antibacterial activity and cytotoxicity of chitosan

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PACCON2011 (Pure and Applied Chemistry International Conference 2011)

Antibacterial Activity and Cytotoxicity of Chitosan and Quaternized Chitosans

P. Tanjak, P. Ngamviriyavong and W. Janvikul*

National Metal and Materials Technology Center, Pathumthani, Thailand 12120

*E-Mail: [email protected]

Abstract: Antibacterial activity and cytotoxicity of chitosan (CS) and quaternized chitosans, i.e., chitosan-graft-poly(2-methacryloyloxy ethyl trimethylammonium chloride) (CS-g-PMEA) and N,O-[(2-hydroxy-3-trimethyl ammonium) propyl] chitosan chloride (CS-GTMAC), were comparatively studied. CS-g-PMEA with a degree of PMEA-grafting about 0.78 and CS-GTMAC with a degree of quaternization about 1.04 were used in this study. The antibacterial activity and cytotoxicity of CS, CS-g-PMEA and CS-GTMAC were assessed, in terms of minimum inhibitory concentration (MIC) and half maximal inhibitory concentration (IC50), respectively. The antibacterial activity was evaluated against both Gram-positive, i.e., Staphylococcus epidermidis RP62A and Staphylococcus aureus ATCC 25923, and Gram-negative, i.e., Escherichia coli ATCC 11775 and Pseudomonas aeruginosa DMST4739, bacteria, using a shake flask method. It was noted that CS-g-PMEA could most effectively inhibit the growth of the tested bacteria. Its MIC values were in the range of 32-128 g/mL. The MIC values of CS-GTMAC were in the range of 64-1000 g/mL, indicating that the trimethylammonium chloride in the substituted chitosan (CS-GTMAC) was less capable of inhibiting the bacteria than that in the grafted chitosan (CS-g-PMEA). Chitosan, on the other hand, demonstrated the least antibacterial activity. Its MIC values were found in the range of 512-1000 g/mL against the Gram-positive bacteria and greater than 5000 g/mL against the Gram-negative bacteria. The in vitro cytotoxicity of all three materials was assessed by an MTT assay, using L929 mouse fibroblasts and normal human fibroblasts (NHF). The results revealed that all the tested materials appeared non-cytotoxic to the L929 and NHF cells. Moreover, the IC50 values of both quaternized chitosans were greater than their MIC values. Introduction

Chitosan is a natural, nontoxic biopolymer composed of N-acetylated glucosamine and glucosamine units linked by (1-4) glycosidic bonds. It exhibits various promising biological activities, e.g., antimicrobial activity, biodegradability and biocompatibility [1]. The antibacterial activity of chitosan was found to be influenced by a number of factors, including molecular weight and environmental pH [2]. In general, chitosan can be dissolved in aqueous acidic solution (pH<6.5), restricting its antibacterial activity under neutral aqeous conditions.

To improve the antibacterial activity of chitosan in aqueous media, a number of chitosan derivatives were synthesized. The antibacterial activities of chitosan salts from amino acids [3] and some quaternized chitosans, e.g., quaternized N-alkyl chitosans [4-5] and N,O-[(2-hydroxy-3-trimethyl ammonium) propyl] chitosan chloride (CS-GTMAC) [6], were reported. These quaternized chitosans were water-soluble and possessed greater antibacterial activities than chitosan even under neutral aqueous conditions.

A graft copolymer of chitosan and poly(2-methacryloyloxy ethyl trimethylammonium chloride), another form of quaternized chitosan, was also prepared and used as a flocculant [7]. Its antibacterial activity has, however, not yet been explored.

The main objective of this study was to study the antibacterial activity of chitosan-graft-poly(2-methacryloyloxy ethyl trimethylammonium chloride) (CS-g-PMEA) against Staphylococcus epidermidis RP62A, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 11775, and Pseudomonas aeruginosa DMST4739, in comparison with those of a quaternized chitosan (2-hydroxy-3-trimethyl ammonium chloride propyl group substituted chitosan, CS-GTMAC) and chitosan. The cytotoxicity of all studied materials in contact with L929 mouse fibroblasts or normal human fibroblasts was also evaluated.

Materials and Methods

Materials: Chitosan (CS), from shrimp shells,

having MW ≈ 700 kDa (determined by GPC) and % deacetylation ≈ 95.5%, (determined by solid state 13CNMR), was purchased from A.N. lab. Glycidyl trimethyl ammonium chloride (GTMAC) and [2-(methacryloyloxy) ethyl] trimehtyl ammonium chloride (MEA), 80 wt% solution in water, were purchased from Aldrich. Potassium persulfate and acetic acid were purchased from Fluka and RCI Labscan, respectively. All reagent-grade chemicals were used as received. Staphylococcus epidermidis RP62A and Escherichia coli ATCC 11775 were received from Thailand Biodiversity Center, National Center for Genetic Engineering and Biotechnology, whereas Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa DMST4739 were purchased

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from the National Institute of Health, Department of Medical Sciences, Thailand.

Preparation of chitosan-graft-poly(2-methacryloyloxy ethyl trimethylammonium chloride) (CS-g-PMEA): CS (2.0 g) was first dissolved in 40 mL of 2% v/v acetic acid aqueous solution. To the chitosan solution, 12.16 mL of 80 wt% solution in water of MEA (4 mole equivalent based on CS unit) and 97.5 mg of potassium persulfate, used as an initiator, (0.75% mole of MEA used) were added. The whole solution was stirred at 80oC for 1 h before being precipitated with methanol. The resulting precipitate was repeatedly washed with methanol and dried under reduced pressure.

Preparation of N,O-[(2-hydroxy-3-trimethyl ammonium) propyl] chitosan chloride (CS-GTMAC): CS (2.0 g) was initally dissolved in 40 mL of 2% v/v acetic acid aqueous solution. Then, 6.29 mL of GTMAC (4 mole equivalent based on CS unit) was added. The whole solution was stirred at 60oC for 6 h before being precipitated with 50% v/v acetone in methanol. The resulting precipitate was repeatedly washed with acetone and dried under reduced pressure.

Chemical structure analysis: The chemical structure, degree of substitution, and degree of grafting of the modified chitosans were directly determined by 1H NMR spectroscopy (Bruker DPX-300 spectrometer).

Antibacterial activity assessment: The antimicrobial activities of chitosan and the quaternized chitosans were evaluated using a shake flask method. S. epidermidis, S. aureus, E. coli, and P. aeruginosa bacteria were individually first overnight cultured in nutrient broth (NB) (Difco) at 37oC before use. The bacterial cultures were subsequently diluted with NB to a concentration of approximately 106 CFU/mL. Meanwhile, CS, CS-g-PMEA or CS-GTMAC was dispersed in NB at various concentrations in flasks. NB without a tested sample was used as a blank. After 24 h of incubation, the whole dispersions of each tested sample in the bacteria cultures were analyzed by a plate count technique, to determine a minimum inhibitory concentration (MIC) of each tested material. MIC was defined as the lowest concentration of an antimicrobial agent which could completely inhibit the growth of a given microorganism after overnight bacterial incubation with the material.

Cytotoxicity assessment: L929 mouse fibroblasts and normal human fibroblasts (NHF) were individually seeded into 96-well microplates at a concentration of 1103 and 3103 cells/well, respectively, in Dubelco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and incubated at 37oC. After 48 h of incubation, the medium was replaced with a fresh medium containing various concentrations of CS, CS-g-PMEA or CS-GTMAC and reincubated for an additional 48 h. Subsequently, a fresh medium and 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were added into the wells, and the whole microplates were again incubated at 37oC for 4 h. The collected purple formazan products were ultimately dissolved in

dimethyl sulfoxide (DMSO) and glycine buffer for the measurement of optical density values, later converted to the percentage of viability of cells (% cell viability), using a Microplate Reader at 570 nm. Cytotoxicity of all tested materials were reported in terms of the half maximal inhibitory concentration (IC50), defined as the maximal concentration of a material which could inhibit the growth of cells at 50% cell viability.

Results and Discussion

Two different modified chitosans, containing trimethyl ammonium salt groups, were prepared via two different approaches in this study. Figure 1 displays the reaction schemes for grafting PMEA onto chitosan (Figure 1 (a)), yielding CS-g-PMEA, and quaternizing chitosan with GTMAC (Figure 1 (b), yielding CS-GTMAC.

The presence of trimethyl ammonium protons

(-N(CH3)3+) in both CS-g-PMEA and CS-GTMAC

were observed in their 1H NMR spectra (not shown here) at 3.10 and 3.13 ppm, respectively. The degree of PMEA-grafting and the degree of quaternization of chitosan calculated from the 1H NMR spectra were about 0.78 and 1.04, respectively.

The antibacterial activities of CS-g-PMEA, CS-GTMAC and chitosan against Gram-positive bacteria, i.e., S. epidermidis and S. aureus, and Gram-negative bacteria, i.e., E. coli and P. aeruginosa, were evaluated at the neutral pH and reported in terms of MIC values in Tables 1 and 2, respectively.

Table 1: MIC values of CS-g-PMEA, CS-GTMAC and chitosan tested against S. epidermidis and S. Aureus

Samples* MIC (g/mL) S. epidermidis S. aureus

CS-g-PMEA 32 32 CS-GTMAC 64 256 CS 1000 512

*n=2

Figure 1: Schematic preparations of (a) CS-g-PMEA and (b) CS-GTMAC

(a)

(b)

O

O

N

Cl+

2%v/v acetic acid

80oC, 1 h

O

HONH

O

OH

(MEA)(CS)

(CS-g-PMEA)

K2S2O8

OO

NCl

O

HONH2

OH

OO

HONH2

OH

O

n

O

HONH2

OH

ON

O

Cl

+O

HONH

OH

O60oC, 6h

OH

NCl

(CS) (GTMAC)

(CS-GTMAC)

O

HONH2

OH

O

2%v/v acetic acid

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Table 2: MIC values of CS-g-PMEA, CS-GTMAC and chitosan tested against E. coli and P. aeruginosa

Samples* MIC (g/mL) E. coli P. aeruginosa

CS-g-PMEA 32 128 CS-GTMAC 256 1000 CS >5000 >10000

*n=2 As revealed in Tables 1 and 2, the MIC values of

CS-g-PMEA and CS-GTMAC were 32 g/mL and in the range of 64-256 g/mL, respectively, when they were tested against the Gram-positive bacteria. On the other hand, the values became greater when they were in contact with the Gram-negative bacteria: in the ranges of 32-128 g/mL for CS-g-PMEA and 256-1000 g/mL for CS-GTMAC. Nevertheless, the MIC values of both quaternized chitosans appeared much lower than those of CS: in the ranges of 512-1000 g/mL against the Gram-positive bacteria and greater than 5000 g/mL against the Gram-negative bacteria. It was previously reported that chitosan could inhibit Gram-positive bacteria better than Gram-negative bacteria [8].

The greater antibacterial activities of CS-g-PMEA and CS-GTMAC, compared with that of chitosan, was attributed to the presence of the positive charges of quaternary ammonium salts in their structures. The introduction of the quaternary ammonium salts onto the chitosan backbone via grafting reaction and subsitution not only enhanced the water solubility of chitosan but also increased its antimicrobial activity at the neutral media condition. However, the trimethylammonium chloride in the substituted chitosan (CS-GTMAC) was found to be less effective in inhibiting the antibacterial growth, compared with that in the grafted chitosan (CS-g-PMEA).

The structures of the substituted chitosan and the grafted chitosan were depicted in Figure 2(a) and 2(b), respectively. The positive charges of 2-hydroxy-3-trimethyl ammonium propyl groups, in CS-GTMAC, were homogeneously distributed on the chitosan units, whereas, in CS-g-PMEA, the ammonium positive charges were mainly located at the grafting chains of poly(2-methacryloyloxy ethyl trimethyl ammonium chloride). The long grafting branches of CS-g-PMEA much more readily enable the positive charges to attach to the bacterial cell walls than the short chains of the substituted positive groups in CS-GTMAC, resulting in the relatively lower MIC values of CS-g-PMEA.

Table 3 shows the IC50 values of each tested material after being exposed to mouse fibroblasts (L929) or normal human fibroblasts for 48 h. It was noted that CS-g-PMEA, CS-GTMAC and chitosan appeared non-toxic to the tested fibroblast cells. Moreover, the IC50 values of both CS-g-PMEA and CS-GTMAC were found to be greater than their MIC values.

