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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
Abstract: Antibacterial activity and cytotoxicity of chitosan (CS) and quaternized chitosans, i.e., chitosangraft-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 Gramnegative, 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.
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  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) , 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(2methacryloyloxy ethyl trimethylammonium chloride), another form of quaternized chitosan, was also prepared and used as a flocculant . 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(2methacryloyloxy 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 13 CNMR), 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
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 . The antibacterial activity of chitosan was found to be influenced by a number of factors, including molecular weight and environmental pH . In general, chitosan can be dissolved in aqueous acidic solution (pH<6.5), restricting its antibacterial activity under neutral aqeous conditions.
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470 from the National Institute of Health, Department of Medical Sciences, Thailand. Preparation of chitosan-graft-poly(2methacryloyloxy 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 1 H 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 CSGTMAC 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.
OH O HO (CS) O NH 2 (MEA) + O O N Cl 2%v/v acetic acid HO K2S2 O8 80oC, 1 h OH O O NH 2 HO NH
OH O O
OH O HO O NH 2 (CS) + O
OH 2%v/v acetic acid 60o C, 6h HO O O NH 2 HO
OH O O NH OH Cl N
Figure 1: Schematic preparations of (a) CS-g-PMEA and (b) 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, CSGTMAC 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* CS-g-PMEA CS-GTMAC CS
MIC (g/mL) S. epidermidis S. aureus 32 32 64 256 1000 512
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471 Table 2: MIC values of CS-g-PMEA, CS-GTMAC and chitosan tested against E. coli and P. aeruginosa Samples
+ + +
+ + +
+ + +
+ + +
+ + + +
CS-g-PMEA CS-GTMAC CS * n=2
MIC (g/mL) E. coli P. aeruginosa 32 128 256 1000 >5000 >10000
+ + + + + + + + + + +
+ + + + +
+ + + + +
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 2561000 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 . 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-3trimethyl 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-gPMEA. 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.
Figure 2: The structures of (a) the substituted chitosan (CS-GTMAC) and (b) the grafted chitosan (CS-gPMEA) Table 3: IC50 values of CS-g-PMEA, CS-GTMAC and chitosan Samples* CS-g-PMEA CS-GTMAC CS * n=8 Conclusions The antibacterial activity of CS-g-PMEA, evaluated against both Gram-positive and Gramnegative 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  C. Qin, H. Li, Q. Xiao, Y. Liu, J. Zhu and Y. Du, Carbohydr. Polym., 63 (2006), pp. 367–374.  H.K. No, N.Y. Park, S.H. Lee and S.P. Meyers, Int. J. Food Microbiol., 74 (2002), pp. 65–72.  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.  C.H. Kim, J.W. Choi, H.J. Chun and K.S. Choi, Polym. Bull., 38 (1997), pp. 387–393.  Z. Jia, D. Shen and W. Xu, Carbohydr. Res., 333 (2001), pp. 1–6.  H.S. Seong, H.S. Whang and S.W. Ko, J. App. Polym. Sci., 76 (2000), pp. 2009–2015. IC50 (g/mL) NHF 2000 2000 5000
L929 625 5000 5000
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Liu.  Y.J. 227–232. N.H. Chen. Li. Wang.. 67 (2007).Q. Yu and J. pp. Liu. X. pp. Meng. Liu. Chen.Z.F.G.P.S. X. Yu.G. X. C. Chemosphere. 66 (2007). L. Wu and H.472  J. Y. PACCON2011 (Pure and Applied Chemistry International Conference 2011) . 1752–1757. Kenendy. Polym. C. Carbohydr.
antibiotic loaded calcium phosphate beads which were prepared by phosphorization of three dimensionally printed (3DP) calcium sulfate dihydrate were developed. However. Klong 1. These beads could function as both drug carrier and bone graft which can integrate to the bone. thus. Recently. Thammarakcharoen and J. Recently. investigated. However. Pathumthani 12120 Thailand *E-mail: jintamai@mtec. 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. the effect of varying size of calcium phosphate beads on the antibiotic uptake and release characteristic was . (a) 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. In this study. Drug loading and releasing rate of both antibiotics were also found to increase with decreasing bead size. including the requirement for material removal by a second surgical procedure and poor antibiotic elution properties. changing the bead size may affect the function of antibiotic release.th Abstract: Antibiotic loaded polymethyl methacrylate (PMMA) beads are commercially available for treating patients with osteomyelitis or bone infection. This was related to the difference in microstructure of beads resulted from the processing of different size of the beads. eliminated.) were prepared and loaded with two kinds of antibiotics including gentamicin and vancomycin. PACCON2011 (Pure and Applied Chemistry International Conference 2011) . Suwanprateeb * National Metal and Materials Technology Center. Klongluang. the bone defect size of individual patients can vary. Three sizes of beads (3 mm. Polymethylmethacrylate (PMMA) is one of a widely used implant materials in orthopaedics and has been used successfully with antibiotics impregnation [3-5]. However. 114 Paholyothin Road. without systemically exposing an individual to antibiotic levels that often would result in numerous toxic side effects [1-2]. Microstructure and phase composition were characterized by x-ray diffraction. 5 mm and 7 mm. energy dispersive spectroscopy and mercury porosimeter. several drawbacks have been associated with PMMA use. Drug loading and releasing were analyzed using UV-VIS spectrophotometer. 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 (b) Figure 1. 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. thus.or. one problem inherent in the local placement of antibiotics loaded PMMA is that it requires subsequent surgery for removal and replacement with bone grafts. the effects of varying size of calcium phosphate beads on the antibiotic uptake and releasing characteristic were investigated.473 The effect of bead size on drug loading and releasing characteristic of antibiotic loaded 3DP calcium phosphate bead F. Chemical structures of a) gentamicin and b) vancomycin. antibiotic loaded calcium phosphate beads which were prepared by phosphorization of three dimensionally printed (3DP) calcium sulfate dihydrate were developed . However. It was found that decreasing bead size led to the decrease in density and increase in porosity and pore size of the beads. scanning electron microscopy. National Science and Technology Development Agency. 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. Since calcium phosphate has excellent biocompatibility and bioactivity. These beads provided local. However. since the bone defect size of patients can vary. 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. The need of second injury and use of additional grafting materials were.
P Drug Laboratories (1969) Co.Ltd. Drug release In order to determine the drug release profile. Materials Raw materials used in this study were calcium sulfate hemihydrate (Lafarge Prestia Co. Thailand).. Thailand) and pre-gelatinized starch (Thaiwah Co. calcium sulfate dihydrate which is typically the main phase in all as-fabricated 3DP samples similarly transformed to hydroxyapatite regardless of the bead size.531 0.15 85.5 second and a step angle of 0.26 63. 5. rinsed by distilled water and oven dried.24 0. the drug loaded calcium phosphate beads (S100 : 3 beads. All samples were immersed in the solution and kept at 80 ºC for 48 hours in the oven. These differences are possibly due to the initial pore size. 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.13 0. Material characterization Powder X-ray diffraction was carried out by using a JEOL JDX 3530 X-ray diffractometer with Co Kalpha radiation in the range of 10-80 º 2θ. All samples were gold sputtered prior to the observation. When the specified times for data collection were reached. XRD patterns of as-fabricated and converted 3DP samples.739 0. It can be seen that the as-fabricated sample is highly porous comprising entanglement of rod-like calcium sulfate crystals.61 75.10 0. The concentration of antibiotics in the sample beads were then determined by elution in 2.02 º.12 59. porosity and density of the samples Samples CaS-S50 CaS-S75 CaS-S100 CaP-S50 CaP-S75 CaP-S100 Median pore size (μm) 23. 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. Ltd) and vancomycin (CJ CheilJedang Corporation.Ltd. Antibiotics used were gentamicin (T..789 0. Results and Discussion Figure 2 shows the XRD patterns of as-fabricated and converted 3DP samples by phosphate solution. 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.89 22.. sample CaS-S100 has the lowest pores size.474 medium. 10 ml of SBF was withdrawn by pipetted and replaced immediately with 10 ml of fresh SBF Figure 2. S75 : 6 beads and S50 : 21 beads) were submerged in 100 ml of simulated body fluid (SBF)  at 37 ºC. Table 1: Pore size. After conversion to calcium phosphate. These materials were supplied in the form of powders and used without further sieving.81 17. Materials and Methods 1. lowest porosity and greatest density. 3. After conversion. Samples were then taken out. Korea). In contrast. Microstructure of the specimens were examined by using a scanning electron microscope (JEOL JSM-5410) at an accelerating voltage of 20 kV. 2. sample CaP-S50 seems to have the lowest pore size and highest porosity while sample CaP-S100 has the greatest density after conversion. 1M of disodium hydrogen phosphate solution was prepared. The chemical structures of these drugs are shown in figure 1.838 0. a counting time of 0. 4.07 61. 5 mm (S75) and 7 mm (S100). using vacuum method. Prior to conversion. Drug loading The calcium phosphate beads were loaded with two types of antibiotics. the PACCON2011 (Pure and Applied Chemistry International Conference 2011) . Water-based binder was used as a jetting media. Z Corporation) to print three sizes of spherical specimens including 3 mm (S50).622 Figure 3 shows the microstructure of as-fabricated and converted samples of various bead sizes.92 Density (g/cm3) 0. The as-fabricated samples were then transformed to calcium phosphate (CaP) by phosphorization reaction.620 0. JCPDS files were used to identify the crystalline phase composition. 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. Quantachrome Instruments).15 Porosity (%) 76.4 M HCl and analysed by using UV-VIS spectrophotometer (Jasco V-530). The porosity and pore size of all samples were measured by mercury porosimetry analyser (Pore Master. 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.
