Gellan gumclay hydrogels for tissue engineering application

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(.

Journal of Bioactive and Compatible Polymers

1 –19

© The Author(s) 2016 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0883911516643106

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JOURNAL OF

Bioactive and Compatible Polymers

Gellan gum/clay hydrogels for

tissue engineering application:

Mechanical, thermal behavior,

cell viability, and antibacterial

properties

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 organophilic-montmorillonite (CTAB-MMT) The Na-MMT, CTAB-MMT, and a commercial organophilic-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).

1 School of Fundamental Science, Universiti Malaysia Terengganu, Kuala Terengganu, Malaysia

2 Institute of Marine Biotechnology, Universiti Malaysia Terengganu, Kuala Terengganu, Malaysia

Corresponding author:

Khairul Anuar Mat Amin, School of Fundamental Science, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia

Email: kerol@umt.edu.my

Original Article

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 lion), followed by lacerations (20 million), diabetic ulcers (11 million), and burn wounds (10 mil-lion) 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

exam-ple, chitosan (CH) and gellan gum (GG) derived from chitin and Pseudomonas elodea,

respec-tively, 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 bio-polymer, 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

mate-rials 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 bio-compatibility 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 character-istics 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 antibacte-rial activities Three types of MMT with different basal-spacing values, that is,

sodium-montmoril-lonite (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

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 transmis-sion rates (WVTR) The thermal behavior of the GG hydrogels was examined by using thermo-gravimetric 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,

Gram-positive 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 capac-ity 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 frac-tions and pre-condition for next 24 h prior to any characterizafrac-tions The GG hydrogels containing Na-MMT, CTAB-MMT, and Cloisite15A will be hereafter referred as GG/Na-MMT, GG/CTAB-MMT, 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

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° inci-dent 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 rela-tive 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

Gelfraction % = W /W ( ) ( 2 1) × 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 solu-tion (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 = W( wet−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

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, Perkin-Elmer-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 approxi-mately 4 mg

Cell studies

Routine cell-culture The culture of normal human skin fibroblast cells (CRL-2522-ATCC) was

pre-pared 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 sub-cultured 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 trans-form the films to hydrogels Prior to testing, the hydrogel was sterilized in a laminar airflow cham-ber under UV radiation for 20 min Three replicates were used for each type of hydrogel samples

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-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium), inner salts; MTS (a)] with electron-coupling 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 bac-teria 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 1 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 scis-soring 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%

The XRD patterns of the Na-MMT, CTAB-MMT, and Cloisite15A samples are shown in Figure 2 and summarized in Table 1 The XRD pattern of Na-MMT shows typical features of basic montmoril-lonite 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 montmo-rillonite 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 com-mercial 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)

Figure 1 ATR-FTIR spectra of (a) Na-MMT, (b) commercial Cloisite15A, and (c)

organo-montmorillonite, CTAB-MMT.

Table 1 Elemental analysis, basal spacing (D001 ), and structural data of sodium-montmorillonite (Na-MMT), organophilic montmorillonite (CTAB-(Na-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 D a is the average pore radius).

Sample Element component (%) D 001 (Å) S BET (m 2 g −1 ) V t (cm 3 g −1 ) D a (nm)

Carbon Hydrogen

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.

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 MMT, which correlates well to increase in basal-spacing values of CTAB-MMT 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 3 and Table 2 depict the compression strength of GG/Na-MMT, GG/CTAB-MMT, and GG/Cloisite15A hydrogels at different percent-age loadings It is worth to note that GG hydrogel without any filler shows highest stress-at-break

Figure 3 (a) Stress-at-break and (b) Strain-at-break of GG hydrogel and GG hydrogel incorporated

Na-MMT, CTAB-Na-MMT, and Cloisite15A at different loadings.

(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 alkylam-monium 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 prob-ably due to the improvement of cross-linking behavior among the hydrogel network which creates

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 )

GG 15 ± 1.6 330 ± 20 6.5 ± 1.0 117 ± 3 74 ± 1.8 1193

GG/Na-MMT2 8 ± 0.7 203 ± 21 5.3 ± 0.4 31 ± 5 77 ± 1.7 1890

GG/Na-MMT5 11 ± 0.5 197 ± 13 7.2 ± 0.3 33 ± 8 79 ± 1.7 1533

GG/Na-MMT10 14 ± 1.6 222 ± 6 7.6 ± 0.8 69 ± 12 80 ± 1.1 1484

GG/Na-MMT15 12 ± 0.7 196 ± 10 7.5 ± 0.6 55 ± 4 80 ± 1.2 1429

GG/Na-MMT20 11 ± 0.8 186 ± 19 7.3 ± 0.9 28 ± 1 80 ± 1.8 1106

GG/CTAB-MMT2 9 ± 0.7 202 ± 2 6.1 ± 0.6 62 ± 14 72 ± 2.1 1555

GG/CTAB-MMT5 10 ± 0.4 217 ± 4 5.7 ± 0.7 65 ± 4 76 ± 0.9 1556

GG/CTAB-MMT10 11 ± 0.6 236 ± 13 6.3 ± 0.8 60 ± 1 74 ± 1.7 1576

GG/CTAB-MMT15 8 ± 0.6 195 ± 5 5.8 ± 0.5 50 ± 13 79 ± 0.9 1604

GG/CTAB-MMT20 8 ± 0.5 204 ± 9 5.5 ± 0.6 70 ± 6 80 ± 0.3 1596

GG/Cloisite15A2 11 ± 1.0 291 ± 12 5.4 ± 0.4 35 ± 3 82 ± 0.3 1603

GG/Cloisite15A5 14 ± 0.6 295 ± 7 6.2 ± 0.7 60 ± 11 75 ± 0.7 1649

GG/Cloisite15A10 11 ± 0.6 247 ± 9 5.7 ± 0.5 82 ± 29 69 ± 0.3 1600

GG/Cloisite15A15 12 ± 0.4 271 ± 14 5.7 ± 0.6 79 ± 13 75 ± 14 1634

GG/Cloisite15A20 10 ± 1.1 289 ± 7 4.0 ± 0.7 68 ± 7 74 ± 0.2 1644

Na-MMT: sodium-montmorillonite; CTAB-MMT: organophilic montmorillonite, and Cloisite15A: commercial montmo-rillonite.

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 car-bonyl 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 6 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

Figure 4 FTIR spectrum of hydrogels (a) gellan gum (GG), (b) GG/Na-MMT10, (c) GG/CTAB-MMT10,

and (d) GG/Cloisite15A5.