Table 3: IC50 values of CS-g-PMEA, CS-GTMAC and chitosan

Samples* IC50 (g/mL)

L929 NHF CS-g-PMEA 625 2000 CS-GTMAC 5000 2000 CS 5000 5000

*n=8 Conclusions

The antibacterial activity of CS-g-PMEA, evaluated against both Gram-positive and Gram-negative bacteria, was found to be higher than those of CS-GTMAC and chitosan, respctively. All three materials after being exposed to the skin fibroblasts, i.e., L929 mouse fibroblasts and normal human fibroblasts, were found to be non-cytotoxic. Furthermore, the IC50 values of both quaternized chitosans were greater than their MIC values.

Acknowledgement

This research was financially supported by

National Metal and Materials Technology Center (project code: MT-B-52-BMD-07-174-I).

References [1] C. Qin, H. Li, Q. Xiao, Y. Liu, J. Zhu and Y. Du,

Carbohydr. Polym., 63 (2006), pp. 367–374. [2] H.K. No, N.Y. Park, S.H. Lee and S.P. Meyers,

Int. J. Food Microbiol., 74 (2002), pp. 65–72. [3] P. Tanjak, P. Uppanan, R. Chainoy, P.

Ngamviriyavong, and W. Janvikul, Effect of environmental pH and MW of chitosan salts on their biological properties, Pure and Applied Chemistry International Conf. Proc., Pitsanulok, Thailand, 2009, pp. 271–274.

[4] C.H. Kim, J.W. Choi, H.J. Chun and K.S. Choi, Polym. Bull., 38 (1997), pp. 387–393.

[5] Z. Jia, D. Shen and W. Xu, Carbohydr. Res., 333 (2001), pp. 1–6.

[6] H.S. Seong, H.S. Whang and S.W. Ko, J. App. Polym. Sci., 76 (2000), pp. 2009–2015.

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+ +

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Figure 2: The structures of (a) the substituted chitosan (CS-GTMAC) and (b) the grafted chitosan (CS-g-PMEA)

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(b)

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[7] J.P. Wang, Y.Z. Chen, X. Wu and H.Q. Yu, Chemosphere, 66 (2007), pp. 1752–1757.

[8] Y. Li, X.G. Chen, N. Liu, C.S. Liu, C.G. Liu, X.H. Meng, L.J. Yu and J.F. Kenendy, Carbohydr. Polym., 67 (2007), pp. 227–232.

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The effect of bead size on drug loading and releasing characteristic of antibiotic loaded 3DP calcium phosphate bead

F. Thammarakcharoen and J. Suwanprateeb *

National Metal and Materials Technology Center,

National Science and Technology Development Agency, 114 Paholyothin Road, Klong 1, Klongluang, Pathumthani 12120 Thailand

*E-mail: [email protected]

Abstract: Antibiotic loaded polymethyl methacrylate (PMMA) beads are commercially available for treating patients with osteomyelitis or bone infection. These beads provided local, sustained and high concentrations of antimicrobial agents to the area of infection without systemically exposing an individual to antibiotic levels that often would result in numerous toxic side effects. However, several drawbacks have been associated with PMMA use, including the requirement for material removal by a second surgical procedure and poor antibiotic elution properties. Recently, antibiotic loaded calcium phosphate beads which were prepared by phosphorization of three dimensionally printed (3DP) calcium sulfate dihydrate were developed. These beads could function as both drug carrier and bone graft which can integrate to the bone. The need of second injury and use of additional grafting materials were; thus, eliminated. However, since the bone defect size of patients can vary, the availability of different size of antibiotic loaded calcium phosphate beads would be beneficial for treatment as the defect can be effectively filled leading the fast new bone formation. In this study, the effects of varying size of calcium phosphate beads on the antibiotic uptake and releasing characteristic were investigated. Three sizes of beads (3 mm, 5 mm and 7 mm.) were prepared and loaded with two kinds of antibiotics including gentamicin and vancomycin. Microstructure and phase composition were characterized by x-ray diffraction, scanning electron microscopy, energy dispersive spectroscopy and mercury porosimeter. Drug loading and releasing were analyzed using UV-VIS spectrophotometer. It was found that decreasing bead size led to the decrease in density and increase in porosity and pore size of the beads. Drug loading and releasing rate of both antibiotics were also found to increase with decreasing bead size. This was related to the difference in microstructure of beads resulted from the processing of different size of the beads. Introduction

A major challenge in treating osteomyelitis or bone infection is to obtain high bactericidal concentrations of antibiotic at the local area of infection and to maintain a highly effective concentration of antibiotic in the infected area for a sufficient period of time. It has been shown that placing materials loaded with high concentration of antibiotics in the infected bone area was able to eradicate or suppress the infectious process in the patients with osteomyelitis effectively

without systemically exposing an individual to antibiotic levels that often would result in numerous toxic side effects [1-2]. Polymethylmethacrylate (PMMA) is one of a widely used implant materials in orthopaedics and has been used successfully with antibiotics impregnation [3-5]. However, one problem inherent in the local placement of antibiotics loaded PMMA is that it requires subsequent surgery for removal and replacement with bone grafts. Recently, antibiotic loaded calcium phosphate beads which were prepared by phosphorization of three dimensionally printed (3DP) calcium sulfate dihydrate were developed [6]. Since calcium phosphate has excellent biocompatibility and bioactivity, the beads could function as both drug carrier and bone graft which can integrate to the bone eliminating the need of reconstruction by additional grafting materials. However, the bone defect size of individual patients can vary. The need for different size of antibiotic loaded calcium phosphate beads is foreseen as an advantage for treatment as the bone defect can be effectively filled leading the fast new bone formation. However, changing the bead size may affect the function of antibiotic release. In this study, the effect of varying size of calcium phosphate beads on the antibiotic uptake and release characteristic was ; thus, investigated.

Figure 1. Chemical structures of a) gentamicin and b) vancomycin.

(a)

(b)

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Materials and Methods

1. Materials Raw materials used in this study were calcium

sulfate hemihydrate (Lafarge Prestia Co.,Ltd, Thailand) and pre-gelatinized starch (Thaiwah Co.,Ltd, Thailand). These materials were supplied in the form of powders and used without further sieving. Antibiotics used were gentamicin (T.P Drug Laboratories (1969) Co., Ltd) and vancomycin (CJ CheilJedang Corporation, Korea). The chemical structures of these drugs are shown in figure 1.

2. Specimen preparation Calcium sulfate hemihydrate (CaS) powders was

mixed with pre-gelatinized starch powders using a mechanical blender and loaded into a three dimensional printing machine (Z400, Z Corporation) to print three sizes of spherical specimens including 3 mm (S50), 5 mm (S75) and 7 mm (S100). Water-based binder was used as a jetting media. The as-fabricated samples were then transformed to calcium phosphate (CaP) by phosphorization reaction. 1M of disodium hydrogen phosphate solution was prepared. All samples were immersed in the solution and kept at 80 ºC for 48 hours in the oven. Samples were then taken out, rinsed by distilled water and oven dried.

3. Material characterization Powder X-ray diffraction was carried out by using

a JEOL JDX 3530 X-ray diffractometer with Co K-alpha radiation in the range of 10-80 º 2θ, a counting time of 0.5 second and a step angle of 0.02 º. JCPDS files were used to identify the crystalline phase composition. Microstructure of the specimens were examined by using a scanning electron microscope (JEOL JSM-5410) at an accelerating voltage of 20 kV. All samples were gold sputtered prior to the observation. Bulk density of as-fabricated 3DP was determined by weighing each specimen by a digital balance (Mettler Toledo PB4002-S) and dividing by volume of the specimen which was determined from a vernier caliper (Mitutoyo) measurements. The porosity and pore size of all samples were measured by mercury porosimetry analyser (Pore Master, Quantachrome Instruments).

4. Drug loading The calcium phosphate beads were loaded with two

types of antibiotics; using vacuum method. The concentration of antibiotics in the sample beads were then determined by elution in 2.4 M HCl and analysed by using UV-VIS spectrophotometer (Jasco V-530).

5. Drug release In order to determine the drug release profile, the

drug loaded calcium phosphate beads (S100 : 3 beads, S75 : 6 beads and S50 : 21 beads) were submerged in 100 ml of simulated body fluid (SBF) [7] at 37 ºC. When the specified times for data collection were reached, 10 ml of SBF was withdrawn by pipetted and replaced immediately with 10 ml of fresh SBF

medium. Gentamicin and vancomycin concentration in the collected samples were measured spectrophotometrically by using UV-VIS spectrophotometer (Jasco V-530) at the wavelength of 254 nm and 280 nm respectively.

Results and Discussion

Figure 2 shows the XRD patterns of as-fabricated and converted 3DP samples by phosphate solution. After conversion, calcium sulfate dihydrate which is typically the main phase in all as-fabricated 3DP samples similarly transformed to hydroxyapatite regardless of the bead size. Pore size, porosity and density of all samples generally decrease after phosphorization as shown in table 1 excepting the porosity of S-100 sample which slightly increases after conversion. Prior to conversion, sample CaS-S100 has the lowest pores size, lowest porosity and greatest density. In contrast, sample CaP-S50 seems to have the lowest pore size and highest porosity while sample CaP-S100 has the greatest density after conversion. These differences are possibly due to the initial pore size, porosity and density of as-fabricated sample in coupled with the size of the bead itself which cause the difference in the phosphorization efficiency resulting in the different degree of dissolution/precipitation of new calcium phosphate crystal.

Figure 2. XRD patterns of as-fabricated and converted 3DP samples.

Table 1: Pore size, porosity and density of the samples

Samples Median pore size

(μm)

Porosity (%)

Density (g/cm3)

CaS-S50 23.89 76.15 0.789

CaS-S75 22.81 85.12 0.739

CaS-S100 17.10 59.61 0.838

CaP-S50 0.13 75.07 0.620

CaP-S75 0.24 61.26 0.531

CaP-S100 0.15 63.92 0.622

Figure 3 shows the microstructure of as-fabricated

and converted samples of various bead sizes. It can be seen that the as-fabricated sample is highly porous comprising entanglement of rod-like calcium sulfate crystals. After conversion to calcium phosphate, the

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microstructures of all converted samples appear denser and the formation of new finer needle-like calcium phosphate crystals is evident. This is in accordance with the decrease in pore size values as seen in table 1. However, no significant difference in the microstructure amongst CaP-S50, CaP-S75 and CaP-S100 samples is observed. Figure 4 shows the amount of antibiotics uptake per volume of the bead in different size of calcium phosphate beads. Comparing between two types of antibiotics, gentamicin could be loaded into the CaP beads at greater amount than vancomycin. Gentamicin has smaller molecular size and lower molecular weight than vancomycin. So gentamicin molecule can penetrate through the pore of the CaP beads more readily. Comparing amongst three sizes of beads, antibiotics uptake of CaP-S50 is the greatest followed by CaP-S75 and CaP-S100 respectively. This may be due to the highest porosity of CaP-S50 sample compared to others. In addition, the diameter of the bead is directly related to the distance for antibiotics solution to diffuse into the core of the bead. Therefore, the decrease in the diameter of the bead will increase the rate of antibiotics diffusion. The antibiotics loading efficiency; thus, follows the decreasing order of the bead size.

Figure 3. Microstructures of as-fabricated and converted 3DP samples.

Figure 4. Drug loading per bead volume of 3DP calcium phosphate samples.

Figure 5 shows the drug release profiles from different sizes of calcium phosphate beads. All release profiles show two-phased release patterns; burst release at the initial period and follows by a slow release phase. Comparing between two types of antibiotics, the release rate of gentamicin is higher than that of vancomycin. This is again due to the smaller molecular size and lower molecular weight of gentamicin than vancomycin. So gentamicin molecule can diffuse out of the CaP beads more readily. Comparing amongst three sizes of beads, antibiotics release rate of CaP-S50 is the greatest followed by CaP-S75 and CaP-S100 respectively. Similarly to the drug loading, the diameter of the bead is directly related to the distance for media solution to diffuse into the core of the bead and dissolve the antibiotics. Therefore, the decrease in the diameter of the bead will increase the diffusion rate of media solution and the increase in release rate of antibiotics. The antibiotics release rate; thus, follows the decreasing order of the bead size.

Figure 5. Drug release profiles of antibiotics from three sizes of calcium phosphate beads; a) gentamicin release and b) vancomycin.