Comparing between two types of antibiotics. The antibiotics loading efficiency. the release rate of gentamicin is higher than that of vancomycin.475 microstructures of all converted samples appear denser and the formation of new finer needle-like calcium phosphate crystals is evident. antibiotics uptake of CaP-S50 is the greatest followed by CaP-S75 and CaP-S100 respectively. This is in accordance with the decrease in pore size values as seen in table 1. Drug release profiles of antibiotics from three sizes of calcium phosphate beads. Drug loading per bead volume of 3DP calcium phosphate samples. antibiotics release rate of CaP-S50 is the greatest followed by CaP-S75 and CaP-S100 respectively. PACCON2011 (Pure and Applied Chemistry International Conference 2011) . Figure 5 shows the drug release profiles from different sizes of calcium phosphate beads. Figure 4. follows the decreasing order of the bead size. Microstructures of as-fabricated converted 3DP samples. Figure 4 shows the amount of antibiotics uptake per volume of the bead in different size of calcium phosphate beads. However. (a) (b) Figure 3. thus. no significant difference in the microstructure amongst CaP-S50. This may be due to the highest porosity of CaP-S50 sample compared to others. a) gentamicin release and b) vancomycin. Therefore. follows the decreasing order of the bead size. Gentamicin has smaller molecular size and lower molecular weight than vancomycin. thus. In addition. This is again due to the smaller molecular size and lower molecular weight of gentamicin than vancomycin. All release profiles show two-phased release patterns. The antibiotics release rate. burst release at the initial period and follows by a slow release phase. Therefore. and Figure 5. the decrease in the diameter of the bead will increase the diffusion rate of media solution and the increase in release rate of antibiotics. Comparing amongst three sizes of beads. Comparing between two types of antibiotics. CaP-S75 and CaPS100 samples is observed. So gentamicin molecule can diffuse out of the CaP beads more readily. the diameter of the bead is directly related to the distance for antibiotics solution to diffuse into the core of the bead. 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. So gentamicin molecule can penetrate through the pore of the CaP beads more readily. Comparing amongst three sizes of beads. Similarly to the drug loading. gentamicin could be loaded into the CaP beads at greater amount than vancomycin. the decrease in the diameter of the bead will increase the rate of antibiotics diffusion.
Fürst and J. 518–522. Diefenbeck. Osmon and R. C.  J. Comparative study of antimicrobial release kinetics from polymethylmethacrylate. 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. 69 (1998).H. pp. Chang. Piper. pp. pp.A. pp. Prophylaxis and treatment of implant-related infections by local application of antibiotics. Arthrop. P. Takadama. Kelm.R. Lee and V. Kleijn and M. 21 (2010). Patel. T. 419-429. Res. 2907-2915. J.E. O. K. Wasoontararat. Mückley.H. Walenkamp. Antibioticimpregnated PMMA hip spacers: Current status. Liquid gentamicin and vancomycin in bone cement: A potentially more cost-effective regimen. Jacofsky. Acta Ortho.S. Mater.476 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). PACCON2011 (Pure and Applied Chemistry International Conference 2011) . 77 (2006). Hofmann. Hsieh. 27 (2006).  G.L. Kokubo and H. Tai. pp. How useful is SBF in predicting in vivo bone bioactivity ?. Rel. Biomaterials.  K. M.M. de Leeuw. Ortho. D. Osteomyelitis treated with gentamicin-PMMA beads: 100 patients followed for 1-12 years.O. Acta Ortho.J. Suvannapruk and K.I. Anagnostakos. 24 (2009). Rouse. 239–244. This is related to the difference in microstructure.  P. J. Clin. Injury: International Journal of the Care of the Injured. Both drug loading and release rate increase with the decrease in bead size. W. 628–637.  P.C. Anguita-Alonso. Med. L. 445 (2006). pp. Suwanprateeb. S95S104.H. pp.  T. Low temperature preparation of calcium phosphate structure via phosphorisation of 3D-printed calcium sulfate hemihydrate based material.L. 37 (2006). D. G. References  M.125-130.
2. the disk surface was heated by a laser to the temperatures up to 400 oC.[1. 8-10] With HAMR technique. Both lubricants were purchased from Aldrich Chemical Company. It was found that the thermal degradation of BL-104 started at 300oC and completed at 430oC. The lubricant depletion characteristics due to laser heating in HAMR were found to depend largely on the lubricant film thickness and material. TGA/SDTA 851e.ac. PACCON2011 (Pure and Applied Chemistry International Conference 2011) . Used in Heat Assisted Magnetic Recording (HAMR) Technique.02) were used.477 Effect of Laser Energy. Chemical structure of the lubricants. The sample heated up from 30 to 700 oC at a scan rate of 10 oC/min in nitrogen atmosphere. RTILs have received much attention due to their unique chemical and physical properties. in this paper. In this study. Thailand. an RTIL. 1-butyl-3-methyl imidazolium tetrafluoroborate (BL-104). Kaewpirom* Burapha University/Department of Chemistry. The thickness of the lubricant films were controlled by pulling-up the disks from the lubricant solutions with a velocity of 1. One of the high performance lubricants is room temperature ionic BL-104 Figure 1. 4-6] The most widely used PFPE for the lubrication of magnetic disk surfaces is Zdol3800. the magnetic thin-film disks were dip coated with two kinds of lubricants. Zhao and co-workers expected that BL-104. Suktanarak. Faculty of Science. * E-mail: firstname.lastname@example.org 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. However. such as high thermal stability and non-flammability. The chemical structures of Zdol3800 and BL-104 are given in Figure 1. The outgassing was analyzed by GC-MS. T. Thermal stability of Zdol3800 and BL-104 were examined by thermogravimetric analysis (TGA) in the temperatures between 20 and 700oC. liquids (RTILs). The coated lubricant was also heated and depleted from the disk surface. The laser rapidly heats the disk surface to temperatures up to 400oC. were used. the disadvantage of PFPE are catalytical degradation by strong nucleophilic agents and strong electropositive metals. 3 and 5 mm/s after being immersed in the solutions for 120 s. After laser heating. was good candidates to replace PFPE as a versatile lubricant.800) and 1-butyl-3methyl imidazolium tetrafluoroborate or BL-104 (MW = 226. on the Lubricant Depletion S. we demonstrated the lubricant behaviour after getting heated from laser source for short periods of time. the outgassing was examined by gas chromatography mass spectroscopy (GC-MS). Poly(tetrafluoroethylene oxideco-difluoromethylene oxide) (Zdol3800). There are many researchs reported some tribological properties of RTILs. and high cost. Introduction In magnetic storage industry. where n/m is 2/3. 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. A conventional lubricant.5-inch glass substrate disks. The good performance of BL-104 in terms of thermal stability was nearly comparable to Zdol3800. HO–CH2–CF2O–(CF2O)m–(CF2CF2O)n–CF2–CH2–OH Zdol3800. In order to search for the suitable lubricants that can withstand such high temperature in HAMR technique. Jamnongkan and S. Thermogravimetry analysis (TGA) The TGA tests were conducted in a Mettler Toledo. which consists of a perfluorinated ether backbone terminated at either end by hydroxyl groups. Materials and Methods Materials The disks used in this study were 2. Chonburi.[4. and an ionic liquid. Two kind of lubricants: poly(tetrafluoroethylene oxide-co-difluoromethylene oxide) or Zdol3800 (MW = 3.
280 oC Capillary column HP-5MS (Agilent) Oven Program Initial Temp. In the next step. The media was allowed to dry in air prior to the measurements. The solution concentration of Zdol3800 and BL-104 were 0. 40 oC Initial Time 1 min. In the first step. Zdol was observed to have 2-stepdecomposition started at 200 oC and completed at 500 o C. 250 oC Detector Temp. PACCON2011 (Pure and Applied Chemistry International Conference 2011) . These observations indicated that the lubricant molecules spreaded evenly on the hard disk surface with the averaged surface roughness values between 1-2 nm.1% (w/v) and 0. Then the media was dipped into and pulled up from the solution with a velocity 1. In this experiment. thermal effects of two kind lubricants. 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. after being immersed in the solution for 120 s.3-dihydrodecafluoropentane.05% (w/v). respectively. Figure 3 (b) shows TGA thermogram of BL-104. After that the out-gassing was characterized by gas chromatography mass spectroscopy (HP 6890) with the following conditions in Table 1. Surface roughness measurement The average surface roughness measured by atomic force microscopy (AFM.478 Film preparations The solution of Zdol3800 and BL-104 were prepared in 2. The white spots in the figures are due to dust particles. Results and Discussion Thermal effects on lubricants In TGA test. Experimental setup for the measurement system for the assessment of the lubricant performance in a HDD with HAMR technology. Mass spectrometer HP 5972 (Agilent) In figure 3 (a). commercial name Vertrel®.mass spectroscopy technique. XE-70) from Park Systems in ambient conditions and a scan rate of 0. its thermal degradation started at 300 oC and completed at 430 oC. TGA thermogram of (a) Zdol and (b) BL104 Surface roughness of lubricants Figure 4 shows 3D surface morphology of Zdol3800 and BL-104 scanned over an area of 5x5 µm2. A laser beam with a wavelength of 532 nm and the energy of 340 mW. from Dupont (Thailand) Ltd. Therfore. 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 . were examined and the results are shown in Figure 3. BL104 had one step decomposition. the test disk was rotated while laser irradiated.5 Hz with dimensions of scan size 5x5 µm2. the decomposition of the lubricant was caused by evaporation of the fragments and and intact molecules remained at 480 oC. (a) Laser source Vacuum chamber Sampling station Magnetic disk media (b) Figure 3. Table 1: GC-MS conditions Gaschromatography HP 6890 (Agilent) Carrier Gas Helium Injector Temp. irradiated on disk surface which the lubricant film deposited. it had catalytic decomposition to scission of the PFPE backbone. 3 and 5 mm/s. BL-104 showed better performance than Zdol3800 in terms of thermal stability. The GC chromatograms of the outgassing collected after Figure 2. 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. the resulting fragments could reasonably be expected to be much more volatile than the intact molecules at 360 oC.
