × 2 cm) were immersed in a sealed beaker containing phosphate buffer solution (pH 7.03) in a water bath and temperature was set at 37°C The weight of wet samples (Wwet) was measured after 24 h to achieve the equilibrium swelling The swelling ratio of each sample was calculated as below Swelling ratio = ( Wwet − Wdry ) / Wdry × 100% WVTRs The WVTRs were measured following a modified ASTM International standard method.22 The hydrogels were dried to a film and fixed on the circular opening of a permeation bottle with the Downloaded from jbc.sagepub.com by guest on May 17, 2016 Mohd et al effective transfer area (A) of 1.33 cm2 The permeation bottle was placed in the humidity chamber (Memmert, HCP108) and the temperature was set to 21°C and relative humidity to 50% ± 5% The WVTR was then determined by measuring the rate of change of mass (m) in permeation bottles at exposure time of 24 h using equation as follows WVTR = (m / A∆t ) where, m/Δt is the amount of water gain per unit time of transfer and A is the area exposed to water transfer (m2) Scanning electron microscopy The morphological characterization of the hydrogel samples was carried out by examining the cross section of the samples using scanning electron microscopy (SEM; JOEL LA-6360) Two types of methodology were used to observe the cross section of the hydrogel samples (1) hydrogel samples were freeze-dried in liquid nitrogen (−160°C), fractured at −150°C, and subsequently imaged for SEM (2) Hydrogel samples were prepared by freezing in −80°C, at least for 24 h and transferred into freeze-drying vessel (Eyela FD-550) for 3 days to obtain porous structure Then, the porous samples were broken down in liquid nitrogen (−160°C) to observe their cross section area and coated with gold using a sputter coater (JFC-1600) TGA Thermal gravimetric (TGA) measurements were taken using a thermal analyzer Pyris 6, PerkinElmer-TGA6 The samples were analyzed at a heating rate of 10°C min−1 under N2 flow at 50 mL min−1 DSC DSC studies of the samples were characterized using a Pyris6, Perkin-Elmer-DSC7 at a heating rate of 10–300°C min−1 in an N2 atmosphere at flow rate of 50 mL min−1 Sample was approximately 4 mg Cell studies Routine cell-culture. The culture of normal human skin fibroblast cells (CRL-2522-ATCC) was prepared by using the Eagle’s Minimum Essential Medium (EMEM, ATCC, USA) with 10% (v/v) fetal bovine serum (FBS, Sigma Aldrich, USA) and 1% (v/v) antibiotic (Penicillin/Streptomycin, Sciencell, USA) Cells were cultured at 37°C in a humidified 5% CO2 atmosphere and were subcultured every 3 days as established protocols and harvested at 60%–80% confluence Cell viability For this testing, the hydrogel samples were dried in an oven at 40°C for 24 h to have a film with approximate thickness of 50 µm The films (diameter ~6 mm) were then placed into the 96-well culture plates (Nunc, Germany) containing EMEM medium and leaved overnight in order to transform the films to hydrogels Prior to testing, the hydrogel was sterilized in a laminar airflow chamber under UV radiation for 20 min Three replicates were used for each type of hydrogel samples Downloaded from jbc.sagepub.com by guest on May 17, 2016 Journal of Bioactive and Compatible Polymers The CRL2522 cells (5000 cells/well) were seeded into wells containing samples and cultured at 37°C in 5% CO2 atmosphere Tissue culture polystyrene plates (TCPP) were used as control for cell adhesion and growth After 24, 48, and 72 h of incubation, cell viability was observed by using an Olympus TH4-200 microscope equipped with an Olympus U-RFL-T UV pack stained with calcein-AM Cell proliferation Cell proliferation was quantified by using a CellTiter 96 aqueous one solution assay (Promega, USA) which contained tetrazolium compound [3-(4, 5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium), inner salts; MTS (a)] with electroncoupling reagent (phenazine ethosulfate) Prior to the addition of the assay solution (20 µL in each wells), the media in all wells that contained hydrogels, except for the positive control, were replaced with fresh media and later incubated for 3 h at 37°C in an atmosphere containing 5% CO2 Then, 100 µL of the inoculants were transferred into new wells, and the absorbance