(a)

(b)

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Conclusions

It is concluded that varying sizes of calcium phosphate beads which were prepared by the phosphorization of 3DP samples can affect the loading and release rate of antibiotics (gentamicin and vancomycin). Both drug loading and release rate increase with the decrease in bead size. This is related to the difference in microstructure, pore characteristics and density of beads resulted from the processing of different size of the beads and also from geometrical effect due to the diameter of the beads themselves. References [1] M. Diefenbeck, T. Mückley, G.O. Hofmann,

Prophylaxis and treatment of implant-related infections by local application of antibiotics, Injury: International Journal of the Care of the Injured. 37 (2006), pp. S95-S104.

[2] G.H.I.M. Walenkamp, L.L.A. Kleijn and M. de Leeuw, Osteomyelitis treated with gentamicin-PMMA beads: 100 patients followed for 1-12 years, Acta Ortho. 69 (1998), pp. 518–522.

[3] P. Anguita-Alonso, M.S. Rouse, K.E. Piper, D.J. Jacofsky, D.R. Osmon and R. Patel, Comparative study of antimicrobial release kinetics from polymethylmethacrylate, Clin. Ortho. Rel. Res. 445 (2006), pp. 239–244.

[4] K. Anagnostakos, O. Fürst and J. Kelm, Antibiotic-impregnated PMMA hip spacers: Current status, Acta Ortho. 77 (2006), pp. 628–637.

[5] P.H. Hsieh, C.L. Tai, P.C. Lee and V.H. Chang, Liquid gentamicin and vancomycin in bone cement: A potentially more cost-effective regimen, J. Arthrop. 24 (2009), pp.125-130.

[6] J. Suwanprateeb, W. Suvannapruk and K. Wasoontararat, Low temperature preparation of calcium phosphate structure via phosphorisation of 3D-printed calcium sulfate hemihydrate based material, J. Mater. Med. 21 (2010), pp. 419-429.

[7] T. Kokubo and H. Takadama, How useful is SBF in predicting in vivo bone bioactivity ?, Biomaterials. 27 (2006), pp. 2907-2915.

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Effect of Laser Energy, Used in Heat Assisted Magnetic Recording (HAMR)

Technique, on the Lubricant Depletion

S. Suktanarak, T. Jamnongkan and S. Kaewpirom*

Burapha University/Department of Chemistry, Faculty of Science, Chonburi, Thailand.

* E-mail: [email protected]

Abstract: Heat assisted magnetic recording (HAMR) is a

promising recording technique that could increase areal

storage densities beyond 1 Tb/in2 by using a tiny laser

light spot focuses onto a small region of the disk surface.

The laser rapidly heats the disk surface to temperatures

up to 400oC. The lubricant depletion characteristics due

to laser heating in HAMR were found to depend largely

on the lubricant film thickness and material. In this

study, the magnetic thin-film disks were dip coated with

two kinds of lubricants. The thickness of the lubricant

films were controlled by pulling-up the disks from the

lubricant solutions with a velocity of 1, 3 and 5 mm/s

after being immersed in the solutions for 120 s. A

conventional lubricant, Poly(tetrafluoroethylene oxide-

co-difluoromethylene oxide) (Zdol3800), and an ionic

liquid, 1-butyl-3-methyl imidazolium tetrafluoroborate

(BL-104), were used. Thermal stability of Zdol3800 and

BL-104 were examined by thermogravimetric analysis

(TGA) in the temperatures between 20 and 700oC. It was

found that the thermal degradation of BL-104 started at

300oC and completed at 430oC. The good performance of

BL-104 in terms of thermal stability was nearly

comparable to Zdol3800. After laser heating, the out-

gassing was examined by gas chromatography mass

spectroscopy (GC-MS).

Introduction In magnetic storage industry, magnetic layers of hard disks are usually coated with a thin films of perfluoropolyalkyl ether (PFPE) lubricants by dip coating process to guard against damage due to contact with the read-write head.[1,2, 4-6] The most widely used PFPE for the lubrication of magnetic disk surfaces is Zdol3800, which consists of a perfluorinated ether backbone terminated at either end by hydroxyl groups.

However, the disadvantage of PFPE are catalytical degradation by strong nucleophilic agents and strong electropositive metals, and high cost. One of the high performance lubricants is room temperature ionic

liquids (RTILs). RTILs have received much attention due to their unique chemical and physical properties, such as high thermal stability and non-flammability. Zhao and co-workers expected that BL-104, an RTIL, was good candidates to replace PFPE as a versatile lubricant.[8] There are many researchs reported some tribological properties of RTILs.[4, 8-10]

With HAMR technique, the disk surface was heated by a laser to the temperatures up to 400 oC. The coated lubricant was also heated and depleted from the disk surface. In order to search for the suitable lubricants that can withstand such high temperature in HAMR technique, in this paper, we demonstrated the lubricant behaviour after getting heated from laser source for short periods of time. The outgassing was analyzed by GC-MS.

Materials and Methods Materials

The disks used in this study were 2.5-inch glass substrate disks. Two kind of lubricants: poly(tetrafluoroethylene oxide-co-difluoromethylene oxide) or Zdol3800 (MW = 3,800) and 1-butyl-3-methyl imidazolium tetrafluoroborate or BL-104 (MW = 226.02) were used. The chemical structures of Zdol3800 and BL-104 are given in Figure 1. Both lubricants were purchased from Aldrich Chemical Company.

HO–CH2–CF2O–(CF2O)m–(CF2CF2O)n–CF2–CH2–OH

Zdol3800, where n/m is 2/3.

BL-104

Figure 1. Chemical structure of the lubricants. Thermogravimetry analysis (TGA) The TGA tests were conducted in a Mettler Toledo, TGA/SDTA 851e. The sample heated up from 30 to 700 oC at a scan rate of 10 oC/min in nitrogen atmosphere.

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Film preparations The solution of Zdol3800 and BL-104 were

prepared in 2,3-dihydrodecafluoropentane, commercial name Vertrel®, from Dupont (Thailand) Ltd. The solution concentration of Zdol3800 and BL-104 were 0.1% (w/v) and 0.05% (w/v), respectively. Then the media was dipped into and pulled up from the solution with a velocity 1, 3 and 5 mm/s, after being immersed in the solution for 120 s. The media was allowed to dry in air prior to the measurements. Surface roughness measurement

The average surface roughness measured by atomic force microscopy (AFM, XE-70) from Park Systems in ambient conditions and a scan rate of 0.5 Hz with dimensions of scan size 5x5 µm2. Depletion of lubricants

Experimental setup for the measurement system for the assessment of the lubricant performance in a HDD with the heat assisted magnetic recording technology is shown in Figure 2. A laser beam with a wavelength of 532 nm and the energy of 340 mW, irradiated on disk surface which the lubricant film deposited. In this experiment, the test disk was rotated while laser irradiated. After that the out-gassing was characterized by gas chromatography mass spectroscopy (HP 6890) with the following conditions in Table 1. Table 1: GC-MS conditions Gaschromatography HP 6890 (Agilent) Carrier Gas Helium Injector Temp. 250 oC Detector Temp. 280 oC Capillary column HP-5MS (Agilent) Oven Program Initial Temp. 40 oC Initial Time 1 min. Mass spectrometer HP 5972 (Agilent)

Figure 2. Experimental setup for the measurement system for the assessment of the lubricant performance in a HDD with HAMR technology. Results and Discussion Thermal effects on lubricants

In TGA test, thermal effects of two kind lubricants, were examined and the results are shown in Figure 3.

In figure 3 (a), Zdol was observed to have 2-step-decomposition started at 200 oC and completed at 500 oC. In the first step, it had catalytic decomposition to scission of the PFPE backbone, the resulting fragments could reasonably be expected to be much more volatile than the intact molecules at 360 oC. In the next step, the decomposition of the lubricant was caused by evaporation of the fragments and and intact molecules remained at 480 oC.[3] Figure 3 (b) shows TGA thermogram of BL-104. It was observed that BL-104 showed little weight loss below 300 oC which corresponds to an extremely low vapor pressure and hence meets the demand of high performance.[8] BL-104 had one step decomposition, its thermal degradation started at 300 oC and completed at 430 oC. Therfore, BL-104 showed better performance than Zdol3800 in terms of thermal stability.

(a)

(b) Figure 3. TGA thermogram of (a) Zdol and (b) BL-104 Surface roughness of lubricants Figure 4 shows 3D surface morphology of Zdol3800 and BL-104 scanned over an area of 5x5 µm2. The white spots in the figures are due to dust particles. These observations indicated that the lubricant molecules spreaded evenly on the hard disk surface with the averaged surface roughness values between 1-2 nm. The depletion of lubricants The depletion of Zdol3800 from the hard disk surface after laser heating at different periods of time in form of outgassing was analyzed by gas chromatography - mass spectroscopy technique. The GC chromatograms of the outgassing collected after

Laser source

Magnetic disk media

Vacuum chamber

Sampling station

479


laser heating on the HDD surface are shown in figure 5, and their corresponding mass spectra are shown in figure 6. In figure 5, It was founded that the depletion product had the retention time between 1.110 to 1.155 min, which was the time of fluorocarbon moved from column. Figure 6 shows mass spectra corresponded to the peak of GC chromatogram: m/z = 19 is F-, m/z = 20 is HF, m/z = 69 is CF3

+, m/z = 85 is CF3O+, m/z =

97 is CF2COF+, m/z = 119 is C2F5+, m/z = 147 is

CF2CF2COF+, m/z = 163 is C3F5O2-, m/z = 169 is

C3F7+, m/z = 185 is C3F7O

-, m/z = 219 is C4F9+, m/z =

285 is C5OF11+ and m/z = 335 is C6OF13

+. These results are consistent with the results reported by Gerard et. al.[7]

(a) 1 mm/s

(b) 3 mm/s

(c) 5 mm/s

(d) 1 mm/s

Figure 4. Three-dimensional AFM images of the HDD surface coated by: (a-c) Zdol3800 obtained from dip coating method at different pulling-up speed and (d) BL-104

(a)

(b)

(c)

(d)

(e)

Figure 5. GC chromatograms of the outgassing after laser radiation at (a) 1 s, (b) 5 s, (c) 10 s (d) 30 s and (e) 60 s.

(a)

(b)

(c)

(d)

(e)

Figure 6. Mass spectra of the outgassing after laser radiation at (a) 1 s, (b) 5 s, (c) 10 s (d) 30 s and (e) 60 s.

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Conclusions

1. The magnetic disks were dip coated with Zdol3800 and BL-104 successfully with the average surface roughness values between 1-2 nm.

2. From TGA, BL-104 showed better performance than Zdol3800 in term of thermal stability.

3. After laser heating of Zdol3800 lubricant film, in the measurement system for the assessment of the lubricant performance in a HDD with HAMR technology, at different periods of time, the depletion of Zdol3800 was detected.

4. The molecules obtained from the outgassing analyzed by GC-MS show the mass spectra: m/z = 19 is F-, m/z = 20 is HF, m/z = 69 is CF3

+, m/z = 85 is CF3O

+, m/z = 97 is CF2COF+, m/z = 119 is C2F5+, m/z

= 147 is CF2CF2COF+, m/z = 163 is C3F5O2-, m/z =

169 is C3F7+, m/z = 185 is C3F7O

-, m/z = 219 is C4F9

+, m/z = 285 is C5OF11+ and m/z = 335 is C6OF13

+. 5. The depletion of Zdol3800 occured since the

first second of laser heating. Acknowledgments Financial support from the Industry/University Cooperative Research Center (I/UCRC) in Data Storage Technology and Applications, KMITL, and National Electronics and Computer Technology Center, National Science and Technology Development Agency and the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education is gratefully acknowledged. The acknowledgement also goes to Dupont Co., Ltd. Supporting Vertrel®, Coax Group Corporation for AFM analysis, and the Department of Chemistry, Faculty of Science, Burapha University. References [1] C.-Y. Chen, W. Fong, D. B. Bogy and S. Bhatia, Tribology Letters 8 (2000), pp. 25–34. [2] K.R. Paserba and A.J. Gellman, J. Phys. Chem. B. 105

(2001), pp. 12105–12110. [3] M J. Stirniman, S.J. Falcone and B.J. Marchon,

Tribology Letters 6 (1999), pp. 199-205. [4] M. Palacio and B. Bhushan, Ultramicroscopy 109

(2009), pp. 980-990. [5] P.H. Kasai and C. Spiese, Tribology Letters 17 (2004),

pp. 823-833. [6] R.Z. Lei, A.J. Gellman and P. Jones, Tribology Letters

11 (2001), pp. 1-5. [7] V. Gerard, Z. Raymond and S. David In: Y.