m/z = 97 is CF2COF+. m/z = 169 is C3F7+. These results are consistent with the results reported by Gerard et. Figure 6 shows mass spectra corresponded to the peak of GC chromatogram: m/z = 19 is F-. 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 (e) Figure 6. (a) (b) (c) (d) (a) 1 mm/s (e) Figure 5. m/z = 69 is CF3+. m/z = 85 is CF3O+.479 laser heating on the HDD surface are shown in figure 5. m/z = 163 is C3F5O2-. (c) 10 s (d) 30 s and (e) 60 s. m/z = 185 is C3F7O-. (c) 10 s (d) 30 s and (e) 60 s. and their corresponding mass spectra are shown in figure 6. (b) 5 s.155 min. PACCON2011 (Pure and Applied Chemistry International Conference 2011) . m/z = 147 is CF2CF2COF+.110 to 1. m/z = 20 is HF. In figure 5. which was the time of fluorocarbon moved from column. It was founded that the depletion product had the retention time between 1. m/z = 119 is C2F5+. (b) 5 s. (b) 3 mm/s (a) (b) (c) 5 mm/s (c) (d) (d) 1 mm/s Figure 4. Mass spectra of the outgassing after laser radiation at (a) 1 s. GC chromatograms of the outgassing after laser radiation at (a) 1 s. m/z = 285 is C5OF11+ and m/z = 335 is C6OF13+. al. m/z = 219 is C4F9+.
Acknowledgments Financial support from the Industry/University Cooperative Research Center (I/UCRC) in Data Storage Technology and Applications. m/z = 163 is C3F5O2-. Xue. m/z = 219 is + C4F9 . KMITL. Z. Falcone and B. B. 1-5. Bai. 141-148. Gerard.Z. Raymond and S. J. Bhatia. M. Tribology Letters 6 (1999). Tribology Letters 11 (2001). Ultramicroscopy 109 (2009). Tribology Letters 8 (2000). Chem. Y. 12105–12110.480 Conclusions 1. pp. Bhushan. at different periods of time. L. Marchon. The magnetic disks were dip coated with Zdol3800 and BL-104 successfully with the average surface roughness values between 1-2 nm. Chen.H. After laser heating of Zdol3800 lubricant film. American Chemical Society. Colloid and Surfaces A: Physicochem. 980-990. Kasai and C. Commission on Higher Education. Bhushan and C. B. A.  W. Supporting Vertrel®. Mo. Phys. 823-833. pp. 169-179. Washington. pp.M. Bryan. Tribology Letters 17 (2004). pp.  Z.J. Editors. 105 (2001). Wang.  M. 2. The molecules obtained from the outgassing analyzed by GC-MS show the mass spectra: m/z = 19 is F-. 4.. m/z = 97 is CF2COF+.J.  P. 199-205. m/z = 69 is CF3+. m/z = 20 is HF. in the measurement system for the assessment of the lubricant performance in a HDD with HAMR technology. Palacio and B.  K. Burapha University. m/z = 285 is C5OF11+ and m/z = 335 is C6OF13+. m/z = 169 is C3F7+. PACCON2011 (Pure and Applied Chemistry International Conference 2011) . D. Kajdas. A. 3. From TGA. SURFACE SCIENCE INVESITGATIONS IN TRIBOLOGY.J. National Science and Technology Development Agency and the Center of Excellence for Innovation in Chemistry (PERCH-CIC). Fong. m/z = 119 is C2F5+. References  C.  M J. Eng.C (1999). Bai and Q.  R. Faculty of Science. Gellman. and National Electronics and Computer Technology Center. pp. pp. Wang. Tribology Letters 6 (1999). pp. Paserba and A. Ltd.  W.  V. Coax Group Corporation for AFM analysis. m/z = 185 is C3F7O-. J. The depletion of Zdol3800 occured since the first second of laser heating. Aspects 361 (2010). 5. The acknowledgement also goes to Dupont Co. D. Zhao.-Y. 828-835. Spiese. Pu and M. B. m/z = 147 is CF2CF2COF+. Ministry of Education is gratefully acknowledged. Gellman and P. BL-104 showed better performance than Zdol3800 in term of thermal stability.R. S. Jones. Bogy and S.J. pp. the depletion of Zdol3800 was detected. Y. David In: Y. and the Department of Chemistry. W. 118-125. Chung. m/z = 85 is CF3O+. Homola and G. pp. Stirniman. 25–34. Zhao. Tribology International 42 (2009). Lei. pp. Zhao.
In this study. Therefore. poly(hydroxybutyrate) (PHB) and poly(butylene succinate) (PBS). the expression of detected aggrecan gene was not much different in all scaffolds. 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. Cell culture: The sterilized sample discs were placed into 24-well culture plates. and differentiated cell function. Pathumthani. after 21-day culture Introduction Recently. were directly prepared in our laboratory by a supercritical fluid CO2 method. surface treatments of scaffolds have been conducted to improve the cell-material interfaces. 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. 0.e. tissue engineering has been studied as an alternative method to repair damaged articular cartilage. 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].1 M NaOH. DNA assay: Cell proliferation on the scaffolds was assessed by a DNA assay. glycosaminoglycans (GAGs) and collagen production. Typically. and concentrations of NaOH solution. i. This technique involves the regeneration of tissues by culturing chondrocytes on three-dimensional scaffolds to generate articular cartilage.or.9-dimethylmethylene blue (DMB) dye solution. The culture medium was regularly replaced every 3 days. with three ranges of pore sizes.1 M or 0. i. Thavornyutikarn.. The product code was assigned based on the concentration of NaOH initially used. it was noted that the pore sizes of the scaffolds affected the production of GAGs by chondrocytes. i. Various biodegradable polymers have been extensively explored as tissue engineered scaffolds. Selection of porous three-dimensional scaffolds is an inevitable consideration for tissue engineering study. 350-500 µm. Biodegradable aliphatic polyesters.. W. The pore size. Furthermore. plays an important role in cell adhesion. the resulting HPBS scaffolds could facilitate the secretion of glycosaminoglycans (GAGs) and total collagen by chondrocytes. i.e. cell migration. poly(-caprolactone) (PCL).g. cell growth. 500-700 µm and 700-1000 µm.6 M NaOH solution at 60oC for 30 min. with a range of pore size of 500-700 µm.e. In brief. The results revealed that after hydrolysis. The number of cells was directly relative to the measured intensity. after 28-day culture period. and cell infiltration and distribution observed by histological evaluation. the cells cultured on each scaffold were lyophilized and digested with papain solution (5 mg/ml) at 60oC for 18 h. Thailand 12120 * E-Mail: wanidaj@mtec. Surface chemistry of scaffolds also governs how cultured cells respond to the materials.. articular cartilage-specific gene expression. compared with those cultured on the PBS scaffolds. Human articular chondrocyte suspensions (2x106 cells/specimen) were seeded onto the scaffolds.. in terms of cell proliferation. The PBS scaffolds were subsequently hydrolyzed with 0.g. PACCON2011 (Pure and Applied Chemistry International Conference 2011) . used in the PBS surface hydrolysis on the formation of cartilage-extracellular matrix (ECM) were investigated. The higher the concentration of NaOH solution used in the hydrolysis process. the greater the amount of GAGs and collagen content secreted.e. aggrecan and type II collagen genes. e. e. The effects of ranges of average pore sizes of the scaffolds. 350-500 µm. with 0.481 Study on Hydrolyzed Poly(butylene succinate) Scaffold-Chondrocyte Responses P. large pore sizes could lead to low cell attachment and low intracellular signaling whereas small pore sizes could promote these functions [3-4]. Conversely. 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. Kosorn and W. Uppanan. poly(lactic acid) (PLA). Aliquots of each papain-digested solution were mixed with dye Hoechst 33258 solution (20 µg/ml). 500-700 µm and 700-1000 µm. Materials and Methods Materials: PBS scaffolds.1 M and 0.6 M.. Glycosaminoglycans (GAGs) assay: The total GAGs content secreted from the cells cultured on each scaffold was determined using 1.th Abstract: The responses of chondrocytes on the hydrolyzed poly(butylene succinate) (HPBS) scaffolds were studied. Meesap. B. P. The cells cultured on the scaffolds were incubated under 5% CO2 atmosphere at 37oC for given periods. Janvikul* National Metal and Materials Technology Center. have been extensively studied for potential uses in bone and cartilage tissue engineering applications as cell/tissue culture substrates [1-2]. The histological results demonstrated that the chondrocytes proliferated more effectively both on the surface and inside the pores of the HPBS scaffolds.. However. HPBS-500-0.1 prepared from the hydrolysis of PBS. Greater amounts of total collagen were also yielded when larger pore sizes of the scaffolds were employed. in general. The fluorescence intensity was measured at 355 and 460 nm. poly(glycolic acid) (PGA).