at 490 nm was measured by using a microplate reader (Multiskan Ascent 96/384, USA) The absorbance readings were converted to cell number using calibration curves of CRL2522 cells in 96-well plates under the same condition Antibacterial study Two Gram-positive (S aureus and B cereus) and two Gram-negative (E coli and K pneumoniae) bacterial suspensions were used for the antibacterial assay Meuller-Hinton (MH, Difco, Malaysia) agar was used for the growth of both bacterial types Each Gram-positive and Gram-negative bacteria suspension was evenly spread on the solid MH agar and dried in a laminar flow air chamber The wells were designed for each solid MH agar so that the hydrogel (diameter ~6 mm) can be placed into them The solid LB agar with the hydrogel samples were incubated at 37°C for 24 h The presence of any clear zone around the gels on the LB agar was recorded as an indication of inhibition against the S aureus, B cereus, K pneumoniae, and E coli Result and discussion Synthesis of organophilic-montmorillonite The organophilic-montmorillonite synthesized by using CTAB via cationic exchange reaction was verified by using ATR-FTIR, elemental analyzer and X-ray diffraction (XRD) Figure shows that the ATR-FTIR spectra of synthesized organophilic-montmorillonite (CTAB-MMT) exhibit three characteristic bands which are not distinct in Na-MMT spectrum Absorbance bands at 2918 cm−1 and 2850 cm−1 corresponded to the presence of aliphatic C-H symmetrical and asymmetrical stretching vibrations of CTAB, respectively, and a band at 1450 cm−1 also referred to the CH2 scissoring bending vibrations of CTAB.23 These bands indicated that the intercalation of CTAB into the interlayer space of the Na-MMT was successful As comparison, the ATR-FTIR spectrum of commercial Cloisite15A shows similar peaks to CTAB-MMT at 2920, 2850, and 1469 cm−1 due to the presence of quaternary ammonium salt Different intensity of absorbance bands between CTAB-MMT and Cloisite15A suggested difference in ionization degree of the present groups on an MMT surface.24 The elemental analysis of CTAB-MMT shows the presence of carbon element at ≈30.2%, which is not distinct in Na-MMT (Table 1) The presence of carbon was also detected in commercially modified Cloisite15A at ≈32.4% Downloaded from jbc.sagepub.com by guest on May 17, 2016 Mohd et al Figure 1. ATR-FTIR spectra of (a) Na-MMT, (b) commercial Cloisite15A, and (c) organomontmorillonite, CTAB-MMT Table 1. Elemental analysis, basal spacing (D001), and structural data of sodium-montmorillonite (NaMMT), organophilic montmorillonite (CTAB-MMT), and commercial montmorillonite (Cloisite15A) samples: (SBET is the specific surface area calculated by BET method (2 parameters line); Vt is the total porous volume; and Da is the average pore radius) Sample Na-MMT CTAB-MMT Cloisite15A Element component (%) Carbon Hydrogen 30.2 32.4 1.9 6.5 6.5 D001 (Å) SBET (m2 g−1) Vt (cm3 g−1) Da (nm) 12.72 19.15 28.04 31.416 3.7714 15.471 0.0447 0.0132 0.0373 16.212 42.691 31.408 The XRD patterns of the Na-MMT, CTAB-MMT, and Cloisite15A samples are shown in Figure and summarized in Table The XRD pattern of Na-MMT shows typical features of basic montmorillonite structure which the diffraction plane (d001) indicate the basal spacing at ≈12.72 Å, resulting from the presence of the main counterbalancing ions (Na+ cations) in the interlayer space of montmorillonite structure.24 While the synthesis of organophilic-montmorillonite by using CTAB caused increase in basal-spacing value to 19.2 Å and difference of 2θ angle values from 4.61° (Na-MMT) to 6.94° The increase in basal-spacing and shifting of 2θ shows that the intercalating of CTAB ion into the silicate layers of Na-MMT was successful, resulting in an organophilic montmorillonites The organophilic montmorillonite (CTAB-MMT and Cloisite15A) also shows the crystalline characteristics due to the appearance of sharp peak of the d001 compared to the broad peak of Na-MMT (Figure 2) In theory, the clay layers of the montmorillonites are held loosely with weak Van Der Waals forces, and once the organic molecules are introduced between the clay layers, it shows the difference of interlayers which translated into the difference of basal spacing The