Chung, A.M. Homola and G. Bryan, Editors, SURFACE SCIENCE INVESITGATIONS IN TRIBOLOGY, American Chemical Society,

Washington, D.C (1999), pp. 169-179. [8] W. Zhao, Y. Mo, J. Pu and M. Bai, Tribology

International 42 (2009), pp. 828-835.

[9] W. Zhao, Y. Wang, L. Wang, M. Bai and Q. Xue, Colloid and Surfaces A: Physicochem. Eng. Aspects 361 (2010), pp. 118-125.

[10] Z. Zhao, B. Bhushan and C. Kajdas, Tribology Letters 6 (1999), pp. 141-148.

481


Study on Hydrolyzed Poly(butylene succinate) Scaffold-Chondrocyte Responses

P. Meesap, P. Uppanan, B. Thavornyutikarn, W. Kosorn and W. Janvikul*

National Metal and Materials Technology Center, Pathumthani, Thailand 12120

* E-Mail: [email protected]

Abstract: The responses of chondrocytes on the hydrolyzed poly(butylene succinate) (HPBS) scaffolds were studied. The effects of ranges of average pore sizes of the scaffolds, i.e., 350-500 µm, 500-700 µm and 700-1000 µm, and concentrations of NaOH solution, i.e., 0.1 M and 0.6 M, used in the PBS surface hydrolysis on the formation of cartilage-extracellular matrix (ECM) were investigated. The results revealed that after hydrolysis, the resulting HPBS scaffolds could facilitate the secretion of glycosaminoglycans (GAGs) and total collagen by chondrocytes. The higher the concentration of NaOH solution used in the hydrolysis process, the greater the amount of GAGs and collagen content secreted. Furthermore, it was noted that the pore sizes of the scaffolds affected the production of GAGs by chondrocytes. Greater amounts of total collagen were also yielded when larger pore sizes of the scaffolds were employed. The RT-PCR analysis revealed that the chondrocytes cultured on the HPBS scaffolds with larger pore sizes expressed higher levels of type II collagen gene. However, the expression of detected aggrecan gene was not much different in all scaffolds. The histological results demonstrated that the chondrocytes proliferated more effectively both on the surface and inside the pores of the HPBS scaffolds, compared with those cultured on the PBS scaffolds. Introduction

Recently, tissue engineering has been studied as an alternative method to repair damaged articular cartilage. This technique involves the regeneration of tissues by culturing chondrocytes on three-dimensional scaffolds to generate articular cartilage. Various biodegradable polymers have been extensively explored as tissue engineered scaffolds. Biodegradable aliphatic polyesters, e.g., poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(-caprolactone) (PCL), poly(hydroxybutyrate) (PHB) and poly(butylene succinate) (PBS), have been extensively studied for potential uses in bone and cartilage tissue engineering applications as cell/tissue culture substrates [1-2].

Selection of porous three-dimensional scaffolds is an inevitable consideration for tissue engineering study. The pore size, in general, plays an important role in cell adhesion, cell growth, cell migration, and differentiated cell function. High porosity and large pore sizes could facilitate the diffusion of nutrients and gases and the removal of metabolic wastes while small pore sizes could hinder these activities. Conversely, large pore sizes could lead to low cell attachment and low intracellular signaling whereas small pore sizes could promote these functions [3-4].

Surface chemistry of scaffolds also governs how cultured cells respond to the materials. Therefore, surface treatments of scaffolds have been conducted to improve the cell-material interfaces. The surface hydrolysis of biodegradable polymers using NaOH solution was found to significantly improve cell adhesion and enhance cell proliferation due to their increased surface hydrophilicity [5-6].

In this study, the responses of human articular chondrocytes to the porous hydrolyzed poly(butylene succinate) (HPBS) scaffolds with variable ranges of average pore sizes and variable surface hydrophilicity were examined, in terms of cell proliferation, glycosaminoglycans (GAGs) and collagen production, articular cartilage-specific gene expression, i.e., aggrecan and type II collagen genes, and cell infiltration and distribution observed by histological evaluation. Materials and Methods

Materials: PBS scaffolds, with three ranges of pore sizes, i.e., 350-500 µm, 500-700 µm and 700-1000 µm, were directly prepared in our laboratory by a supercritical fluid CO2 method. The PBS scaffolds were subsequently hydrolyzed with 0.1 M or 0.6 M NaOH solution at 60oC for 30 min. The product code was assigned based on the concentration of NaOH initially used, e.g., HPBS-500-0.1 prepared from the hydrolysis of PBS, with a range of pore size of 500-700 µm, with 0.1 M NaOH.

Cell culture: The sterilized sample discs were placed into 24-well culture plates. Human articular chondrocyte suspensions (2x106 cells/specimen) were seeded onto the scaffolds. The cells cultured on the scaffolds were incubated under 5% CO2 atmosphere at 37oC for given periods. The culture medium was regularly replaced every 3 days.

DNA assay: Cell proliferation on the scaffolds was assessed by a DNA assay. Typically, after 28-day culture period, the cells cultured on each scaffold were lyophilized and digested with papain solution (5 mg/ml) at 60oC for 18 h. Aliquots of each papain-digested solution were mixed with dye Hoechst 33258 solution (20 µg/ml). The fluorescence intensity was measured at 355 and 460 nm. The number of cells was directly relative to the measured intensity.

Glycosaminoglycans (GAGs) assay: The total GAGs content secreted from the cells cultured on each scaffold was determined using 1,9-dimethylmethylene blue (DMB) dye solution. In brief, after 21-day culture

482


period, the scaffolds were lyophilized and digested with papain solution (5 mg/ml) at 60oC for 18 h. Aliquots of 100 l of each papain-digested solution were mixed with 100 l of DMB solution. The absorbance was measured at 525 nm. The amount of GAGs was determined against the chondroitin-6-sulphate standard curve.

Collagen content assay: The total collagen content secreted from the cells cultured on each scaffold was determined using a hydroxyproline assay. In brief, after 28-day culture period, the scaffolds were lyophilized and hydrolyzed with 6 M HCl solution at 100oC for 12 h. The hydrolyzates were then neutralized with 6 M NaOH solution. The solutions of each hydrolyzate were brought to 0.25 ml with water and then mixed with 0.25 ml of 17%w/v NaCl in H2O. The mixtures were sequentially reacted with 0.5 ml freshly prepared chloramine-T reagent at room temperature for 5 min and 0.5 ml Ehrlich’s reagent at 60oC for 20 min, respectively. The absorbance was measured at 550 nm. The amount of total collagen was determined against the hydroxyproline standard curve.

RT-PCR analysis: Articular cartilage-specific genes, i.e., aggrecan and type II collagen, were determined by RT-PCR analysis. In brief, after a given culture period, total RNA was extracted from the chondrocytes cultured on each scaffold using TRIZOL reagent (GibcoBRL). 1 µg of total RNA was reverse-transcribed into cDNA using the RevertAid™ First Strand cDNA synthesis Kit (Fermentas). PCR analysis was conducted for both genes of interest and glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The GAPDH mRNA levels were used as internal controls. The PCR products were identified by electrophoresis on 2% agarose gel.

Histological analysis: The cells cultured on the scaffolds were fixed with 2% paraformaldehyde solution, embedded in paraffin, and sectioned (5 µm thickness). The sections were stained with hematoxylin and eosin (H&E) to observe the cell infiltration and distribution. Results and Discussion

The surface morphology of the scaffolds was examined by scanning electron microscopy, as revealed in Figure 1. All scaffolds appeared highly porous. The pore surface of the PBS scaffold was clearly smooth, as shown in Figure 1(b). After surface treatment with NaOH solution, the scaffold surface became rough (as shown in Figure 1(c-f)). The higher the concentration of NaOH solution used, the rougher the pore surface observed.

The average pore sizes of PBS scaffolds before and after surface hydrolysis using different NaOH concentrations, i.e., 0.1 M and 0.6 M, are reported in Table 1. Three ranges of average pore sizes of PBS scaffolds used in this study were 350-500 µm, 500-700 µm and 700-1000 µm, which were coded as PBS-350, PBS-500 and PBS-700, respectively. It was noted that after surface hydrolysis, the pore sizes of the hydrolyzed

PBS (HPBS) scaffold increased when compared with the initial pore sizes of the PBS scaffolds, especially when a high concentration of NaOH, i.e., 0.6 M NaOH, was employed.

Table 1: The ranges of average pore sizes of the PBS scaffolds before and after hydrolysis with NaOH solutions

a = PBS scaffolds before hydrolysis b = PBS scaffolds after hydrolysis with 0.1 M NaOH solution c = PBS scaffolds after hydrolysis with 0.6 M NaOH solution

Figure 2 demonstrates the results of the proliferation

of chondrocytes cultured on the PBS and hydrolyzed PBS (HPBS) scaffolds with variable average pore sizes. It was found that the number of cells became greater when the chondrocytes were cultured on the surface hydrolyzed scaffolds with the same pore sizes. The improved hydrophilicity of the HPBS scaffolds could facilitate the cell suspension to infiltrate into the inner part of the scaffolds after the cells were seeded. This resulted in the greater number of cells being cultured within the scaffolds; the higher levels of cell proliferation were consequently yielded.

Scaffolds Pore Size (µm)

PBS-350a PBS-500 a PBS-700 a

HPBS-350-0.1b HPBS-500-0.1b HPBS-700-0.1b HPBS-350-0.6c HPBS-500-0.6c HPBS-700-0.6c

350-500 500-700

700-1000 400-500 700-900

800-1000 750-900

1000-1200 1200-1600

Figure 1. SEM micrographs of (a,b) PBS-500, (c,d) HPBS-500-0.1, (e,f) HPBS-500-0.6. The scaffold surface morphology (a,c,e), original magnification x75 and the pore surface morphology (b,d,f), original magnification x1500

(a) (b)

(c) (d)

(e) (f)

500 m

500 m

500 m

30 m

30 m

30 m

483


The effect of pore size of the scaffolds on the cell proliferation was also investigated. As displayed in Figure 2, the number of cells significantly increased with an increasing pore size of the HPBS scaffolds used. Larger pores could more readily facilitate cell migration, nutrient flow, and waste removal, leading to higher cell growth.

The amounts of GAGs and total collagen secreted

from the chondrocytes cultured on the scaffolds are reported in Figures 3 and 4, respectively. The results revealed that after surface hydrolysis, the HPBS scaffolds could promote the secretion of GAGs and total collagen by chondrocytes. The higher the concentration of NaOH solution used in the hydrolysis process, the greater the amount of GAGs and collagen content secreted.

The effect of pore size of the scaffolds on the secretion of GAGs and collagen was also studied. It was noted that, overall, the amount of GAGs was increased with an increasing pore size of the scaffolds, especially observed in the HPBS scaffolds prepared with a high NaOH concentration, i.e., 0.6 M NaOH (Figure 3). Likewise, the secretion of collagen was apparently increased with an increasing pore size of the scaffolds (Figure 4). These results were in good accordance with the proliferation results. The hydrophilic surface and the large pores of the HPBS scaffolds could enhance the level of the cell proliferation; the secretion of more GAGs and total collagen by chondrocytes were consequently resulted.

Figure 5 presents the mRNA expression of aggrecan and type II collagen secreted by the chondrocytes after being cultured with scaffolds, compared with that of GAPDH as a housekeeping gene. The mRNA of GAPDH was vividly expressed in all scaffolds. Aggrecan gene expression was observed in every scaffold with slightly different levels. Interestingly, type II collagen gene expression was detected only in the hydrolyzed PBS scaffolds which had the average pore size within the range of 700 µm to 1200 µm. No type II collagen gene expression was found when the cells were cultured on HPBS-350-0.1 (lane 4 in Figure 5) and HPBS-700-0.6 (lane 9 in Figure 5), whose pore sizes

were in the ranges of 400-500 µm and 1200-1600 µm, respectively. The results implied that both surface hydrophilicity and pore size of the scaffolds directly influenced the cellular function of the chondrocytes.