aggrecan and type II collagen. the rougher the pore surface observed. 0. The GAPDH mRNA levels were used as internal controls.d) HPBS500-0. 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.e. PACCON2011 (Pure and Applied Chemistry International Conference 2011) . as shown in Figure 1(b).1b HPBS-700-0.1 M NaOH solution c = PBS scaffolds after hydrolysis with 0. (e. the higher levels of cell proliferation were consequently yielded. especially when a high concentration of NaOH. The amount of GAGs was determined against the chondroitin-6-sulphate standard curve.6c a Pore Size (µm) 350-500 500-700 700-1000 400-500 700-900 800-1000 750-900 1000-1200 1200-1600 a = PBS scaffolds before hydrolysis b = PBS scaffolds after hydrolysis with 0. i. The solutions of each hydrolyzate were brought to 0. The pore surface of the PBS scaffold was clearly smooth. The hydrolyzates were then neutralized with 6 M NaOH solution.6 M NaOH. The PCR products were identified by electrophoresis on 2% agarose gel. as revealed in Figure 1. after 28-day culture period. the pore sizes of the hydrolyzed PBS (HPBS) scaffold increased when compared with the initial pore sizes of the PBS scaffolds. Results and Discussion The surface morphology of the scaffolds was examined by scanning electron microscopy. embedded in paraffin. 500-700 µm and 700-1000 µm.d. which were coded as PBS-350.482 period.c. The sections were stained with hematoxylin and eosin (H&E) to observe the cell infiltration and distribution. respectively. Three ranges of average pore sizes of PBS scaffolds used in this study were 350-500 µm. The amount of total collagen was determined against the hydroxyproline standard curve. It was noted that after surface hydrolysis. The absorbance was measured at 525 nm.25 ml of 17%w/v NaCl in H2O. SEM micrographs of (a.1. PCR analysis was conducted for both genes of interest and glyceraldehydes-3-phosphate dehydrogenase (GAPDH). All scaffolds appeared highly porous. Collagen content assay: The total collagen content secreted from the cells cultured on each scaffold was determined using a hydroxyproline assay.6.6c HPBS-700-0. 1 µg of total RNA was reverse-transcribed into cDNA using the RevertAid™ First Strand cDNA synthesis Kit (Fermentas). i.. total RNA was extracted from the chondrocytes cultured on each scaffold using TRIZOL reagent (GibcoBRL). This resulted in the greater number of cells being cultured within the scaffolds. the scaffold surface became rough (as shown in Figure 1(c-f)).. After surface treatment with NaOH solution.e). were determined by RT-PCR analysis.6c HPBS-500-0.f) HPBS-500-0. The absorbance was measured at 550 nm. 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. The higher the concentration of NaOH solution used. Histological analysis: The cells cultured on the scaffolds were fixed with 2% paraformaldehyde solution. RT-PCR analysis: Articular cartilage-specific genes. The scaffold surface morphology (a.6 M. after a given culture period. In brief. original magnification x1500 Table 1: The ranges of average pore sizes of the PBS scaffolds before and after hydrolysis with NaOH solutions Scaffolds PBS-350 PBS-500 a PBS-700 a HPBS-350-0.1 M and 0. Aliquots of 100 l of each papain-digested solution were mixed with 100 l of DMB solution.1b HPBS-500-0. original magnification x75 and the pore surface morphology (b.e. the scaffolds were lyophilized and digested with papain solution (5 mg/ml) at 60oC for 18 h.25 ml with water and then mixed with 0. PBS-500 and PBS-700.b) PBS-500. i. are reported in Table 1. The average pore sizes of PBS scaffolds before and after surface hydrolysis using different NaOH concentrations. and sectioned (5 µm thickness). The mixtures were sequentially reacted with 0.f).5 ml Ehrlich’s reagent at 60oC for 20 min.1b HPBS-350-0. 500 m (a) 30 m (b) 500 m (c) 30 m (d) 500 m (e) 30 m (f) Figure 1. (c. In brief.e. the scaffolds were lyophilized and hydrolyzed with 6 M HCl solution at 100oC for 12 h.. was employed. 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.5 ml freshly prepared chloramine-T reagent at room temperature for 5 min and 0. respectively.
(lane 4) HPBS-350-0.0 0 300 250 250 0.6 0. The cells more highly proliferated and homogeneously infiltrated through the inner hydrophilic HPBS scaffold than those cultured with the less hydrophilic PBS scaffold.6 and M=marker The histological results illustrated the infiltration and distribution of the cells after cultured with the PBS and HPBS scaffolds for 28 days. No type II collagen gene expression was found when the cells were cultured on HPBS-350-0. Figure 5 presents the mRNA expression of aggrecan and type II collagen secreted by the chondrocytes after being cultured with scaffolds. and waste removal. The mRNA of GAPDH was vividly expressed in all scaffolds.2 .6. the number of cells significantly increased with an increasing pore size of the HPBS scaffolds used. as revealed in PACCON2011 (Pure and Applied Chemistry International Conference 2011) . respectively. 0. 6 Aggrecan Type II Collagen Figure 5. the HPBS scaffolds could promote the secretion of GAGs and total collagen by chondrocytes. RT-PCR results of genes encoding GAPDH. i. 0- 0- 0- S- S- S- 0- 50 PB PB PB 35 S- 70 S- S- 35 S- PB PB PB PB H PB S- H Figure 4.6. the greater the amount of GAGs and collagen content secreted.0 0 1 1 6 1 6 00.8 0. 400 GAGs (g/g scaffold) 350 Fluorescence Intensity 1000 800 600 400 200 200 0 .6 M NaOH (Figure 3).4 0. 0. 00.2 0. 0. 0. compared with that of GAPDH as a housekeeping gene. These results were in good accordance with the proliferation results.. (lane 6) HPBS-700-0.1. (lane 9) HPBS-700-0.6 . Aggrecan gene expression was observed in every scaffold with slightly different levels. 050 PB S70 0 0 0 00. 35 50 70 0- 0- 0- 0- 0- S- S- S- 50 35 70 35 50 PB PB PB S- S- S- S- S- S- 70 0- 0. H 6 0.6 (lane 9 in Figure 5). 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.1 (lane 4 in Figure 5) and HPBS-700-0. the secretion of collagen was apparently increased with an increasing pore size of the scaffolds (Figure 4).0 0 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 implied that both surface hydrophilicity and pore size of the scaffolds directly influenced the cellular function of the chondrocytes. overall.6 0.8 . 0.4 0. (lane 7) HPBS-350-0. (lane 8) HPBS-500-0.483 The effect of pore size of the scaffolds on the cell proliferation was also investigated.8 0. aggrecan and type II collagen secreted by chondrocytes cultured on scaffolds. The hydrophilic surface and the large pores of the HPBS scaffolds could enhance the level of the cell proliferation. whose pore sizes 1 H 1 6 PB S50 PB PB PB 00. (lane 1) PBS-350. Total collagen content secreted from chondrocytes cultured on various scaffolds at 28-day incubation period M 1 2 3 4 5 6 7 8 9 GAPDH 700 500 400 700 500 400 700 500 400 H H H H 0- 0. the amount of GAGs was increased with an increasing pore size of the scaffolds. The results revealed that after surface hydrolysis. Fluorescence intensity of culture media of chondrocytes incubated with each tested scaffold for 28 days Total Collagen (g/g scaffold) H PB Figure 3. the secretion of more GAGs and total collagen by chondrocytes were consequently resulted. 6 120 100 80 60 40 20 20 0. Interestingly. The effect of pore size of the scaffolds on the secretion of GAGs and collagen was also studied. Larger pores could more readily facilitate cell migration. S- S- S- S50 S70 S35 S35 PB PB PB PB H H H H PB PB PB PB PB H H H H H Figure 2.1. (lane 2) PBS-500. leading to higher cell growth. The higher the concentration of NaOH solution used in the hydrolysis process. GAGs content secreted from chondrocytes cultured on various scaffolds at 21-day incubation period 1 1 1 6 6 0 0 0 0. 0. Likewise. 00.1.2 0. As displayed in Figure 2. PB S70 35 50 70 6 0 0 0 1 0. 00. 0. 1400 1200 were in the ranges of 400-500 µm and 1200-1600 µm. nutrient flow.4 . (lane 3) PBS-700. (lane 5) HPBS500-0. especially observed in the HPBS scaffolds prepared with a high NaOH concentration. 35 50 70 0. It was noted that.e.
the chondrocytes proliferated more homogeneously throughout the entire HPBS scaffolds. pp. This led to greater cell proliferation and ECM production.Y. 200 µm (a) 200 µm (b) Figure 6.H. H&E staining of cross-sections of the scaffolds: (a) PBS-700 and (b) HPBS-350-0. I.K. Acta Biomater. Yu.P. Kim and J. 5 (2009). Cai.J. Zhang. Yang. L.6. 1664-1671. Chang. Ho. 1937-1947. e108-e111. Chen and X. pp. Biomaterials. 5 (2009). Lee.H. Ko and T. Jing. N. 670-679. 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. pp.484 Figure 6.W. Biomaterials. pp.Y. Acknowledgement This research was financially supported by National Metal and Materials Technology Center (project code: MT-B-52-BMD-07-175-I).M. Lee.Y. 28 (2007).Q. J.H. Huang. Wang and J. 30 (2009).T. Huang and Y.  Y. Moreover. 7S1 (2007). References  K. Acta Biomater. Hong. Zhang. X. Liu and S. pp. G.D. Y. Z. 44 (2009). L. 992-998. Curr.  P. Ng. pp. PACCON2011 (Pure and Applied Chemistry International Conference 2011) . 58-70. Lien.  R. Oh. Appl Phys.M.  S. compared with those cultured on the starting PBS scaffolds. Park. after 28-day cell culture 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. T. Process Biochem. X.  S. Cheng.