commercial montmorillonite (Cloisite15A) shows the highest basal-spacing at 28.04 Å compared to its counterparts with 2θ at 3.15° For the porous structural data of Na-MMT, CTAB-MMT, and Cloisite15A as summarized in Table 1, CTAB-MMT shows that specific area (SBET = 3.7714 m2 g−1), microporous surface area (Smicro = 6.4837 m2 g−1), total porous volume (Vt = 0.0132 cm3 g−1), and microporous volume (Vmicro = 0.0031 cm3 g−1) was lower than to its counter parts, that is, Cloisite15A and Na-MMT In contrast, due to low specific area of CTAB-MMT compared to others, the sample exhibits highest average pore radius (Da) at ≈42.691 nm, while Na-MMT recorded the highest specific area (SBET) Downloaded from jbc.sagepub.com by guest on May 17, 2016 Journal of Bioactive and Compatible Polymers Figure 2. XRD patterns of (a) CTAB-MMT particle, (b) Na-MMT particle, (c) Cloisite15A particle, (d) GG/CTAB-MMT10 hydrogel, (e) GG/Na-MMT10 hydrogel, and (f) GG/Cloisite15A5 hydrogel Figure 3. (a) Stress-at-break and (b) Strain-at-break of GG hydrogel and GG hydrogel incorporated NaMMT, CTAB-MMT, and Cloisite15A at different loadings at 31.416 m2 g−1 and exhibits lowest average pore radius (Da) at 16.212 nm (Table 1) A reasonable interpretation of the BET result is that the modification of Na-MMT with CTAB allows some small size of CTAB species to enter into the interlayer region, and simultaneously increases the average pore radius of CTAB-MMT, which correlates well to increase in basal-spacing values of CTABMMT sample To conclude, we show that the intercalation of CTAB to replace the Na ions within the silicate layers of MMT was successful judging from the additional peak observed in ATR-FTIR spectra, the presence of carbon element, and increase in basal-spacing of CTAB-MMT samples Compression of hydrogel The application of hydrogels are highly depends on its mechanical properties to bear or withstand the maximum force applied on it prior to failure Figure and Table depict the compression strength of GG/Na-MMT, GG/CTAB-MMT, and GG/Cloisite15A hydrogels at different percentage loadings It is worth to note that GG hydrogel without any filler shows highest stress-at-break Downloaded from jbc.sagepub.com by guest on May 17, 2016 Mohd et al Table 2. Summary of the stress at break (σ), modulus (E), strain at break ( ε ), swelling ratio, gel fraction and water vapor transmission rates (WVTR) of gellan gum hydrogel containing Na-MMT, CTAB-MMT, and Cloisite15A at different loadings Hydrogel samples (%) σ (kPa) E (kPa) ε (%) Swelling (%) Gel Fraction (%) WVTR (g m−2 d−1) Blank GG GG/Na-MMT2 GG/Na-MMT5 GG/Na-MMT10 GG/Na-MMT15 GG/Na-MMT20 GG/CTAB-MMT2 GG/CTAB-MMT5 GG/CTAB-MMT10 GG/CTAB-MMT15 GG/CTAB-MMT20 GG/Cloisite15A2 GG/Cloisite15A5 GG/Cloisite15A10 GG/Cloisite15A15 GG/Cloisite15A20 – 330 ± 20 203 ± 21 197 ± 13 222 ± 6 196 ± 10 186 ± 19 202 ± 2 217 ± 4 236 ± 13 195 ± 5 204 ± 9 291 ± 12 295 ± 7 247 ± 9 271 ± 14 289 ± 7 – 6.5 ± 1.0 5.3 ± 0.4 7.2 ± 0.3 7.6 ± 0.8 7.5 ± 0.6 7.3 ± 0.9 6.1 ± 0.6 5.7 ± 0.7 6.3 ± 0.8 5.8 ± 0.5 5.5 ± 0.6 5.4 ± 0.4 6.2 ± 0.7 5.7 ± 0.5 5.7 ± 0.6 4.0 ± 0.7 – 117 ± 3 31 ± 5 33 ± 8 69 ± 12 55 ± 4 28 ± 1 62 ± 14 65 ± 4 60 ± 1 50 ± 13 70 ± 6 35 ± 3 60 ± 11 82 ± 29 79 ± 13 68 ± 7 – 15 ± 1.6 8 ± 0.7 11 ± 0.5 14 ± 1.6 12 ± 0.7 11 ± 0.8 9 ± 0.7 10 ± 0.4 11 ± 0.6 8 ± 0.6 8 ± 0.5 11 ± 1.0 14 ± 0.6 11 ± 0.6 12 ± 0.4 10 ± 1.1 – 74 ± 1.8 77 ± 1.7 79 ± 1.7 80 ± 1.1 80 ± 1.2 80 ± 1.8 72 ± 2.1 76 ± 0.9 74 ± 1.7 79 ± 0.9 80 ± 0.3 82 ± 0.3 75 ± 0.7 69 ± 0.3 75 ± 14 74 ± 0.2 5407 1193 1890 1533 1484 1429 1106 1555 1556 1576 1604 1596 1603 1649 1600 1634 1644 Na-MMT: sodium-montmorillonite; CTAB-MMT: organophilic montmorillonite, and Cloisite15A: commercial montmorillonite (σ ) and strain-at-break (ε ) values compared to GG hydrogels with fillers Nevertheless, the content (%) of the MMT in the GG hydrogels significantly affected the mechanical performance of the materials As shown in Figure 3(a), the stress (kPa) versus the concentration of MMT exhibits that the GG hydrogel for Na-MMT and CTAB-MMT (at ≈10% w/w) reach an optimum σ at 14 ± 1.6 kPa and 11 ± 0.6 kPa, respectively The addition of higher concentrations (≥10% w/w) of Na-MMT and CTAB-MMT into GG decreased the σ of the hydrogel However, GG/Cloisite15A