The histological results illustrated the infiltration and distribution of the cells after cultured with the PBS and HPBS scaffolds for 28 days. The cells more highly proliferated and homogeneously infiltrated through the inner hydrophilic HPBS scaffold than those cultured with the less hydrophilic PBS scaffold, as revealed in

Figure 2. Fluorescence intensity of culture media of chondrocytes incubated with each tested scaffold for 28 days

PBS-350

PBS-500

PBS-700

HPBS-350-0

.1

HPBS-500-0

.1

HPBS-700-0

.1

HPBS-350-0

.6

HPBS-500-0

.6

HPBS-700-0

.60 .00.20.40.60.8200

400

600

800

1000

1200

1400

Fluo

resc

ence

Int

ensi

ty

200 0

500 700

Figure 5. RT-PCR results of genes encoding GAPDH, aggrecan and type II collagen secreted by chondrocytes cultured on scaffolds; (lane 1) PBS-350, (lane 2) PBS-500, (lane 3) PBS-700, (lane 4) HPBS-350-0.1, (lane 5) HPBS-500-0.1, (lane 6) HPBS-700-0.1, (lane 7) HPBS-350-0.6, (lane 8) HPBS-500-0.6, (lane 9) HPBS-700-0.6 and M=marker

GAPDH

M 1 2 3 4 5 6 7 8 9

Aggrecan

500 700

400

Type II Collagen

500 700

400

400

Figure 3. GAGs content secreted from chondrocytes cultured on various scaffolds at 21-day incubation period

PBS-350

PBS-500

PBS-700

HPBS-350

-0.1

HPBS-500-

0.1

HPBS-700-

0.1

HPBS-350

-0.6

HPBS-500

-0.6

HPBS-700

-0.6

0.00.20.40.60.8250

300

350

400

GA

Gs

(g/

g sc

affo

ld)

250 0

Figure 4. Total collagen content secreted from chondrocytes cultured on various scaffolds at 28-day incubation period

PBS-350

PBS-500

PBS-700

HPBS-350-

0.1

HPBS-500

-0.1

HPBS-700-0

.1

HPBS-350-

0.6

HPBS-500

-0.6

HPBS-700-0

.60 .00.20.40.60.820

40

60

80

100

120

Tot

al C

olla

gen

(g/

g sc

affo

ld)

20 0

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Figure 6. This led to greater cell proliferation and ECM production.

Conclusions

The reponses of the human articular chondrocytes to the hydrolyzed PBS scaffolds were directly influenced by both the suface hydrophilicity and the pore size of the scaffolds. The enhanced hydrophilicity and the suitable range of pore size of the HPBS scaffolds could promote the cell proliferation and facilitate the secretion of GAGs and total collagen and the expression of cartilage-specific genes. Moreover, the chondrocytes proliferated more homogeneously throughout the entire HPBS scaffolds, compared with those cultured on the starting PBS scaffolds. Acknowledgement

This research was financially supported by National Metal and Materials Technology Center (project code: MT-B-52-BMD-07-175-I). References [1] K.Y. Chang, L.W. Cheng, G.H. Ho, Y.P. Huang and Y.D.

Lee, Acta Biomater. 5 (2009), pp. 1937-1947. [2] P. Zhang, Z. Hong, T. Yu, X. Chen and X. Jing,

Biomaterials. 30 (2009), pp. 58-70. [3] S.H. Oh, I.K. Park, J.M. Kim and J.H. Lee, Biomaterials.

28 (2007), pp. 1664-1671. [4] S.M. Lien, L.Y. Ko and T.J. Huang, Acta Biomater. 5

(2009), pp. 670-679.

[5] Y.Q. Wang and J.Y. Cai, Curr. Appl Phys. 7S1 (2007), pp. e108-e111.

[6] R. Ng, X. Zhang, N. Liu and S.T. Yang, Process Biochem. 44 (2009), pp. 992-998.

Figure 6. H&E staining of cross-sections of the scaffolds: (a) PBS-700 and (b) HPBS-350-0.6, after 28-day cell culture

(b) 200 µm

(a) 200 µm

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TiO2-Coated Zn2SnO4 Nanowires for Dye-Sensitized Solar Cells

R. Tuayjareon 1,2*, T. Nootong 1, 2, T. Rattana 1, P. Suapadkron 1, 2, P. Chinvetkitvanich 1,2, P. Limsuwan 1,2, and T. Jutarosaga 1,2

1 Department of Physics, Faculty of Science, King Mongkut’s University of Technology Thonburi, 126 Pracha-uthit Rd, Bangmod, Tungkru, Bangkok, Thailand 10140 2 Thin Film Technology Research Laboratory, ThEP Center, CHE, 328 Si Ayutthaya Road, Ratchathewi, Bangkok, 10400 *E-mail:[email protected]

Abstract: Metal oxide nanowires were successfully fabricated via vapor-solid-liquid (VLS) mechanism at 630oC on indium-tin oxide/borosilicate glass for a dye-sensitized solar cell (DSSC) application. The crystallinity and the morphology of synthesized nanostructure were verified using X-ray diffraction (XRD) technique and scanning electron microscopy (SEM), respectively. XRD patterns confirmed that the synthesized material consists of face-center cubic Zn2SnO4, hexagonal ZnO and tetragonal SnO2. SEM images showed wire-like nanostructures having the height and the diameter of about 61 m and 61±22 nm, respectively. Before applying as electrode for DSSCs, the nanowires were then coated with TiO2 thin film via DC reactive magnetron sputtering process. The electrical characteristics of the fabricated cell under AM 1.5 solar simulation of TiO2-coated nanowire were studied. The performances of these composite solar cells were compared with a pure nanowire cell. The solar cell with only nanowires has the short-circuit current density JSC of 0.64 mA/cm2, while the cells with TiO2-coated nanowires show a significant improvement of JSC to 2.4 mA/cm2. The increase of JSC is possibly due to the better adsorption of dye molecules on the TiO2 surface compared to nanowire surface. Introduction Dye-sensitized solar cells (DSSCs) based on nanocrystalline semiconductor film [1] have attracted much attention by many research groups for twenty years because their low production cost and environmental friendly. Basically, what make DSSC difference from a conventional p-n junction solar cell is the functional element (dye) inside the cell which absorbs photon from sunlight to excite electrons to the excited state of dye molecules. Then free electrons from the dye molecules are injected into the conduction band of the semiconductor. So, the increase of dye molecules in solar cell and the improvement of semiconductor morphology are the key to enhance the efficiency of DSSCs. One-dimensional (1D) nanocrystalline of metal oxide was purposed to use as transparent electrode for DSSCs to enhance the rate of electron transportation [2]. In order to synthesize metal oxide nanowires, the most well-known method, Vapor-Liquid-Solid (VLS) mechanism, has been developed. This process was first described by Wagner and Ellis in 1964[3]. They used Au particles as catalysts to grow semiconductor whiskers on the Si wafer from the gas source. Metal oxide nanowhiskers are grown on substrates by several techniques such as chemical vapor deposition (CVD),

metal organic chemical vapor deposition and molecular beam epitaxy (MBE) [4]. The vapor transport technique was selected for our study due to its simplicity. As mentioned earlier, the electron conduction rate could be enhanced by using 1D nanostructure. However, due to the lack of surface area adding nanoparticles into the nanowire matrix will possibly improve the carrier generation. Therefore, in our study, TiO2 nanostructure film was coated on nanowire via DC reactive magnetron sputtering process. The TiO2-coated nanowires on ITO/borosilicate glass were then used as electrodes for dye-sensitize solar cells. Using this TiO2 coated on metal oxide nanowire, the observe efficiency was greatly improved when it is compared to conventional metal oxide nanowires. Materials and Methods Metal oxide nanowires were grown on ITO/borosilicate glass substrates. First, a thin layer of gold was sputtered on the desired substrates by Polaron SC7620 sputter coater at a sputtering pressure of 9.0×10-2 mbar and a sputtering current of 4 mA for 4 min. The controlled synthesis of nanowires was achieved by evaporation of a mixture of SnO and Zn powder at a relative low pressure of 10 mbar and temperature of 630˚C. Mixture of 0.25 g SnO powder (99%, Aldrich) and 0.25 g Zn powder (99%, Ajax) was placed in one end of the alumina boat while substrates were placed in the other end. The alumina boat was then inserted into a horizontal quartz tube. The furnace was then heated to 750 ˚C at source with a constant ramping rate of 24oC/min under N2 atmosphere with a flow rate of 10 cm3/min to obtain a temperature of 630 ˚C at substrate. Preparation conditions of nanowire are set in Table 1. After synthesizing nanowires on ITO/glass substrates, 2 nanowire of specimens were then coated with TiO2 via DC reactive magnetron sputtering using the mixture of Ar and O2. Before TiO2 deposition, the chamber was evacuated to a base pressure of 6.01x10-6 mbar. The admission of O2 (the reactive gas) to the chamber was independently controlled using mass flow controller until the pressure in the chamber reached 6.67x10-4 mbar. After that the Ar was flowed into the chamber until the pressure reached 5.61x10-3 mbar. A pre-sputtering process was employed for 300s to clean the target surface. During the sputtering process, the distance between the Ti target and the substrates was

486


set to 5.9 cm and the sputter power was set to 160W. The sputtering times were 560s and 1120s corresponding to TiO2 film thickness of ~20nm (thin) and ~40nm (thick), respectively. Preparation conditions of TiO2 coating are given in Table 2. Table 1: Preparation conditions of metal oxide nanowire coated with three different TiO2 thicknesses

Source 0.25g SnO + 0.25g Zn

Substrate Au/ITO/borosilicate glass

Source temperature (oC)

750

Substrate temperature (oC)

630

Table 2: preparation conditions of TiO2 coating by reactive DC magnetron sputtering

Preparation conditions

Sputtering Parameters

K.Aeimpanakit [5] This work

Sputter power (W) 160 160 Base pressure (mBar)

6.00 x10-6 6.01 x10-6

Sputtering pressure (mBar)

6.00 x10-3 5.61 x10-3

Ar:O2 flow rate (sccm)

40:5 88:38

The obtained products were characterized with X-ray diffraction (XRD,Miniflex/Cu/ 30kV /15mA) and scanning electron microscopy (SEM, S4700 Hitachi). Then, the three different thicknesses of TiO2 coated on metal oxide (MO) nanowires on ITO/glass substrates were immersed in the solution of sensitizer, ruthenium 535 bis-TBA (N719), at room temperature for 4 hours. They were used as working electrodes. The counter electrode which was prepared from electrochemically platinized on FTO/soda-lime glass. The MPN-100 SOLARONIX was used as electrolyte

3/ II in the

fabricated cell. The cell assembly procedure followed Ponken et al.[6]. The current density versus voltage characteristics of the solar cells were characterized under standard radiation AM 1.5 (100 mW/cm2). The typical active cell area was about 0.25 cm2. Result and Discussion The X-ray diffraction spectra of the TiO2-coated and uncoated metal oxide (MO) nanowire on Au coated ITO/borosilicate glass is shown in Figure 1. XRD

spectra show characteristic peaks of three phases which are tetragonal SnO2, face-center cubic Zn2SnO4 and hexagonal ZnO (wurtzite) but do not show phase of TiO2. The morphology of synthesized one-dimensional nanostructures grown on Au-coated ITO/borosilicate glass was examined using a scanning electron microscopy (SEM) and the result is shown in figure 2(a-f). Figure 2(a-c) displays the surface of synthesized nanowires for (a) without Tio2 coating b) with thin TiO2 coating and c) with thick TiO2 coating. The morphology of synthesized nanowire was investigated by considering SEM images. It was found that the synthesized nanowires are in round wire-like shape. Figure 2 (d-f) displays the cross sectional SEM micrographs of synthesized nanowire for (d) without TiO2 coating (e) with thin TiO2 coating and (f) with thick TiO2 coating. According to SEM images, we can measure diameters and heights of nanowire and result are shown in the Table 3. It is seem that the heights of uncoated, thin coated and thick coated nanowires are 9.26, 61.0 and 40.3 µm while their diameters are 62, 61 and 78 nm, respectively. The three different thicknesses of TiO2–coating nanowire on ITO/glass substrates as listed in Table 2 were used in dye-sensitized solar cell assembly. The cell efficiency was then measured and the current density v.s. voltage (J-V) characteristics was obtained.