64 mA/cm2. 2. XRD patterns confirmed that the synthesized material consists of face-center cubic Zn2SnO4. Then free electrons from the dye molecules are injected into the conduction band of the semiconductor. Thailand 10140 2 Thin Film Technology Research Laboratory.5 solar simulation of TiO2-coated nanowire were studied. while the cells with TiO2-coated nanowires show a significant improvement of JSC to 2. One-dimensional (1D) nanocrystalline of metal oxide was purposed to use as transparent electrode for DSSCs to enhance the rate of electron transportation . First. The solar cell with only nanowires has the short-circuit current density JSC of 0. So. Rattana 1.25 g Zn powder (99%. 328 Si Ayutthaya Road. After synthesizing nanowires on ITO/glass substrates. The electrical characteristics of the fabricated cell under AM 1. The alumina boat was then inserted into a horizontal quartz tube. The performances of these composite solar cells were compared with a pure nanowire cell. Metal oxide nanowhiskers are grown on substrates by several techniques such as chemical vapor deposition (CVD).2. Materials and Methods Metal oxide nanowires were grown on ITO/borosilicate glass substrates. They used Au particles as catalysts to grow semiconductor whiskers on the Si wafer from the gas source. Ratchathewi. the electron conduction rate could be enhanced by using 1D nanostructure. Tuayjareon 1. hexagonal ZnO and tetragonal SnO2. Therefore. the nanowires were then coated with TiO2 thin film via DC reactive magnetron sputtering process. This process was first described by Wagner and Ellis in 1964. 1 metal organic chemical vapor deposition and molecular beam epitaxy (MBE) . P. The TiO2coated nanowires on ITO/borosilicate glass were then used as electrodes for dye-sensitize solar cells. The vapor transport technique was selected for our study due to its simplicity. 2. Jutarosaga 1. The increase of JSC is possibly due to the better adsorption of dye molecules on the TiO2 surface compared to nanowire surface. P. 2 nanowire of specimens were then coated with TiO2 via DC reactive magnetron sputtering using the mixture of Ar and O2. due to the lack of surface area adding nanoparticles into the nanowire matrix will possibly improve the carrier generation. Nootong 1. respectively. Mixture of 0.2*. CHE. has been developed.th Abstract: Metal oxide nanowires were successfully fabricated via vapor-solid-liquid (VLS) mechanism at 630oC on indium-tin oxide/borosilicate glass for a dyesensitized solar cell (DSSC) application. and T. Aldrich) and 0. 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.0×10-2 mbar and a sputtering current of 4 mA for 4 min. Faculty of Science. However. In order to synthesize metal oxide nanowires.61x10-3 mbar. Suapadkron 1. 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. 10400 *E-mail:s1400503@kmutt. A pre-sputtering process was employed for 300s to clean the target surface. Preparation conditions of nanowire are set in Table 1. TiO2 nanostructure film was coated on nanowire via DC reactive magnetron sputtering process. P. During the sputtering process.67x10-4 mbar.485 TiO2-Coated Zn2SnO4 Nanowires for Dye-Sensitized Solar Cells R. Before TiO2 deposition. PACCON2011 (Pure and Applied Chemistry International Conference 2011) . the chamber was evacuated to a base pressure of 6. Tungkru. T. Limsuwan 1. The crystallinity and the morphology of synthesized nanostructure were verified using X-ray diffraction (XRD) technique and scanning electron microscopy (SEM).4 mA/cm2. The admission of O2 (the reactive gas) to the chamber was independently controlled using mass flow controller until the pressure in the chamber reached 6.2. Before applying as electrode for DSSCs.ac. 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. ThEP Center. After that the Ar was flowed into the chamber until the pressure reached 5. Bangkok. in our study. SEM images showed wire-like nanostructures having the height and the diameter of about 61 m and 61±22 nm. King Mongkut’s University of Technology Thonburi. Using this TiO2 coated on metal oxide nanowire.01x10-6 mbar. Basically. Bangkok. Ajax) was placed in one end of the alumina boat while substrates were placed in the other end. the observe efficiency was greatly improved when it is compared to conventional metal oxide nanowires. the increase of dye molecules in solar cell and the improvement of semiconductor morphology are the key to enhance the efficiency of DSSCs.2 Department of Physics. Bangmod. respectively. Vapor-Liquid-Solid (VLS) mechanism. As mentioned earlier. 126 Pracha-uthit Rd.25 g SnO powder (99%. T. a thin layer of gold was sputtered on the desired substrates by Polaron SC7620 sputter coater at a sputtering pressure of 9. Chinvetkitvanich 1. the most well-known method. the distance between the Ti target and the substrates was Introduction Dye-sensitized solar cells (DSSCs) based on nanocrystalline semiconductor film  have attracted much attention by many research groups for twenty years because their low production cost and environmental friendly.
we can measure diameters and heights of nanowire and result are shown in the Table 3. Table 1: Preparation conditions of metal oxide nanowire coated with three different TiO2 thicknesses Source Substrate Source temperature (oC) Substrate temperature (oC) 0. Then.25g Zn Au/ITO/borosilicate glass 750 630 spectra show characteristic peaks of three phases which are tetragonal SnO2.3 µm while their diameters are 62. 61.00 x10-3 40:5 The obtained products were characterized with Xray diffraction (XRD. face-center cubic Zn2SnO4 and hexagonal ZnO (wurtzite) but do not show phase of TiO2. the three different thicknesses of TiO2 coated on metal oxide (MO) nanowires on ITO/glass substrates were immersed in the solution of sensitizer.. XRD SnO(1):Zn(1) Tsub 630 C Tsou 750 C coated TiO2 40 nm Degree (2) Figure 1. The cell assembly procedure followed Ponken et al. at room temperature for 4 hours. They were used as working electrodes.Miniflex/Cu/ 30kV /15mA) and scanning electron microscopy (SEM. respectively. The three different thicknesses of TiO2–coating nanowire on ITO/glass substrates as listed in Table 2 were used in dyesensitized solar cell assembly.26. According to SEM images. The typical active cell area was about 0. SnO(1):Zn(1) Tsub 630 C Tsou 750 C without TiO2 Table 2: preparation conditions of TiO2 coating by reactive DC magnetron sputtering Preparation conditions Sputtering Parameters Sputter power (W) Base pressure (mBar) Sputtering pressure (mBar) Ar:O2 flow rate (sccm) K.5 (100 mW/cm2). 61 and 78 nm. The MPN-100 SOLARONIX was used as electrolyte I / I 3 in the fabricated cell. 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. respectively.Aeimpanakit  160 6.25g SnO + 0. ruthenium 535 bis-TBA (N719). The current density versus voltage characteristics of the solar cells were characterized under standard radiation AM 1. S4700 Hitachi). 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.9 cm and the sputter power was set to 160W.01 x10-6 5.61 x10-3 88:38 6. 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). 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 1.00 x10 -6 This work 160 6. 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 PACCON2011 (Pure and Applied Chemistry International Conference 2011) .25 cm2. The morphology of synthesized nanowire was investigated by considering SEM images. It was found that the synthesized nanowires are in round wire-like shape.486 set to 5.s.0 and 40. Preparation conditions of TiO2 coating are given in Table 2. The counter electrode which was prepared from electrochemically platinized on FTO/soda-lime glass. The sputtering times were 560s and 1120s corresponding to TiO2 film thickness of ~20nm (thin) and ~40nm (thick). The cell efficiency was then measured and the current density v. thin coated and thick coated nanowires are 9. voltage (JV) characteristics was obtained. It is seem that the heights of uncoated.
32 600 2.40 and 1.7 480. While the short-circuit current density combined the uncoated nanowire electrode is 0.33%.74 308.09 36.5 1379 With thin TiO2 coating 61. The short-circuit current density of solar cell coated with thin and thick TiO2 are 2.09%.8 PACCON2011 (Pure and Applied Chemistry International Conference 2011) .cm2) Without with TiO2 9. which is generated from electrode whisker coated with thin and thick TiO2 are 0.5 illumination.3 78±38 0.41 28.487 a) d) b) e) c) f) Figure 2. thick TiO2 and without TiO2 are 600.6 326. 555 and 410 mV. respectively.88 mA/cm2. 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.4 With thick TiO2 coating 40.0 61±22 0. respectively. While the efficiency of DSSC combined the uncoated nanowire electrode is quite low which is 0.66 555 1.40% and 0. The short-circuit current density.88 187.18 410 0. It was found that the efficiency of photovoltaic cell. 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. 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 1.74 mA/cm2.cm2) Rsh (Ohm.40 172. The open-circuit voltage of solar cell coated with thin. respectively. Table 3: Chareristics of dye-sensitized solar cell with different TiO2 thicknesses DSSC Height (µm) Diameter (nm) Efficiency (%) Fill Factor (%) Voc (mV) Jsc (mA/cm2) Rs (Ohm.33 31.29 62±38 0.
488 Onreabroy for their kind supports on laboratory instruments and chemicals used in this experiment. 2004. S. 9-10  T. =0. R.31% TiO2 uncoating. C. 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 DyeSensitized Solar cell depends on the TiO2 coating and the length of nanowire. King Mongkut’s University of Technology Thonburi for X-Ray Diffractometer. G sele. Applied Physics Letters. 4. P.28% Thick TiO2 coating. Department of Physics. Semiconductor Physics Laboratory. National Graduate Research Conference CG and Khon Kaen University. Nature. Faculty of Science. Greene. Chatraphorn. Ponken. Master Thesis. the efficiency of solar cell is higher than that of without coating with TiO2.Yang. 353. Chulalongkorn University for fabrication of DSSCs and the I-V measurement. 4  N. Werner. Aeimpanakit. ZnO metal oxide nanowires were synthesized and coated with monolayer of TiO2 nanoparticle at three different thicknesses which are uncoated. When considering the working electrode coated with TiO2. Physica E. 737-738. FF=0. J. Zn2SnO4. Zakharov_. Moreover. Gra··tzel. FF=0. C. Department of Physics Faculty of Science King Mongkut’s University of Technology Thonburi. 632-638 Thin TiO2 coating. Nat. Wandee PACCON2011 (Pure and Applied Chemistry International Conference 2011) . Mater. L. U.4%. Wagner and W. C.33%. L. O’Regan. 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. Law.  R.09%. 2005. FF=0. 1991. In addition to effect of TiO2 coating thickness. S.36 Figure 3. P. Ellis. 2007. the lenght of nanowire also relates to the increasing of the cell efficiancy because of an increasing surface area of dye adsorption. In conclusion. =0. M. thin coated and thick coated layers. Acknowledgements: We would like to thank Department of Physics and The Scientific Instrument Center for Standard and Industry. Reference  B. and Assistant Prof. Using these coated nanowires as a part of working electrode of DSSC. 1964. the cells were assembled and measured the cell efficiency and then the TiO2 coating cells were compared with a pure nanowire cell. =0. Chityuttakon. 148-152  K. Saykally. Sokolov. TiO2 coating may possibly improves an electron transport in solar cell which leads to an increase of the cell efficiency. 37. Johnson.  M. Conclusion Mixture of SnO2. 2009. Thai Microelectronics Center for SEM characterization. E.