hydrogel shows an optimum σ at lower concentration than Na-MMT and CTAB-MMT, that is, at 5% (w/w) The same behavior was observed when adding higher concentration of Cloisite15A (≥5% w/w) which led to decrease in the σ values In addition, the strain-at-break (ε ) of GG/Na-MMT, GG/CTAB-MMT, and GG/Cloisite 15A hydrogels exhibit a similar trend of σ , which increased their elasticity with the increase in MMT and reached an optimum at 7.6% ± 0.8% (GG/Na-MMT10), 6.3% ± 0.8% (GG/CTAB-MMT10), and 5.7% ± 0.7% (GG/Cloisite15A5), respectively (Figure 3(b)) The addition of more MMT above the concentrations mentioned decreased the ε values Our results show that the σ and ε values of GG/CTAB-MMT and GG/Cloisite15A hydrogels were comparable to GG/Na-MMT hydrogels, which slightly contradict a previous studies.25 In their study, the author reported that the tensile strength of polyvinyl alcohol (PVA)-Na-MMT hydrogel was higher than those modified clays due to the well-dispersed MMT Na-MMT clay is claimed to disperse easily in polymer than alkylammonium ion exchanged montmorillonite because of its hydrophilic characteristic In our study, the increased values of σ and ε at 10% (w/w) loading of the GG/Na-MMT and GG/CTAB-MMT hydrogels compared to 5% (w/w) for the GG/Cloisite15A hydrogels were probably due to the improvement of cross-linking behavior among the hydrogel network which creates Downloaded from jbc.sagepub.com by guest on May 17, 2016 10 Journal of Bioactive and Compatible Polymers Figure 4. FTIR spectrum of hydrogels (a) gellan gum (GG), (b) GG/Na-MMT10, (c) GG/CTAB-MMT10, and (d) GG/Cloisite15A5 the strong hydrogen bond interactions FTIR was used to confirm the chemical interaction of GG hydrogels with the MMT (Figure 4) In the spectra, two major peaks were present in GG polymer with the MMT, that is, at λ = 3421–3452 cm−1 which corresponded to the hydroxyl group (O–H) stretching absorption and 1631–1637 cm−1 which corresponded to the stretching vibrations of carbonyl group (C=O) The shifting wavelength of C=O group of GG hydrogel with MMT is expected due to the withdrawal of electrons from hydroxyl moiety (deformation of carbonyl group) for the formation of hydrogen bonding between the polymer and the clays.19 The changes in interlayer basal-spacing of the GG/Na-MMT, GG/CTAB-MMT, and GG/ Cloisite15A hydrogels were examined by XRD analysis (Figure 2) The XRD spectra for GG hydrogel with MMT did not show any peak in the range of 2θ = 3°–10° compared to those for MMT in pure forms (Na-MMT, CTAB-MMT and Cloisite15A) (Figure 2) This might be because the MMT fillers uniformly dispersed into the GG hydrogel network by either being disordered/ exfoliated completely.26 These results were supported by the cross-sectional morphological study of the internal structure of GG hydrogels by using SEM (Figure 5) To observe the homogeneity of the MMT fillers in GG hydrogel network, the hydrogel samples were freeze-dried and the images are shown in Figure The results show that the MMT dispersed homogeneously into the hydrogel network with an identical porous structure for each sample This special morphological characteristic is believed to give a good sign in terms of the compression strength as well as on water absorption capacity.27,28 The compact and homogenously distributed MMT could contribute to enhance the compression strength of hydrogels.29 The author also reported that the larger size of pores might result in the loose network of cross linkages thus contributing to Downloaded from jbc.sagepub.com by guest on May 17, 2016 11 Mohd et al Figure 5. Scanning electron microscopy of cross-sectional images of hydrogel (a) GG/Na-MMT10, (b) GG/CTAB-MMT10, and (c) GG/Cloisite15A5 Dotted-line on the image represents the cross-section of the hydrogel Figure 6. Scanning electron microscopy of cross-sectional images of hydrogel by using freeze-dried technique (a) gellan gum (GG), (b) GG/Na-MMT10, (c) GG/CTAB-MMT10, and (d) GG/Cloisite15A5 the brittleness, whereas the decrease in pore sizes of hydrogel incorporated MMT supposedly increased the strength due to the formation of sub-networking polymer clay’s among the crosslinkages However, the following reduction in stress-at-break and strain-at-break values were