Figure 1. XRD spectra of Sn and Zn composite on Au/ITO/borosilicate glass (a) without TiO2 (b) with thick TiO2-coating to increase surface area of semiconductor

SnO(1):Zn(1) Tsub 630 C Tsou 750 C without TiO2

SnO(1):Zn(1) Tsub 630 C Tsou 750 C coated TiO2 40 nm

Degree (2)

487


Figure 2. SEM top view and cross sectional image of nanowire: (a) and (d) without TiO2 coating (b) and (e) thin TiO2 coating and (c) and (f) with thick TiO2 coating which are synthesized according to the conditions listed in Table 1

The J-V characteristics of three cases were measured under the AM 1.5 illumination. The J-V curve of each solar cell generated from different electrodes was obtained as shown in Figure 3 and the efficiency and other characteristics such as open circuit voltage and fill factor are shown in Table 3. It was found that the efficiency of photovoltaic cell, which is generated from electrode whisker coated with thin and thick TiO2 are 0.40% and 0.33%, respectively. While the efficiency of DSSC combined the uncoated nanowire electrode is quite low which is 0.09%. The short-circuit current density of solar cell coated with thin and thick TiO2 are 2.40 and 1.88 mA/cm2, respectively. While the short-circuit current density combined the uncoated nanowire electrode is 0.74 mA/cm2. The open-circuit voltage of solar cell coated with thin, thick TiO2 and without TiO2 are 600, 555 and 410 mV, respectively. The short-circuit current density, open-circuit voltage and efficiency of solar cell show that the DSSC using the nanowire coated with thin TiO2 as a part of working electrode is the best of DSSC in these three conditions.

Table 3: Chareristics of dye-sensitized solar cell with different TiO2 thicknesses

DSSC Without

with TiO2

With thin TiO2

coating

With thick TiO2

coating Height (µm)

9.29 61.0 40.3

Diameter (nm)

62±38 61±22 78±38

Efficiency (%)

0.09 0.41 0.33

Fill Factor (%)

36.18 28.32 31.66

Voc (mV) 410 600 555 Jsc

(mA/cm2) 0.74 2.40 1.88

Rs (Ohm.cm2)

308.5 172.6 187.7

Rsh (Ohm.cm2)

1379 326.4 480.8

a)

b)

c)

e)

d)

f)

488


Figure 3. The comparison of J-V curve for each solar cell generated from the different electrodes (a) electrode coated with TiO2 at a thickness ~20 nm (b) electrode which coated with TiO2 at a thickness of about 40 nm and (c) electrode without coating The results indicate that the efficiency of Dye-Sensitized Solar cell depends on the TiO2 coating and the length of nanowire. When considering the working electrode coated with TiO2, the efficiency of solar cell is higher than that of without coating with TiO2. In addition to effect of TiO2 coating thickness, it was found that the efficiency of cell with longer nanowires is higher than the shorter nanowire due to the possibility of increasing amount of dye absorbed on nanowire which lead to the increase of generated photoelectrons. Conclusion Mixture of SnO2, Zn2SnO4, ZnO metal oxide nanowires were synthesized and coated with monolayer of TiO2 nanoparticle at three different thicknesses which are uncoated, thin coated and thick coated layers. Using these coated nanowires as a part of working electrode of DSSC, the cells were assembled and measured the cell efficiency and then the TiO2 coating cells were compared with a pure nanowire cell. In conclusion, TiO2 coating may possibly improves an electron transport in solar cell which leads to an increase of the cell efficiency. Moreover, the lenght of nanowire also relates to the increasing of the cell efficiancy because of an increasing surface area of dye adsorption. Acknowledgements: We would like to thank Department of Physics and The Scientific Instrument Center for Standard and Industry, Faculty of Science, King Mongkut’s University of Technology Thonburi for X-Ray Diffractometer, Thai Microelectronics Center for SEM characterization, Semiconductor Physics Laboratory, Department of Physics, Chulalongkorn University for fabrication of DSSCs and the I-V measurement, and Assistant Prof. Wandee

Onreabroy for their kind supports on laboratory instruments and chemicals used in this experiment. Reference [1] B. O’Regan, M. Gra··tzel, Nature, 1991, 353, 737-738. [2] M. Law, L. E. Greene, J. C. Johnson, R. Saykally, P.Yang, Nat. Mater, 2005, 4. [3] R. S. Wagner and W. C. Ellis, Applied Physics Letters, 1964, 4 [4] N. Zakharov_, P. Werner, L. Sokolov, U. G�sele, Physica E, 2007, 37, 148-152 [5] K. Aeimpanakit, Master Thesis, Department of Physics Faculty of Science King Mongkut’s University of Technology Thonburi, 2004, 9-10 [6] T. Ponken, C. Chityuttakon, S. Chatraphorn, National Graduate Research Conference CG and Khon Kaen University, 2009, 632-638

Thin TiO2 coating, =0.4%, FF=0.28%

Thick TiO2 coating, =0.33%, FF=0.31%

TiO2 uncoating, =0.09%, FF=0.36

489


Preparation of Injection-Molded Polypropylene/Reclaimed Tire Rubber/Sawdust Blends

S. Sunaree 1* and T. Supawan 2

1 Program of Petrochemistry and Polymer Science, Faculty of Science, Chulalongkorn University, Bangkok, Thailand

2Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand

* [email protected]

Abstract: In this research, reclaimed tire rubber (RTR) and sawdust were used as a filler in polypropylene (PP) composite. The composites of reclaimed tire rubber (RTR) /sawdust/ polypropylene (PP) were prepared by injection molding dynamic vulcanization. The RTR and sawdust content were varied from 0% to 30% by weight, while PP content was kept content at 70% by weight. The effect of RTR /sawdust/ PP on properties of the blends. Increasing the RTR and sawdust content to more than resulted in a decrease in the impact strength and the tensile strength of blend, most likely due to the increasing carbon black content. The composites vulcanization show slightly higher tensile strength and impact strength than composites with unvulcanization. Results from solvent swelling ratio, water absorption and morphology of the blends was also proved by scanning electron microscopy (SEM). Introduction The present used tire and has become one of the largest scale rubber wastes. Recycle of the waste rubber is thus a great challenge for both environmental and economic reasons. The tire rubber part can be ground to small particles, known as ground rubber tire (GRT) can further be devulcanized to be come reclaimed tire rubber (RTR). [1], The polymer–sawdust fibre composites utilize sawdust fibres as a reinforcing filler in the polymer matrix and are known to be advantageous over the neat polymers in terms of the materials cost and some mechanical properties such as stiffness and strength. [2], and sawdust fibers besides the environmental concern (environmental friendly and biodegradability) are low cost, and low density. Using RTR, having a non-cross-linked rubber structure, as filler should be a more effective choice to impart elastic property to the thermoplastics. We earlier reported the dynamic vulcanization of RTR and PP blends, and found that sulphur vulcanization blend of 30/70 RTR/PP had higher impact strength than that of GRT/PP blend and of PP alone [3]. The purpose of this work to present another attempt to obtain value added products from injection-molded reclaimed tire rubber (RTR) /sawdust/polypropylene (PP) composites blends.

Materials and Methods 2.1 Materials Sawdust (60 mesh) was purchased from Artowood Co.Ltd., Thailand. Reclaimed tire rubber (RTR) (68.22% rubber, 25.07% carbonblack and 6.71% residue with particle size of 500-600 mm), was purchased from Union Commercial Development Co. Ltd., Thailand. An injection grade PP (1100NN), was provided by IRPC Co. Ltd., Thailand, dibenzothiazole disulfide (MBTS), tetramethyl thiuram disulfide (TMTD), zinc oxide (ZnO), stearic acid were all of Fluka reagent grades. Sulphur was purchased from Merck. 2.2. Mixing and injection molding Sawdust was prepared into 60 mesh. After that was dried in an oven at 100 °C for 1 hr. The compositions of reclaimed tire rubber (RTR) /sawdust/ polypropylene (PP) constants and composites with sulphur system are indicated in Table 1.The composites were prepared by injection molding dynamic vulcanization and unvulcanization. The RTR and sawdust content were varied from 0% to 30% by weight, while PP content was kept content at 70% by weight. The mixing temperature was 180 °C and rotor speed was 50 rpm. with 15 min of mixing time. Table 1. Composition of tested RTR/sawdust/PP.

PP RTR Sawdust ZnO Stearic acid

MBTS TMTD Sulphur

70 0 30 0 0 0 0 0

70 5 25 0.25 0.1 0.25 0.075 0.25

70 10 20 0.5 0.2 0.5 0.1 0.5

70 15 15 0.75 0.3 0.75 0.125 0.75

70 20 10 1 0.4 1 0.3 1

70 25 5 1.25 0.5 1.25 0.375 1.25

70 30 0 1.5 0.6 1.5 0.45 1.5

490


2.3. Tensile strength Tensile strength measurement was performed on dumbbell specimens at ambient temperature according to ASTM D468 using universal testing machine. 2.4 Impact strength Notch-Izod impact strength was measured according to ASTM D256. Impact specimens had dimensions of 63.5 x1.27 x3 mm. The results are reported in Kg-cm/cm notch unit. 2.5 Water absorption test For the water absorption test study was carried out by cutting a sample into discs of 1 cm in diameter and 0.3 cm in thickness. The specimens are dried in an oven temperature 40 °C for 24 hours. They were weighed accurately before being immersed into water kept for 7 days at room temperature. Specimens are removed, patted dry with a lint free cloth, and weighed. Water absorption is expressed as increase in weight percent. Percent Water Absorption = [(Wet weight - Dry weight)/ Dry weight] x 100 2.6 Solvent swelling Solvent swelling study was carried out by cutting a sample into discs of 1 cm in diameter and 0.3 cm in thickness. They were weighed accurately before being immersed into toluene. The samples were kept in the dark for 7 days at room temperature. 2.7 Morphological property The blend morphology was examined by a scanning electron microscope (JEOL model JSM 5800 SEM). Dumbbell shaped samples after tensile testing were immersed in liquid nitrogen, then fractured and gold-coated prior to analysis. Results and Discussion 3.1. Mechanical properties In this study, RTR and sawdust content were varied from 0% to 30% by weight, while PP content was kept content at 70% by weight the and two types of vulcanization by Sulphur and unvulcanization. The mechanical properties of all these blends were then compared. Fig. 1 shows that the tensile strength of the blends. The tensile strength of each blend, decrease with increasing reclaimed tire rubber content and sawdust content in the blend. Further, agglomeration and hence particle–particle interaction of the rubber powder accounts for the observed decrease in tensile strength. [1] Fig. 2 shows the impact strength of the blends. The impact strength of each blend, decrease with increasing reclaimed tire rubber content and sawdust content in the blend. Its high content could induce a split in the layer structure of the blend

providing a shorter path for fracture propagation, thereby causing the sudden decrease in impact strength. [2]. But, composites in the formula to find unvulcanized RTR/Sawdust/PP (25/5/70) with vulcanized (25/5/70), impact strength increase to find may be involved in both molecular entangling and interphase sulfur crosslinking mechanisms which all composites.Yield a better interfacial bonding between the RTR particle and sawdust in the matrix PP.

0/30/70 5/25/70 10/20/70 15/15/70 20/10/70 25/5/70 30/0/70

16

17

18

19

20

21

22

23

Ten

sile

str

engt

h(M

Pa)

RTR/Sawdust/PP

Unvulcanization Vulcanization

Fig. 1. Tensile strength of vulcanized and unvulcanized RTR/Sawdust/PP blends.

0/30/70 5/25/70 10/20/70 15/15/70 20/10/70 25/5/70 30/0/70

40

60

80

100

120

140

Imp

ac

t st

ren

gth

(Kg

-cm

/cm

)

RTR/Sawdust/PP

Vulcanization Unvulcanization

Fig. 2. Impact strength of vulcanized and unvulcanized RTR/Sawdust/PP blends. 3.2 Morphology properties The morphology of RTR/sawdust/PP blends with vulcanization and unvulcanization are shown in Fig. 3 The RTR particles and sawdust can disperse well in PP matrix when the blend was vulcanized with sulphur. This indicates that re-cross-linking is more effective in blending with these systems leading to the improvement of physical properties. It is also found that with (c) unvulcanized RTR/Sawdust/PP (25/5/70) (d) with vulcanized (25/5/70) RTR/HDPE blends at ratios of 30/70 to 70/30,

491


(a)

(b)

(c)

(d)

(e)

(f) Fig. 3. Scanning electron micrograph (a) with unvulcanized RTR/Sawdust/PP (0/30/70) (b) with vulcanized RTR/Sawdust/PP (0/30/70) vulcanized (c ) with unvulcanized RTR/Sawdust/PP (25/5/70) (d) with vulcanized (25/5/70) (e) with unvulcanized RTR/Sawdust/PP (30/0/70) (f) with vulcanized RTR/Sawdust/PP (30/0/70)

Conclusions In this research, reclaimed tire rubber (RTR) and sawdust were used as a filler in polypropylene (PP) composite. The composites of reclaimed tire rubber (RTR) /sawdust/ polypropylene (PP) (25/5/70) were prepared by injection molding dynamic vulcanization. The composites vulcanization show slightly higher tensile strength and impact strength than composites with unvulcanization. References

[1] P. Punnarak ,T Supawan., T Varawut, Polymer Degradation and Stability, 91, 3456-3462 (2006).