The compositions of reclaimed tire rubber (RTR) /sawdust/ polypropylene (PP) constants and composites with sulphur system are indicated in Table 1. .25 1. Composition of tested RTR/sawdust/PP. was purchased from Union Commercial Development Co.25 0.1 Materials Sawdust (60 mesh) was purchased from Artowood Co.5 Introduction The present used tire and has become one of the largest scale rubber wastes. while PP content was kept content at 70% by weight. The effect of RTR /sawdust/ PP on properties of the blends.4 0. 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. Faculty of Science.1 0..75 1 1. Mixing and injection molding Sawdust was prepared into 60 mesh. 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. dibenzothiazole disulfide (MBTS). PP 70 70 70 70 70 70 70 RTR 0 5 10 15 20 25 30 Sawdust 30 25 20 15 10 5 0 ZnO 0 0. The RTR and sawdust content were varied from 0% to 30% by weight. Thailand * Sunaree. Bangkok. Bangkok. while PP content was kept content at 70% by weight. was provided by IRPC Co.6 MBTS 0 0. The composites of reclaimed tire rubber (RTR) /sawdust/ polypropylene (PP) were prepared by injection molding dynamic vulcanization.3 0.25 0.75 1 1. and sawdust fibers besides the environmental concern (environmental friendly and biodegradability) are low cost. Chulalongkorn University.25 1. known as ground rubber tire (GRT) can further be devulcanized to be come reclaimed tire rubber (RTR).125 0.Ltd. 25. Reclaimed tire rubber (RTR) (68.1 0. tetramethyl thiuram disulfide (TMTD). Faculty of Science. stearic acid were all of Fluka reagent grades. as filler should be a more effective choice to impart elastic property to the thermoplastics.075 0.The composites were prepared by injection molding dynamic vulcanization and unvulcanization. Thailand. Chulalongkorn University. 2. Recycle of the waste rubber is thus a great challenge for both environmental and economic reasons. Materials and Methods 2.2 0. The mixing temperature was 180 °C and rotor speed was 50 rpm. Sunaree 1* and T. most likely due to the increasing carbon black content.25 0. Sulphur was purchased from Merck. Results from solvent swelling ratio.To@Student.45 Sulphur 0 0..5 0. reclaimed tire rubber (RTR) and sawdust were used as a filler in polypropylene (PP) composite.489 Preparation of Injection-Molded Polypropylene/Reclaimed Tire Rubber/Sawdust Blends S.25 1.chula. After that was dried in an oven at 100 °C for 1 hr.75 1 1. Using RTR. Increasing the RTR and sawdust content to more than resulted in a decrease in the impact strength and the tensile strength of blend.22% rubber. Table 1. The RTR and sawdust content were varied from 0% to 30% by weight.5 Stearic acid 0 0.3 0. . Thailand.5 TMTD 0 0.375 0. Thailand.th Abstract: In this research. 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 . Thailand 2 Department of Chemistry. We earlier reported the dynamic vulcanization of RTR and PP blends. The tire rubber part can be ground to small particles.. Ltd.71% residue with particle size of 500-600 mm). The composites vulcanization show slightly higher tensile strength and impact strength than composites with unvulcanization.ac.5 0. having a non-cross-linked rubber structure. zinc oxide (ZnO). PACCON2011 (Pure and Applied Chemistry International Conference 2011) . water absorption and morphology of the blends was also proved by scanning electron microscopy (SEM). Supawan 2 1 Program of Petrochemistry and Polymer Science. An injection grade PP (1100NN).2. with 15 min of mixing time.5 0. Ltd.07% carbonblack and 6. and low density.5 0.
and weighed. Percent Water Absorption = [(Wet weight Dry weight)/ Dry weight] x 100 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. 2 shows the impact strength of the blends.2 Morphology properties The morphology of RTR/sawdust/PP blends with vulcanization and unvulcanization are shown in Fig. Tensile strength of vulcanized and unvulcanized RTR/Sawdust/PP blends. Impact strength of vulcanized and unvulcanized RTR/Sawdust/PP blends. But.490 2. Impact specimens had dimensions of 63. 1 shows that the tensile strength of the blends. 1. impact strength increase to find may be involved in both molecular entangling and interphase sulfur crosslinking mechanisms which all composites. then fractured and goldcoated prior to analysis. agglomeration and hence particle–particle interaction of the rubber powder accounts for the observed decrease in tensile strength. decrease with increasing reclaimed tire rubber content and sawdust content in the blend. Mechanical properties In this study. The tensile strength of each blend. 2. They were weighed accurately before being immersed into toluene. Fig.3 cm in thickness. 2. PACCON2011 (Pure and Applied Chemistry International Conference 2011) . Water absorption is expressed as increase in weight percent. Dumbbell shaped samples after tensile testing were immersed in liquid nitrogen.4 Impact strength Notch-Izod impact strength was measured according to ASTM D256. The mechanical properties of all these blends were then compared. 3 The RTR particles and sawdust can disperse well in PP matrix when the blend was vulcanized with sulphur. 140 Vulcanization Unvulcanization Impact strength(Kg-cm/cm) 120 100 80 60 40 0/30/70 5/25/70 10/20/70 15/15/70 20/10/70 25/5/70 30/0/70 RTR/Sawdust/PP Fig.27 x3 mm. RTR and sawdust content were varied from 0% to 30% by weight. Specimens are removed. Unvulcanization Vulcanization 23 22 21 20 19 18 17 16 0/30/70 5/25/70 10/20/70 15/15/70 20/10/70 25/5/70 30/0/70 RTR/Sawdust/PP Fig. Tensile strength Tensile strength measurement was performed on dumbbell specimens at ambient temperature according to ASTM D468 using universal testing machine. composites in the formula to find unvulcanized RTR/Sawdust/PP (25/5/70) with vulcanized (25/5/70). 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. Its high content could induce a split in the layer structure of the blend Tensile strength(MPa) providing a shorter path for fracture propagation. The specimens are dried in an oven temperature 40 °C for 24 hours.1.3 cm in thickness.  Fig. The samples were kept in the dark for 7 days at room temperature.7 Morphological property The blend morphology was examined by a scanning electron microscope (JEOL model JSM 5800 SEM). patted dry with a lint free cloth. thereby causing the sudden decrease in impact strength. Results and Discussion 3.3. . The impact strength of each blend.Yield a better interfacial bonding between the RTR particle and sawdust in the matrix PP. This indicates that re-cross-linking is more effective in blending with these systems leading to the improvement of physical properties. The results are reported in Kgcm/cm notch unit. 2. Further.6 Solvent swelling Solvent swelling study was carried out by cutting a sample into discs of 1 cm in diameter and 0. 3. decrease with increasing reclaimed tire rubber content and sawdust content in the blend. They were weighed accurately before being immersed into water kept for 7 days at room temperature. 2.5 x1. while PP content was kept content at 70% by weight the and two types of vulcanization by Sulphur and unvulcanization.
Polymer Degradation and Stability. 91.  H Ismail . Polymer Testing... The composites of reclaimed tire rubber (RTR) /sawdust/ polypropylene (PP) (25/5/70) were prepared by injection molding dynamic vulcanization. 137-144 (2002).T Supawan. (a) References  P. 76. Polymer Degradation and Stability. 3.. 21. 389–395 (2002) (c) (d) (e) (f) Fig. 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) PACCON2011 (Pure and Applied Chemistry International Conference 2011) . Karger-Kocsis J.491 Conclusions In this research. reclaimed tire rubber (RTR) and sawdust were used as a filler in polypropylene (PP) composite. Fuhrmann I. (b) C K Radhesh. T Varawut. The composites vulcanization show slightly higher tensile strength and impact strength than composites with unvulcanization. Punnarak . 3456-3462 (2006).