then caused by the limited network formation and poor adhesion of cross-links thus changing the hydrogel characteristics, which then reduced the stiffening effect and increased the brittleness.30 Gel fraction and swelling of hydrogel The gel fraction and swelling ratio of GG hydrogels with Na-MMT, CTAB-MMT, and Cloisite15A as a function of the concentrations are summarized in Table It can be seen that the gel fraction Downloaded from jbc.sagepub.com by guest on May 17, 2016 12 Journal of Bioactive and Compatible Polymers of GG/Na-MMT, GG/CTAB-MMT, and GG/Cloisite15A hydrogels at all concentrations was higher than the control (GG hydrogel = 74% ± 1.8%) The addition of MMT shows an improvement in hydrogel cross-linking density compared to GG hydrogel and is in agreement with a previous study.31 This reveals that the Na-MMT, CTAB-MMT, and Cloisite15A as cross-linking agents in hydrogel network develop some interactions between the MMT layers and GG chains in hydrogel as previously shown by the FTIR results The same observations were reported by other studies which confirmed the function of MMT as physical cross-linking agent in hydrogel.32,33 Generally, the lower gel fraction value was the weakest mechanical stability and less flexible of the gel was.21 It is important to produce a balance gelling fraction to avoid poor mechanical characteristics and low swelling property For swelling results, it seems that the water uptake ratio for GG hydrogel increases with the increase of Na-MMT, CTAB-MMT, and Cloisite15A content until it reaches the equilibrium state (Table 2) This is perhaps why the clay’s sheets are well dispersed in GG hydrogel matrix, and the pores in hydrogel are bigger and are suitable for water absorbency The increase of swelling also was attributed to the reaction of –OH groups of MMT with the –OH and –COOH of the polymeric chains as indicated in IR spectra for the hydrogel MMT.19,34,35 The presence of hydroxyl group and stretching vibrations of carbonyl groups were detected in all clay hydrogels at different wavelength as depicted in Figure (FTIR result) These functional groups are able to relieve the entanglement of polymeric chains thus weakening the hydrogen bonding among the hydrophilic groups This will decrease the degree of physical cross-linking and therefore improve the water uptake of the hydrogels After reaching the equilibrium, the swelling ratio decreases with the additional percentage of MMT This could be due to the increase in cross-linking density with the increase in MMT and the decrease in pores size that not fit the movement of water into the hydrogel samples.36 Zhang et al.37 also reported the effect of MMT content on the swelling capacity of hydrogel and revealed that the maximum water absorbency achieved at a certain weight ratio of MMT Excess clays could also decrease the hydrophilicity due to the osmotic pressure difference then resulting in shrinkage of the composite.38 For that reason, introducing moderate amount of MMT could enhance the water absorbency of the hydrogels WVTRs The WVTR values of GG hydrogels containing MMT were in the range of 1106–1890 g m−2 d−1 while the control (without sample) was recorded at 5407 g m−2 d−1 (Table 2) For GG/Na-MMT hydrogel, the addition of Na-MMT significantly decreased the WVTR values from 1890– 1106 g m−2 d−1 The WVTR values decreased upon addition of higher concentration of Na-MMT which could be due to the tight packing of the clays in GG hydrogels, which then interrupt water vapor diffusion rates However, this behavior was not observed for GG/ CTAB-MMT and GG/Cloisite15A hydrogels The WVTR values of GG/CTAB-MMT were in the range of 1555–1596 g m−2 d−1, while for GG/Cloisite15A in the range of 1600– 1649 g m−2 d−1 For commercial dressings, Hyalofil®, Promogram®, and CBC also showed the same WVTR values, which were 1635 g m−2 d−1, 1549 g m−2 d−1, and 1623 g m−2 d−1, respectively.39 Another study reported that the WVTR value of hydrogel from gelatin, oxidized alginate, and borax was found to be 2686 ± 124 g m−2 d−1,40 which was higher than our WVTR values These differences in WVTR values were due to the change in polymer hydrogel, and their additives thus provide the different transmission rates Nevertheless, these