[2]C K Radhesh, Fuhrmann I., Karger-Kocsis J.,

Polymer Degradation and Stability, 76, 137-144 (2002). [3] H Ismail , Polymer Testing, 21, 389–395 (2002)

492


Synthesis of Sulphur Bentonite as an Effective Liquid Sulphur Derivative

S. Nuntawat1, A. Luengnaruemitchai1, R. Magaraphan1, and H. Manuspiya1,2*

1 The Petroleum and Petrochemical College, Chulalongkorn University, Phayathai road, Pathumwan, Bangkok, Thailand 10330 2 Center of Excellence for Petroleum, Petrochemical, and Advance Materials, Phayathai road, Pathumwan, Bangkok, Thailand

10330

* E-mail: [email protected]

Abstract: Elemental sulphur fertilizer is the one of the most effective outlets of liquid sulphur based on the availability of technology providers, the highest expected margin, innovation perspective, and the largest percentage of liquid sulphur requirement. The elemental sulphur is oxidized to sulphate form under typical condition before taking up by plant. Among the variables studied, the particle size of sulphur is the most significant factor because the smaller particle sizes are easier to disperse and oxidize to sulphate form. Sulphur bentonite mixtures, prepared by blending molten sulphur and bentonite, offer a method of applying high concentration of elemental sulphur (70–90 % S) of fine particle size in a safe-handing product. The purpose of this work is to formulate a novel sulphur bentonite by using liquid sulphur produced from a local refinery plant mixed with bentonite, organomodified bentonite, and porous clay heterostructure bentonite (PCH) in various ratios. Introduction Every year, crop is harvested, a portion of the available sulphur is depleted and a portion is returned to soil as residue and converted into organic matter. But in the cropping system, it tends to remove more sulphur than being replaced. So the sulphur fertilizers are required for soil supplement. There are various chemical and physical forms of sulphur fertilizer such as sulfate fertilizes (example ammonium sulfate and potassium sulfate) and another one is elemental sulphur in different physical forms. For the sulphate fertilizer, it provides an immediate source of sulfate to the plant, but sulfate is easy to leaching loss. The elemental sulphur fertilizer has high concentration of 70-100% wt sulphur and greatly physical characteristic. Element sulphur is oxidized to sulfate before it can be taken up by plant. Release of an available sulfate form sulphur pills depends on 2; process 1) physical dispersion of the pill and 2) oxidation to sulfate. The benefits of elemental sulphur are a continual releasing of sulfate during the growth season and minimal sulfate leaching loses. However, the oxidation process depends on soil moisture, temperature, bacteria activity, time, and particle size. The particle size of sulphur is the most crucial factor, especially the small particle sizes are easily dispersed and oxidized [1]. For preparing sulphur bentonite, a molten sulphur has been mixed with bentonite to form a safe and easy to apply product. The principle is that when the clay

absorbs water and swell, it makes pill fracture and disperses to small particles of sulphur. From the previous work [2], the yield of crops, total sulphur uptake and extractable soil sulfate of sulpher bentonite were less than ammonium sulfate and micronized sulphur fertilizer, because the sulphur particles were not small enough and slow dispersion. The purpose of this work aims to formulate a novel sulphur bentonite by using liquid sulphur that was provided by a local refinery plant and our modified bentonite clay in order to reduce the particle size of sulphur and to control the release of sulphur. Experimental Materials and Preparation Na-bentonite fertilizer grade (CEC = 100), was supplied by Thai Nippon Co., Ltd. Liquid sulphur supplied from Thaioil Public Co., Ltd. Cetyltrimethylammonium [C16H31N

+(CH3)3] bromide 99 % (CTAB) from Acros organic. Dodecylamine 98% from Sigma-Aldrich. Tetraetyoxysilane (TEOS) reagent grade from Sigma Aldrich. Start with clay preparation, bentonite was converted into a quaternary ammonium exchange form by ion exchange with cetyltrimethylammonium bromide (CTAB) 0.1 M for 24 hr, 50 C to obtained organoclay. A portion of organoclay was synthesized to PCH by adding co-surfactant (Dodecylamine), then stirred until mixed completely. TEOS was immediately added in the mixture at room temperature for 4 hr to obtain PCH. An obtaining PCH was calcined for removing surfactant at 600 C. There were 3 formulas of sulphur bentonite: (1) bentonite+sulphur (BS˚), (2) organoclay+sulphur (OS˚), (3) PCH+sulphur (PCHS˚). Each formula was divided into 3 compositions of clay: 10, 20, 30, % of clay (w/w). A process to produced pills of clays and sulphur mixture was mixed by mixing stirrer in a chamber at 140˚C for 2 hour. Then forming droplets of the mixture immediately, the droplets of mixture was dipped on stainless steel plate to obtained sulphur bentonite fertilizer [3]. Characterization Clay characterizations were conducted by X-ray driffractometer (XRD) and Fourier transform infrared

493


spectroscopy (FTIR). XRD measured for low angle (2θ from 1� to 10�) and scan speed at 0.5 sec/step. Clay structure was presented by S-4800 field emission scanning electron microscope (FE-SEM). The measurement was carried out at 5 kV. BET method was used to determine pore size, pore volume and surface area of clay. In case of sulphur bentonite fertilizers, the prill structure was examined with SEM and SEM-EDX. Density maps had showed the dispersion of bentonite and PCH in sulphur matrix. Result and Discussion Bentonite, Organoclay and PCH were initially characterized by using FTIR method to confirm the present of surfactant in Organoclay and the change of structure of PCH. The broad peak around 3500 cm-1 can be assigned to the stretching vibration of the silanol associated with the silica structure. The peak at 1100, 1000 and 800 cm-1 can be assigned to the stretching vibration of the SiO4 units, the asymmetric and symmetric stretching vibrations of the Si-O-Si linkage, respectively. The sharp peaks at 2935 and 2860 cm-1 in organoclay can be assigned to asymmetric and symmetric vibrations of methyl and methylene groups of surfactant. The FTIR spectra of PCH was different from starting bentonite indicated by the shift of peak at 1000 cm-1to 1100 cm-1 and the absent of peaks 2935 and 2860 cm-1were confirmed that the surfactant was removed after the calcination. It roughly infers that the structure of starting bentonite was changed after the modification.

Figure 1. FTIR spectra of Bentonite (a), Organoclay (b) and PHC (C) XRD pattern of the samples are shown in Figure 2, the basal spacing of BTN is 1.21 nm. After BTN was treated with CTAB to obtain organoclay, d-value at 2 = 2.28 increased to 3.88 nm. Two strong peaks were observed at lower angle as a result of the successful intercalation of cationic surfactant in the interlayer of bentonite [4]. PCH was observed board peak at 2 = 2.21 (d = 3.82 nm) and the pattern was different from Organoclay. A possible reason might be due to the disordered structure of silica framework which was formed in the galleries of clay shielding a highly regular interstratifications of the clay layers but it still

had some pattern order structure that presenting in PCH.

2 theta

0 2 4 6 8 10 12

Lin

ear in

tensi

ty (C

ounts

)

0

10000

20000

30000

40000

50000

(a)

(b)

(C)

d=1.21

d=3.88

d=3.82

Figure 2. The XRD patterns of Bentonite (a), Organoclay (b), PCH (c) BET surface area of BTN was 55 m2/g. After modification of organoclay, it showed a decreasing of surface area and pore volume while increasing of pore diameter. A possible reason might be due to the presented of surfactant in the interlayer clay, it blocked N2 gas to adsorb into the pore. After modification of PCH, the BET surface area PCH was significantly increased from 55 to 594 m2/g that confirmed the structure of clay was changed from plate structure to porous structure. The evidences from SEM (figure 3.) confirmed the structure of clay was changed from plate structure to porous structure, higher surface roughness and higher void. Table 1: Porosity characteristics of Bentonite, Organoclay and PCH

Sample

Total

surface

area (m2/g)

pore

diameter

(nm)

pore

volume

(cc/g)

Bentonite 55 8.7 0.120

Organoclay 8 11.0 0.022

PCH 594 5.0 0.737

(a) (b) Figure 3. SEM image of Bentonite (a) and PCH (b) Sulphur forms the matrix of the prill and the varying amounts of bentonite were held within it. Almost all evidence of density map suggests that the dispersion of bentonite in the sulphur matrix was

494


relatively uniform and free form aggregation [5]. Sulphur matrix and bentonite was clearly separated and sulphur matrix just covered around bentonite particle (figure 4. a) .In case of Sulphur-PCH, micrograph was different because a portion of sulphur matrix going inside pore and void of PCH. A portion remained coat on surface of PCH (figure 4. b)

(a) (b) Figure 4. SEM-EDX density map of sulphur, sulphur bentonite (a) and sulphur-PCH (b) (x700) Conclusions This work successfully prepared sulphur bentonite and sulphur-PCH. While, the sulphur-organoclay was not successfully prepared because of the degradation of existing surfactant. Fertilizers derived by sulphur bentonite and sulphur-PCH will be test the mechanical property, the dispersibility and the control release of sulphur in future works. Acknowledgements The author would like to thank The petroleum and petrochemical college, Chulalongkorn University, Thaioil Plublic Company Limited and the National Excellence Center for Petroleum, Petrochemicals, and Advance Materials for research funding. In addition, the authors wish to thank Thai-Nipon chemical Co., Ltd. for supported bentonite clay. References [1] J.R. Bettany, and H.H. Janzen. Proceedings of

Sulphur 84 (1984), pp. 817-812. [2] N.G. Riley, F.J. Zhao, and S.P. McGrath. Plant and

Soil 222 (2000). pp. 139-147. [3] R.J. Zaharko. U.S. Patent 4,469,859 (1986). [4] A. Mattayan, , R. Magaraphanand and H. Manuspiya, the 15th PPC Symposium on

Petroleum, Petrochems, and Polymers (2009). [5] C.C. Bosswell, W.R. Owers, B. Swanney, and H.P.

Rothbaum, Fertilizer research 15 (1988), pp. 13-31.

495


Effect of Nanoclay on Mechanical Properties and Flame Retardancy of Sisal Fiber/Pp Composites

W. Chanprapanon1,2, N. Suppakarn1,2 and K. Jarukumjorn1,2*

1 Suranaree University of Technology /Institute of Engineering /School of Polymer Engineering, Nakhon Ratchasima, Thailand

2 Center for Petroleum, Petrochemicals, and Advanced Materials /Chulalongkorn University, Bangkok, Thailand

* [email protected]

Abstract: Natural fiber reinforced polypropylene (PP) has gained a lot of interests due to low cost, low density, acceptable strength and stiffness, and fiber biodegradability. However, shortcomings of this composite are relatively low modulus, low notched impact resistance, large thermal expansion, and substantial creep. Moreover, its high flammability is one of the critical drawbacks of the composite. In this study, to improve the mechanical properties and flame retardancy of sisal fiber (Si)/PP composites, an organoclay (OMMT) as a nanosize filler was added into the composites. PP composites containing 30 phr of sisal fiber were prepared using an internal mixer. The test specimens were molded using an injection molding machine. In addition, maleic anhydride grafted polypropylene (MAPP) was used to enhance the interfacial adhesion between PP matrix and sisal fiber and also to improve the dispersion of the organoclay in PP matrix. It was observed that the tensile properties of PP and PP/Si composite were further increased with the addition of 3 phr of OMMT. Moreover, the incorporation of OMMT into PP and PP/Si composite also improved their flame retarding properties and thermal stability. X-ray diffraction analysis revealed that PP/OMMT composite exhibited the intercalated nanostructure. However, PP/Si/OMMT composite formed the exfoliated nanostructure. Introduction

Nowadays, the use of natural fibers to reinforce polymer has received considerable attention, particularly in structural and automotive industry due to cost effectiveness and eco-friendliness. By comparing with inorganic fillers, the main advantages of natural fibers are light weight, low cost, biodegradability, renewable nature, easiness of processing, the absence of toxic by-products, and high specific strength and modulus. The addition of natural fibers to polymers can cause a change in properties of the derived composites, which depends on properties of the natural materials and those of the polymers.