The particle size of sulphur is the most crucial factor. Start with clay preparation. A process to produced pills of clays and sulphur mixture was mixed by mixing stirrer in a chamber at 140˚C for 2 hour. Cetyltrimethylammonium [C16H31N+(CH3)3] bromide 99 % (CTAB) from Acros organic. 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. total sulphur uptake and extractable soil sulfate of sulpher bentonite were less than ammonium sulfate and micronized sulphur fertilizer. Phayathai road. Petrochemical. Liquid sulphur supplied from Thaioil Public Co.1 M for 24 hr.ac. Ltd. temperature. innovation perspective. The elemental sulphur fertilizer has high concentration of 70-100% wt sulphur and greatly physical characteristic. and H. absorbs water and swell. R. process 1) physical dispersion of the pill and 2) oxidation to sulfate. Thailand 10330 * E-mail: Hathaikarn. There were 3 formulas of sulphur bentonite: (1) bentonite+sulphur (BS˚). Nuntawat1. A portion of organoclay was synthesized to PCH by adding co-surfactant (Dodecylamine). Element sulphur is oxidized to sulfate before it can be taken up by plant. But in the cropping system. 50 C to obtained organoclay. So the sulphur fertilizers are required for soil supplement. Manuspiya1. (3) PCH+sulphur (PCHS˚). and porous clay heterostructure bentonite (PCH) in various ratios. Characterization Clay characterizations were conducted by X-ray driffractometer (XRD) and Fourier transform infrared Introduction Every year.. because the sulphur particles were not small enough and slow dispersion.M@chula. but sulfate is easy to leaching loss. Each formula was divided into 3 compositions of clay: 10. From the previous work . Experimental Materials and Preparation Na-bentonite fertilizer grade (CEC = 100). and particle size. a portion of the available sulphur is depleted and a portion is returned to soil as residue and converted into organic matter. Dodecylamine 98% from Sigma-Aldrich. However. then stirred until mixed completely. Pathumwan. Release of an available sulfate form sulphur pills depends on 2. and the largest percentage of liquid sulphur requirement. A. For the sulphate fertilizer. The principle is that when the clay PACCON2011 (Pure and Applied Chemistry International Conference 2011) . especially the small particle sizes are easily dispersed and oxidized . 20. Luengnaruemitchai1. Among the variables studied. it tends to remove more sulphur than being replaced. Bangkok. time. 30. was supplied by Thai Nippon Co. it provides an immediate source of sulfate to the plant. Pathumwan. Thailand 10330 Center of Excellence for Petroleum. offer a method of applying high concentration of elemental sulphur (70–90 % S) of fine particle size in a safe-handing product. the oxidation process depends on soil moisture. a molten sulphur has been mixed with bentonite to form a safe and easy to apply product. Then forming droplets of the mixture immediately. the particle size of sulphur is the most significant factor because the smaller particle sizes are easier to disperse and oxidize to sulphate form.th Abstract: Elemental sulphur fertilizer is the one of the most effective outlets of liquid sulphur based on the availability of technology providers. For preparing sulphur bentonite.2* 1 2 The Petroleum and Petrochemical College. TEOS was immediately added in the mixture at room temperature for 4 hr to obtain PCH. crop is harvested. the yield of crops. % of clay (w/w).. The benefits of elemental sulphur are a continual releasing of sulfate during the growth season and minimal sulfate leaching loses. the droplets of mixture was dipped on stainless steel plate to obtained sulphur bentonite fertilizer . bentonite was converted into a quaternary ammonium exchange form by ion exchange with cetyltrimethylammonium bromide (CTAB) 0. Sulphur bentonite mixtures. and Advance Materials. Ltd. Chulalongkorn University. An obtaining PCH was calcined for removing surfactant at 600 C. Magaraphan1. Phayathai road. The elemental sulphur is oxidized to sulphate form under typical condition before taking up by plant. bacteria activity. Tetraetyoxysilane (TEOS) reagent grade from Sigma Aldrich. organomodified bentonite. it makes pill fracture and disperses to small particles of sulphur. prepared by blending molten sulphur and bentonite. 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. Bangkok.492 Synthesis of Sulphur Bentonite as an Effective Liquid Sulphur Derivative S. the highest expected margin. (2) organoclay+sulphur (OS˚). 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.
Density maps had showed the dispersion of bentonite and PCH in sulphur matrix. the asymmetric and symmetric stretching vibrations of the Si-O-Si linkage. 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.21 (a) 0 2 4 6 8 10 12 2 theta Figure 2.120 0.28 increased to 3.0 pore volume (cc/g) 0. A possible reason might be due to the presented of surfactant in the interlayer clay. had some pattern order structure that presenting in PCH. it blocked N2 gas to adsorb into the pore. BET method was used to determine pore size. Result and Discussion Bentonite. d-value at 2 = 2.7 11. 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. After BTN was treated with CTAB to obtain organoclay. XRD measured for low angle (2θ from 1 to 10 ) and scan speed at 0.82 nm) and the pattern was different from Organoclay. The evidences from SEM (figure 3. Clay structure was presented by S-4800 field emission scanning electron microscope (FE-SEM). Organoclay (b). pore volume and surface area of clay. SEM image of Bentonite (a) and PCH (b) (b) Sulphur forms the matrix of the prill and the varying amounts of bentonite were held within it. The broad peak around 3500 cm-1 can be assigned to the stretching vibration of the silanol associated with the silica structure. 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 (a) Figure 3. After modification of organoclay. In case of sulphur bentonite fertilizers.493 spectroscopy (FTIR). higher surface roughness and higher void. Two strong peaks were observed at lower angle as a result of the successful intercalation of cationic surfactant in the interlayer of bentonite .737 Figure 1. 1000 and 800 cm-1 can be assigned to the stretching vibration of the SiO4 units.21 nm. 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. it showed a decreasing of surface area and pore volume while increasing of pore diameter. The XRD patterns of Bentonite (a). PCH (c) BET surface area of BTN was 55 m2/g.) confirmed the structure of clay was changed from plate structure to porous structure.0 5. Table 1: Porosity characteristics of Bentonite.022 0.82 20000 10000 (C) (b) 0 d=1. Almost all evidence of density map suggests that the dispersion of bentonite in the sulphur matrix was PACCON2011 (Pure and Applied Chemistry International Conference 2011) . PCH was observed board peak at 2 = 2. the basal spacing of BTN is 1. Organoclay and PCH Total Sample Bentonite Organoclay PCH surface area (m /g) 55 8 594 2 pore diameter (nm) 8.88 Linear intensity (Counts) 30000 d=3. After modification of PCH. It roughly infers that the structure of starting bentonite was changed after the modification.21 (d = 3.5 sec/step. The measurement was carried out at 5 kV.88 nm. Organoclay (b) and PHC (C) XRD pattern of the samples are shown in Figure 2. respectively. 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. 50000 40000 d=3. The peak at 1100. the prill structure was examined with SEM and SEM-EDX. FTIR spectra of Bentonite (a).
Sulphur matrix and bentonite was clearly separated and sulphur matrix just covered around bentonite particle (figure 4. the dispersibility and the control release of sulphur in future works. Petrochemicals. Fertilizer research 15 (1988). micrograph was different because a portion of sulphur matrix going inside pore and void of PCH. Manuspiya. SEM-EDX density map of sulphur. Swanney. Owers. McGrath. Fertilizers derived by sulphur bentonite and sulphur-PCH will be test the mechanical property. the sulphur-organoclay was not successfully prepared because of the degradation of existing surfactant. Proceedings of Sulphur 84 (1984).G. and Polymers (2009).J. W. and H. 139-147. pp. References  J.  A.R. Mattayan. Chulalongkorn University. sulphur bentonite (a) and sulphur-PCH (b) (x700) Conclusions This work successfully prepared sulphur bentonite and sulphur-PCH. for supported bentonite clay. 817-812. Magaraphanand and H. pp. b) (a) (b) Figure 4. pp.J.  R. Acknowledgements The author would like to thank The petroleum and petrochemical college.494 relatively uniform and free form aggregation . Janzen. PACCON2011 (Pure and Applied Chemistry International Conference 2011) . and Advance Materials for research funding. U.In case of Sulphur-PCH. a) . the 15th PPC Symposium on Petroleum. . F. Bettany. Bosswell.  C. the authors wish to thank Thai-Nipon chemical Co. Thaioil Plublic Company Limited and the National Excellence Center for Petroleum. 13-31.C.469.. Ltd. Patent 4. and H. R.  N. B. and S. In addition.R. Zhao.P. Plant and Soil 222 (2000). Zaharko. A portion remained coat on surface of PCH (figure 4.P.859 (1986). Rothbaum. While. Petrochems.S.H. Riley.
and high specific strength and modulus. In order to solve it. By comparing with inorganic fillers. The total mixing time was 15 min. Maleic anhydride grafted polypropylene (MAPP. an organoclay (OMMT) as a nanosize filler was added into the composites.495 Effect of Nanoclay on Mechanical Properties and Flame Retardancy of Sisal Fiber/Pp Composites W. Jarukumjorn1. Incorporation of nanoclay at very low loading (less than 5 wt%) can improve properties e. Polypropylene (PP) is particularly interesting thermoplastics used in composites because it has good and extensive properties. and fiber biodegradability.2* 1 Suranaree University of Technology /Institute of Engineering /School of Polymer Engineering. thermal and flame retarding properties of sisal fiber/PP composites. increased strength and heat resistance. PP composites containing 30 phr of sisal fiber were prepared using an internal mixer. Moreover. Thailand. and increased biodegradability of biodegradable polymers . ease of processing. sisal fiber and OMMT were dried in an oven at 70C over night. Sisal fiber (Si) was purchased from the Sisal-Handicraft OTOP group. low cost. In order to achieve the good property enhancement. Another critical drawback of the use of natural fiber in PP composites is their high flammability due to their structure and chemical composition. its high flammability is one of the critical drawbacks of the composite. Thailand 2 Center for Petroleum. Preparation of composites: Prior the composite preparation. Improving their flame retardancy will thus expand the range of their applications. However. Nakhon Ratchasima. Suppakarn1. Dupont) was supplied from Chemical Innovation Co. particularly in structural and automotive industry due to cost effectiveness and eco-friendliness. Thailand * kasama@sut. 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. 3000p). such as extrusion and injection molding [2-4]. The sisal fiber was cut into approximate length of 2 mm. and substantial creep.2 and K. the incompatibility between hydrophobic polymers and hydrophilic natural fibers results in poor interfacial adhesion between the natural fibers and polymers. Bangkok. However. easiness of processing. the incorporation of OMMT into PP and PP/Si composite also improved their flame retarding properties and thermal stability. the use of natural fibers to reinforce polymer has received considerable attention. X-ray diffraction analysis revealed that PP/OMMT composite exhibited the intercalated nanostructure. Materials and Methods Materials: Polypropylene (PP.g. to improve the mechanical properties and flame retardancy of sisal fiber (Si)/PP composites. PP/Si/OMMT composite formed the exfoliated nanostructure. However. the main advantages of natural fibers are light weight. Cloisite®30B. clay particles should be homogeneously dispersed and exfoliated within the polymer matrix . biodegradability. In addition. Introduction Nowadays. The objective of this work was to study the effect of nanoclay on the mechanical. The test specimens were molded using an injection molding machine. nanosized fillers have been good candidates for overcoming the aforementioned drawbacks of polymer composites. P700J) was supplied by SCG Chemicals Co. 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 . USA. Fusabond®P MZ 109D. Organoclay (OMMT.ac. PP compounds were prepared by melt blending using an internal mixer (Haake Rheomix.Ltd.2. shortcomings of this composite are relatively low modulus. which depends on properties of the natural materials and those of the polymers. In recent years. It was observed that the tensile properties of PP and PP/Si composite were further increased with the addition of 3 phr of OMMT. acceptable strength and stiffness. Ltd. Chanprapanon1. renewable nature. Moreover. PACCON2011 (Pure and Applied Chemistry International Conference 2011) .. 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. large thermal expansion.. and low cost also. decreased gas permeability and flammability. The addition of natural fibers to polymers can cause a change in properties of the derived composites..th Abstract: Natural fiber reinforced polypropylene (PP) has gained a lot of interests due to low cost. low notched impact resistance. low density. high modulus. N. Petrochemicals. an originally modified montmorillonite) was supplied from Southern Clay Products Inc. Nakhon Ratchasima. and Advanced Materials /Chulalongkorn University. In this study. the absence of toxic by-products.