values still remain within the range of WVTR values (76–9360 g m−2 d−1) as reported for commercial synthetic wound dressings.22 Downloaded from jbc.sagepub.com by guest on May 17, 2016 13 Mohd et al Table 3. Thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC) properties of gellan gum (GG) hydrogel, GG/Na-MMT10 hydrogel, GG/CTAB-MMT10 hydrogel, and GG/Cloisite15A5 hydrogel Hydrogel GG GG/Na-MMT10 GG/CTAB-MMT10 GG/Cloisite15A5 TGA (°C) DSC (°C) To Tc R (%) Tm 54 49 52 48 477 437 473 516 20.11 23.12 23.46 23.78 224 211 225 228 To: temperature onset, Tc: temperature completion, R: residue, Tm: melting point TGA and DSC The thermal stability of GG hydrogel with MMT was characterized by using thermogravimetric analysis (TGA) and DSC The weight loss of pure GG was started at temperature onset, To = 54°C with the evaporation of water and the temperature completion, Tc = 477°C (Table 3) The addition of Na-MMT, CTAB-MMT, and Cloisite15A into GG hydrogel decreased the temperature onset (To), and temperature completion (Tc) values except for GG/Cloisite15A5 hydrogel, which exhibited increase in Tc value to 516°C The increase in Tc value of GG/Cloisite15A5 by the addition of Cloisite15A is due to the fact that clay layers have good barrier action which prevents heat from transmitting quickly and therefore limiting the continuous degradation of composites The final residue of GG/Cloisite15A5 hydrogels also shows the highest value at 23.78%, suggesting that the samples provide better thermal stability possibly due to strong internal cross-linking network that improves their thermal stability (Table 3) DSC results show that the addition of fillers altered the melting point (Tm) of the hydrogels (Table 3) GG/Cloisite15A5 hydrogels exhibit the highest melting point (Tm = 228°C) followed by GG/CTAB-MMT10 at Tm = 225°C and GG at Tm = 224°C GG/ Na-MMT10 hydrogels recorded the lowest Tm value at 211°C The highest Tm value for GG hydrogel incorporated with Cloisite15A, also shows optimum thermal behavior in TGA thermogram and is contributed by the great barrier property of Cloisite15A, which enhanced the overall hydrogel thermal stability of the samples It can be concluded that increasing the thermal stability of GG hydrogels incorporated clays was attributed to the function of MMT particles which act as a barrier for the mass transportation during the decomposition process and significantly improved the thermal stability of materials.26,41 Cell studies The biocompatibility of GG hydrogel and MMT (clays) was investigated through cell viability and cell proliferation after being incubated for 24, 48, and 72 h Figure 7(a) to (n) illustrates the images of human skin fibroblast cells (CRL2522, ATCC) adhering onto the surface of GG hydrogel, GG/ Na-MMT, GG/CTAB-MMT, and GG/Cloisite15A hydrogels after being cultured for 72 h It clearly can be seen that GG hydrogel is not cytotoxic to the CRL2522 cells as rounded cells were observed throughout the duration of incubation The inclusion of 10wt% Na-MMT into the GG hydrogels (GG/Na-MMT10) shows a good sign in terms of cell growth as the cells transform from rounded shape after incubation for 24 h to elongated cells after 72 h of incubation (Figure 7(g) to (i)) For GG hydrogel containing synthesized organophilic-montmorillonite, that is, CTAB-MMT, it shows the cells were dying due to apoptosis process with limited fluorescence cells observed after being Downloaded from jbc.sagepub.com by guest on May 17, 2016 14 Journal of Bioactive and Compatible Polymers Figure 7. Fluorescence microscope images of the cell viability of (a-c) tissue culture polystyrene plate (TCPP) (d-f) gellan gum (GG) hydrogel (g-i) GG/Na-MMT10 hydrogel (j-l) GG/CTAB-MMT10 hydrogel (mo) GG/Cloisite15A5 hydrogel after incubated for 24, 48, and 72 h cultured for 24 h (Figure 7(j)) The viability of the cells on GG/CTAB-MMT10 was totally disappearing after being incubated for 48 and 72 h (Figure 7(k) and (l)) Meanwhile, for GG/Cloisite15A hydrogel, live cells were observed after being incubated for 24 and 48 h, but no trace of live cells was observed after being cultured for 72 h To quantify the cell numbers on the hydrogels, proliferate study of the samples was done by using Cell