Polypropylene (PP) is particularly interesting thermoplastics used in composites because it has good and extensive properties, ease of processing, and low cost also. However, the incompatibility between hydrophobic polymers and hydrophilic natural fibers results in poor interfacial adhesion between the natural fibers and polymers. In order to solve it, maleic

anhydride grafted polypropylene (MAPP) is generally introduced as a compatibilizer to improve the stress transfer between the polymer matrix and natural fiber at low concentrations [1].

Another critical drawback of the use of natural fiber in PP composites is their high flammability due to their structure and chemical composition. Improving their flame retardancy will thus expand the range of their applications. In recent years, nanosized fillers have been good candidates for overcoming the aforementioned drawbacks of polymer composites. Incorporation of nanoclay at very low loading (less than 5 wt%) can improve properties e.g. high modulus, increased strength and heat resistance, decreased gas permeability and flammability, and increased biodegradability of biodegradable polymers [2]. In order to achieve the good property enhancement, clay particles should be homogeneously dispersed and exfoliated within the polymer matrix [3]. Melt blending is the main method for the preparation of nanocomposite due to its cost effectiveness in using the conventional polymer compounding technique and the compatible with the continuous nature of the industrial process, such as extrusion and injection molding [2-4].

The objective of this work was to study the effect of nanoclay on the mechanical, thermal and flame retarding properties of sisal fiber/PP composites. Materials and Methods

Materials: Polypropylene (PP; P700J) was supplied by SCG Chemicals Co.,Ltd. Sisal fiber (Si) was purchased from the Sisal-Handicraft OTOP group, Nakhon Ratchasima, Thailand. The sisal fiber was cut into approximate length of 2 mm. Organoclay (OMMT; Cloisite®30B, an originally modified montmorillonite) was supplied from Southern Clay Products Inc., USA. Maleic anhydride grafted polypropylene (MAPP; Fusabond®P MZ 109D, Dupont) was supplied from Chemical Innovation Co., Ltd.

Preparation of composites: Prior the composite preparation, sisal fiber and OMMT were dried in an oven at 70C over night. PP compounds were prepared by melt blending using an internal mixer (Haake Rheomix, 3000p). The total mixing time was 15 min.

496


The mixing temperature was 170C and the rotation speed was 60 rpm. Test specimens were molded by an injection molding machine (Chuan Lih Fa, CLF 80T) to produce the specimens according ASTM standard. Composition of PP and PP composites and their designation used are shown in Table 1.

Structure characterization: The structure of OMMT and PP composites were performed by an X-ray diffractometer (XRD; Oxford, D5005) equipped with a Cu K radiation source of wavelength 1.5406 Å operated at 40 kV and 40 mA at room temperature. The scanning rate and step size were 2.0/min and 0.02 with 2 varying from 2 to 10. The XRD patterns were used to calculate the basal spacing or d-spacing from Bragg’s law.

Mechanical test: Tensile properties of PP and PP composites were measured according to ASTM D638 on a universal testing machine (UTM; Instron 5569). Impact test was performed on unnotched PP and PP composites followed ASTM D256 using an impact testing machine (Atlas, BPI). In all cases, the average values of five specimens at least were taken for each sample.

Flammability test: The flame retardancy of PP and PP composites was characterized by a horizontal burning test according to ASTM D635 on a horizontal vertical flame chamber instrument (Atlas, HVUL). The sample was held horizontally and a flame was applied to light one end of the sample. The burning rate of the sample was calculated according to the formula:

V = 60L/t (1) Where V is the burning rate in millimeters per minute; L is the length the flame travels from the first reference mark (25 mm from the end ) to the second reference mark, which is at 100 mm from the end); and t is the time in seconds for the flame to travel. Five measurements per sample were taken.

Thermal analysis: Thermal behaviors of PP and PP composites were carried out on a thermogravimetric analyzer (TGA; TA Instrument, SDT2960) at a constant heating rate of 20C/min under air flow. The temperature ranged from the room temperature to 700C. The weight of each sample was kept within 10-15 mg. Table 1: Formulation of PP and PP composites.

Designation PP

(phr) Sisal (phr)

OMMT (phr)

MAPP (phr)

PP 100 - - - PP/Si 100 30 - 5 PP/OMMT 100 - 3 5 PP/Si/OMMT 100 30 3 5 Results and Discussion XRD patterns of PP, OMMT, PP/OMMT composite, and PP/Si/OMMT composite are shown in Figure 1. XRD pattern of OMMT showed a peak at 2 = 4.77 that corresponds to d-spacing of 1.85 nm.

PP/OMMT composite presented a shift of diffraction peak to a lower angle at 2 = 3.81 which corresponds to d-spacing of 2.32 nm, indicating the formation of an intercalated structure [2]. For PP/Si/OMMT composite, the peak was disappeared. This implied that the silicate layers could be exfoliated and dispersed in PP matrix forming a nanometer scale composite [5]. Table 2: Mechanical properties of PP and PP composites.

Designation Tensile strength (MPa)

Young’s modulus

(GPa)

Impact strength (kJ/mm2)

PP 14.06 0.55 0.92 0.03 78.67 0.94 PP/Si 30.60 0.37 2.32 0.04 11.39 0.25 PP/OMMT 26.53 0.75 1.40 0.05 60.34 0.85 PP/Si/OMMT 31.82 1.60 2.36 0.03 12.79 0.50

The mechanical properties of PP and PP

composites are summarized in Table 2. Tensile properties of PP and PP composites are shown in Figure 2. It was observed that all PP composites exhibited higher tensile strength and Young’s modulus than PP due to the reinforcing effect from sisal fiber and/or organoclay. In addition, adding 3 phr of OMMT into PP/Si composite slightly increased the tensile strength and Young’s modulus due to the formation of exfoliated nanocomposite structures formed at this clay loading [6-8].

Figure 3 represents the unnotched Izod impact strength of PP and PP composites. The addition of fillers decreased the impact strength of PP. It was because the filler particles in PP matrix provided sites for crack initiation [6]. However, the impact strength of PP/OMMT composite was higher than that of PP/Si composite. Riley et al. (1990) reported that the impact strength depends on both the size and the shape of the filler and is also affected by the micromorphology. Impact strength is enhanced by small, low aspect ratio filler particles [7]. Furthermore, the incorporation of OMMT into PP/Si composite slightly increased the impact strength. Similary, Lee and Kim (2008) found the impact strength of wood/PP composite filled 1 phr

2 Theta (degree)

2 3 4 5 6 7 8 9 10

Inte

nsity

(co

unt)

PP OMMT PP/OMMT PP/Si/OMMT

Figure 1. XRD patterns of PP, OMMT, PP/OMMT and PP/Si/OMMT composites.

497


PP PP/Si PP/OMMT PP/Si/OMMT

Ten

sile

str

engt

h (M

Pa)

0

5

10

15

20

25

30

35

40

You

ng's

mod

ulus

(G

Pa)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Tensile strength Young's modulus

Figure 2. Tensile properties of PP and PP composites.


Impa

ct s

tren

gth

(kJ/

mm

2 )

0

5

10

15

2050

60

70

80

90

Figure 3. Impact strength of PP and PP composites.


Bur

ning

rat

e (m

m/m

in)

0

5

10

15

20

25

30

35

Figure 4. Burning rates of PP and PP composites.

Temperature (oC)

100 200 300 400 500 600

Wei

ght (

%)

0

20

40

60

80

100

PP PP/SiPP/OMMTPP/Si/OMMT

Figure 5. TGA curves of PP and PP composites.

of OMMT was slightly higher than that without OMMT, indicating a positive effect of the OMMT inclusion [1].

Burning rates of PP and PP composites measured by a horizontal burning test are shown in Figure 4. PP exhibited the highest burning rate at about 28.89 mm/min. The addition of sisal fiber into PP did not substantially affect the flammability. The incorporation of OMMT into PP showed 16% reduction of the burning rate of PP. The flame retardancy mechanism of OMMT involves a high-performance carbonaceous-silicate char, which builds up on the surface during burning [2]. Moreover, the dramatic reduction of burning rate about 37.5% was observed when OMMT was added into PP/Si composite. Similary, Lee et. al. (2010) reported that the presence of clay at a small amount decreased the burning rate of wood fiber/HDPE composites and also suggested that achieving a higher degree of exfoliation of nanoclay is the key to enhance the flame retarding properties of natural fiber/polymer composites [8]. Generally, improvement in thermal stability is found for which the nanocomposite morphology plays an important role [9]. The incorporation of clay into the polymer matrix enhances thermal stability by acting as a superior insulator and mass transport barrier to the volatile products generated during decomposition [2]. Figure 5 shows TGA curves of PP and PP composites. The temperature at 5% weight loss (T5%) and weight residue at 600C of PP and PP

composites are shown in Table 3. The T5% of PP was at 285C and no char residue left. When compared with PP, PP/Si composite showed no significant change of T5% and left char residue about 1.8%. This was because the sisal fiber as lignocellulosic fillers has a component called lignin, which formed char during thermal degradation. Char reduces the combustion rate of polymeric materials by act as a protective layer to not allowing the oxygen to reach the combustion zone easily resulting in the reduction in thermal degradation in the material [10]. In addition, the incorporation of OMMT into PP and PP/Si composite resulted in a significant improvement in thermal stability by increasing the T5% and char residue. Biswal et al. (2009) also observed a remarkable enhancement in thermal stability of PP/pineapple leaf fiber composite in the presence of OMMT [9]. Table 3: TGA data of PP and PP composites.

Designation T5% (C)

Char residue at 600C (%)

PP 285 0.00 PP/Si 286 1.80 PP/OMMT 304 1.50 PP/Si/OMMT 297 3.59

498


Conclusions Incorporation of sisal fiber increased the tensile properties but reduced the impact strength of PP. Addition of OMMT into PP and PP/Si composites enables to achieve the nanocomposite. XRD patterns showed the intercalated nanostructure for PP/OMMT composite and presented the exfoliated nanostructure for PP/Si/OMMT composite. Tensile properties, flame retardancy, and thermal stability of PP were increased in a presence of OMMT. Moreover, the mechanical properties and flame retardancy of PP/Si composite were improved by incorporating the OMMT. Acknowledgements

The authors express their thanks to Suranaree University of Technology and Center for Petroleum, Petrochemicals, and Advanced Materials for financial supports, SCG Chemicals Co.,Ltd. for supplying P700J, Southern Clay Products Inc., USA for providing Cloisite®30B, and Chemical Innovation Co., Ltd. for supplying Fusabond®P MZ 109D. References [1] H. Lee and D. S. Kim, J. Appl. Polym. Sci. 111 (2009),

pp. 2769-2776. [2] S. Sinha Ray and M. Okamoto, Prog. Polym. Sci. 28

(2003), pp. 1539-1641. [3] Y. Dong and D. Bhattacharyya, Compos. Part A. 39

(2008), pp. 1177-1191. [4] M. Hetzer and D. De Kee, Chem. Eng. Res. Des. 86

(2008), pp. 1083-1093. [5] A. K. Barick and D. K. Tripathy, J. Appl. Polym. Sci.

117 (2010), pp. 639-654. [6] A. Nourbakhsh and A. Ashori, J. Appl. Polym. Sci. 112

(2009), pp. 1386-1390. [7] A. M. Riley, C. D. Paynter, P. M. McGenity and J. M.

Adams. Plast. Rubb. Process. Applic. 14(1990), pp. 85-93.

[8] Y. H. Lee, T. Kuboki, C. B. Park, M. Sain and M. Kontopoulou, J. Appl. Polym. Sci. 118 (2010), pp. 452-461.

[9] M. Biswal, S. Mohanty and S. K. Nayak, J. Appl. Polym. Sci. 114 (2009), pp. 4091-4103.

[10] M. B. A. Bakar, Z. A. M. Ishak, R. M. Taib, H. D. Rozman and S. M. Jani, J. Appl. Polym. Sci. 116 (2010), pp. 2714-2722.

antibacterial activity and cytotoxicity of chitosan

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