Titer 96 aqueous one solution assay The proliferation results show that the inclusion of clays into GG hydrogel significantly affected the cell growth especially with the sample containing CTAB-MMT and Cloisite15A (Figure 8) Our results show that the GG/CTAB-MMT10 hydrogels give no absorbance reading (at 492 nm) indicating all the cells died Better results were observed for GG hydrogel containing commercially treated montmorillonite, that is, Cloisite15A which keep some cells alive with 1000 cells/well after being incubated for 24 h Nevertheless, the cell number on GG/Cloisite15A reduced to below 500 cells/well after being cultured for 48 h and totally died after being cultured for 72 h Without treatment, Na-MMT incorporated GG hydrogels exhibited significant cell growth particularly from 48 to 72 h of incubation The cell number at ≈8200 cells/ well was observed after being cultured for 72 h on GG/Na-MMT hydrogel with increased almost twofold than after being cultured for 48 h Downloaded from jbc.sagepub.com by guest on May 17, 2016 15 Mohd et al Figure 8. Cell proliferation of human skin fibroblast cells (CRL2522, ATCC) on tissue culture polystyrene plate (TCPP), gellan gum (GG), GG containing Na-MMT (GG/Na-MMT10) and GG containing Cloisite15A (GG/Cloisite15A) incubated for 24, 48, and 72 h Error bars indicated standard deviation (n = 3) Statistical analysis using one way ANOVA followed by post hoc test shown by different letters was statistically different (P ≤ 0.05) The low number of cells observed on GG/CTAB-MMT hydrogels results indicated the influence of the toxicity effect of CTAB surfactant which killed the cells Our results contradicted the results reported by Sirousazar and co-workers, which claimed the erythroleukemia cell line (K562, ATCC) is non-toxic on poly(vinyl alcohol) containing wt% modified montmorillonite with CTAB surfactant.13 The physical and mechanical properties, including hardness and roughness of hydrogel containing clays could be another reason cells were dying.17,42 In addition, the solubility of clays in biopolymer solutions also plays an important role which may influence the growth of cell as reported by a previous study.30 They demonstrated that the reduction of weight percentage of polyethylene glycol (PEG) in polypeptide-PEG exhibits better cell adhesion However, the poor water solubility of extremely hydrophobic CTAB-MMT in gels was inhibiting the cell adhesion and thus did not provide the suitable environment for cell growth Antibacterial activities The bacterial activity assays were carried out against four bacteria, that is, two Gram positive (S aureus and B cereus) and two Gram negative (E coli and K pneumoniae) through disk susceptibility test In this qualitative study, GG/CTAB-MMT10 demonstrated the sign of inhibition zones against the two species of Gram-positive bacteria, that were, S aureus (d = 14 ± 0.1 mm) and B cereus (d = 14 ± 0.1 mm) (Figure 9) However, no inhibition was observed on MH agar plates of GG, GG/Na-MMT10, and GG/Cloisite15A5 hydrogels through same agar diffusion method on all bacteria For comparison, the penicillin disk (positive control) shows inhibition zones of d = 15 ± 0.1 mm, d = 13 ± 0.2 mm for both Gram-positive bacteria S aureus and B cereus, respectively However, K pneumoniae did not show any inhibition sign for control penicillin disk compared to the weak inhibitory activity of E coli which found d = 10 ± 0.1 mm inhibition zone In fact, the difference in inhibition of hydrogel-containing clays against Gram-positive and Gram-negative bacteria mostly related to the Downloaded from jbc.sagepub.com by guest on May 17, 2016 16 Journal of Bioactive and Compatible Polymers Figure 9. Images of qualitative results of hydrogels against (a) B cereus, (b) S aureus, (c) K pneumoniae and (d) E coli on (i) penicillin disc, (ii) GG/Cloisite15A5 hydrogel, (iii) GG/CTAB-MMT10 hydrogel, (iv) GG/Na-MMT10 hydrogel, and (v) GG hydrogel structure of bacterial cell The Gram-negative bacteria has an outer membrane, including the thin membrane of peptide polyglicogen, compared to Gram-positive bacteria with the thick cell wall with no outer membrane.43 The complicated membrane of Gram-negative bacteria with a potential barrier against foreign molecules made it hard to be killed However, the possible caused inhibition of GG/CTAB-MMT10 hydrogel may be due to the presence of synthesized CTAB montmorillonite that showed toxicity effect This suggested that the presence of CTAB surfactant in hydrogel was able to interact with the outer lipid layer thus altering the permeability of bacteria cell membrane and allowing the movement of intercellular component and other metabolites to diffuse out of the cell membrane and finally may cause the death of bacteria cell.44–46 The presence of clays with the intermediate specific surface area could adsorb the bacteria and immobilized them on the clay surface Wang and co-workers have reported that the antimicrobial study of CH incorporated modified MMT gave the highest inhibition against Grampositive bacteria due to large specific surface area of clays presented compared to free standing CH and CH unmodified MMT.47 However, another study reported the presence of long-chain hydrophobic alkyl and cationic charge of quaternary ammonium group in modified CTAB-MMT strongly inhibit the growth of bacteria.48 As the conclusion, CTAB-MMT showed a better antibacterial activity than other GG free standing, GG/Na-MMT10 and GG/Cloisite15A5 against Gram-positive compared to Gram-negative bacteria Downloaded from jbc.sagepub.com by guest on May 17, 2016 17 Mohd et al Conclusion This study revealed the effect of addition of Na-MMT, CTAB-MMT and Cloisite15A into GG hydrogels to their compression strength, physical, biocompatibility and antibacterial activities The compression strengths of the GG hydrogels were optimum at (wt %) for Closite15A and 10 (wt %) for both Na-MMT and CTAB-MMT, respectively Not limited to that, the swelling properties of hydrogels show an optimum concentration at the same percentage as observed in compression strength Thermal behavior shows an improvement in degradation stability for GG hydrogel incorporated Cloisite15A than Na-MMT and CTAB-MMT hydrogels For biocompatibility studies, Na-MMT is non-cytotoxic with limited cell growth observed after 72 h incubation The presence of CTAB-MMT (even at low concentration, i.e., 10% (w/w) or 0.1 g in 100 mL solution) does not successfully support the cell growth due to the toxicity effect of CTAB-MMT and the hydrophobicity of modified MMT However, GG/CTAB-MMT10 hydrogel shows bactericidal effect against Gram-positive and Gram-negative bacteria and none for other samples To conclude, this study revealed that the GG hydrogel incorporated Na-MMT has a promising property to be improved and used as a wound dressing material Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article Funding This work was supported by a research grant from Ministry of Higher Education (Malaysia) under Exploratory Research Grant Scheme (ERGS, Grant No: 55094) References Worldwide surgical sealants, glues, wound closure and anti-adhesion markets, 2010-2017 Foothill Ranch, CA: MedMarket Diligence, LLC, 2010, p 262 Cojocariu A, Profire L, Aflori M, et al In vitro drug release from chitosan/Cloisite 15A hydrogels Appl Clay Sci 2012; 57: 1–9 Kabiri K, Mirzadeh H, Zohuriaan-Mehr MJ, et al Chitosan-modified nanoclay–poly(AMPS) nanocomposite hydrogels with improved gel strength Polym Int 2009; 58: 1252–1259 Kabiri K, Mirzadeh H and Zohuriaan-Mehr M Chitosan modified MMT-poly(AMPS) nanocomposite hydrogel: heating effect on swelling and rheological behavior J Appl Polym Sci 2010; 116: 2548–2556 Williams LB, Holland M, Eberl DD, et al Killer clays! 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131: 40079 Downloaded from jbc.sagepub.com by guest on May 17, 2016 ... JT, Santos TC, Martins L, et al Gellan gum injectable hydrogels for cartilage tissue engineering applications: in vitro studies and preliminary in vivo evaluation Tissue Eng Part A 2009; 16: 343–353... Administration (US FDA) and the European Union (EU) for use in the food industry It is also used as a scaffold material for tissue engineering application. 14,15 GG has been reported to have good... Cloisite15A hydrogels were examined by XRD analysis (Figure 2) The XRD spectra for GG hydrogel with MMT did not show any peak in the range of 2θ = 3°–10° compared to those for MMT in pure forms (Na-MMT,