79 0. D5005) equipped with a Cu K radiation source of wavelength 1. The addition of fillers decreased the impact strength of PP.53 0. Oxford. which is at 100 mm from the end). and PP/Si/OMMT composite are shown in Figure 1.67 0. For PP/Si/OMMT composite.34 0.36 0. TA Instrument. the impact strength of PP/OMMT composite was higher than that of PP/Si composite.0/min and 0. PP/OMMT and PP/Si/OMMT composites.06 0. CLF 80T) to produce the specimens according ASTM standard. the peak was disappeared. Table 2: Mechanical properties of PP and PP composites. BPI).85 nm.04 1. the average values of five specimens at least were taken for each sample. In addition. low aspect ratio filler particles .81 which corresponds to d-spacing of 2.496 The mixing temperature was 170C and the rotation speed was 60 rpm.03 2. Impact test was performed on unnotched PP and PP composites followed ASTM D256 using an impact testing machine (Atlas. The weight of each sample was kept within 10-15 mg.60 0. SDT2960) at a constant heating rate of 20C/min under air flow. the incorporation of OMMT into PP/Si composite slightly increased the impact strength.60 Young’s modulus (GPa) 0. PP/OMMT composite. PP/OMMT composite presented a shift of diffraction peak to a lower angle at 2 = 3. 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. Test specimens were molded by an injection molding machine (Chuan Lih Fa.32 0. OMMT.75 31.37 26. Impact strength is enhanced by small. Thermal analysis: Thermal behaviors of PP and PP composites were carried out on a thermogravimetric analyzer (TGA. 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]. Designation PP PP/Si PP/OMMT PP/Si/OMMT Tensile strength (MPa) 14. XRD pattern of OMMT showed a peak at 2 = 4.05 2. The mechanical properties of PP and PP composites are summarized in Table 2. Tensile properties of PP and PP composites are shown in Figure 2.50 Results and Discussion XRD patterns of PP.94 11. Similary.77 that corresponds to d-spacing of 1. Furthermore.03 Impact strength (kJ/mm2) 78.5406 Å operated at 40 kV and 40 mA at room temperature.85 12. indicating the formation of an intercalated structure .40 0. Riley et al. L is the length the flame travels from the first reference mark (25 mm from the end ) to the second reference mark. The scanning rate and step size were 2.55 30. It was because the filler particles in PP matrix provided sites for crack initiation . Instron 5569). Five measurements per sample were taken. 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. The sample was held horizontally and a flame was applied to light one end of the sample.02 with 2 varying from 2 to 10. Composition of PP and PP composites and their designation used are shown in Table 1. 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.82 1. The XRD patterns were used to calculate the basal spacing or d-spacing from Bragg’s law. In all cases. This implied that the silicate layers could be exfoliated and dispersed in PP matrix forming a nanometer scale composite .32 nm. OMMT. Designation PP PP/Si PP/OMMT PP/Si/OMMT PP (phr) 100 100 100 100 Sisal (phr) 30 30 OMMT (phr) 3 3 MAPP (phr) 5 5 5 PP OMMT PP/OMMT PP/Si/OMMT Intensity (count) 2 3 4 5 6 7 8 9 10 2 Theta (degree) Figure 1. XRD patterns of PP.39 0. Figure 3 represents the unnotched Izod impact strength of PP and PP composites. Table 1: Formulation of PP and PP composites.92 0. The temperature ranged from the room temperature to 700C. However. HVUL). Structure characterization: The structure of OMMT and PP composites were performed by an X-ray diffractometer (XRD. Mechanical test: Tensile properties of PP and PP composites were measured according to ASTM D638 on a universal testing machine (UTM.25 60. and t is the time in seconds for the flame to travel. Lee and Kim (2008) found the impact strength of wood/PP composite filled 1 phr PACCON2011 (Pure and Applied Chemistry International Conference 2011) . (1990) reported that the impact strength depends on both the size and the shape of the filler and is also affected by the micromorphology.
This was because the sisal fiber as lignocellulosic fillers has a component called lignin. Burning rates of PP and PP composites measured by a horizontal burning test are shown in Figure 4.497 40 35 30 Tensile strength (MPa) 25 20 15 10 5 0 PP PP/Si PP/OMMT PP/Si/OMMT 0.0 Tensile strength Young's modulus 3. which builds up on the surface during burning .80 1.5 1. (2009) also observed a remarkable enhancement in thermal stability of PP/pineapple leaf fiber composite in the presence of OMMT . The addition of sisal fiber into PP did not substantially affect the flammability. PP exhibited the highest burning rate at about 28. 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.5% was observed when OMMT was added into PP/Si composite.5 Young's modulus (GPa) 35 30 Burning rate (mm/min) 25 20 15 10 5 0 PP PP/Si PP/OMMT PP/Si/OMMT Figure 2. of OMMT was slightly higher than that without OMMT.0 1. (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 . Tensile properties of PP and PP composites. Moreover. the dramatic reduction of burning rate about 37. Burning rates of PP and PP composites. Designation PP PP/Si PP/OMMT PP/Si/OMMT T5% (C) 285 286 304 297 Char residue at 600C (%) 0. Biswal et al. 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 .89 mm/min.8%. Generally. Similary. PP/Si composite showed no significant change of T5% and left char residue about 1. In addition. Lee et. The flame retardancy mechanism of OMMT involves a highperformance carbonaceous-silicate char. Impact strength of PP and PP composites.00 1. Table 3: TGA data of PP and PP composites. indicating a positive effect of the OMMT inclusion . which formed char during thermal degradation.50 3. Figure 5 shows TGA curves of PP and PP composites.0 2. The incorporation of OMMT into PP showed 16% reduction of the burning rate of PP. The T5% of PP was at 285C and no char residue left. 90 80 70 Impact strength (kJ/mm2) 60 Figure 4. composites are shown in Table 3. The temperature at 5% weight loss (T5%) and weight residue at 600C of PP and PP Temperature ( C) Figure 5. improvement in thermal stability is found for which the nanocomposite morphology plays an important role . TGA curves of PP and PP composites.59 PACCON2011 (Pure and Applied Chemistry International Conference 2011) .0 2.5 0. 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 . When compared with PP. 100 PP PP/Si PP/OMMT PP/Si/OMMT 80 15 10 5 0 PP PP/Si PP/OMMT PP/Si/OMMT Weight (%) 50 20 60 40 20 0 100 200 300 400 o 500 600 Figure 3. al.
.  M. and Advanced Materials for financial supports. Sain and M. A. pp. Chem. K. Ltd.  A. Acknowledgements The authors express their thanks to Suranaree University of Technology and Center for Petroleum. Adams. T. H. Moreover. Applic.  M. Compos. Kuboki. M. Mohanty and S. Appl. Appl. J. 1386-1390. Barick and D. Taib. Polym. B. 4091-4103. Lee. Des. pp. Plast.  A. Sci. Polym.  M. 112 (2009). H. K. Sci. M. Park. K. References  H.. Biswal. Sci. Process. Tensile properties. Hetzer and D. pp. Bhattacharyya. Prog. pp. Z.Ltd. 14(1990). 2769-2776. 39 (2008). Sci. Riley. pp. Nourbakhsh and A. Polym. pp. Rubb. Polym. S. C. pp.  Y. 114 (2009). D. 639-654. Paynter. 2714-2722. Res.  Y.  A. M. Jani. Appl. Eng. Addition of OMMT into PP and PP/Si composites enables to achieve the nanocomposite. Kim. Tripathy. J. Bakar. Okamoto. M.. Polym. J. Sci. M. 1539-1641. 28 (2003). pp. pp.498 Conclusions Incorporation of sisal fiber increased the tensile properties but reduced the impact strength of PP. Ashori. Petrochemicals. S. Sci. A. PACCON2011 (Pure and Applied Chemistry International Conference 2011) . Rozman and S. flame retardancy. 452461. M. 8593. SCG Chemicals Co. Dong and D. Polym. 86 (2008). C. Kontopoulou. Southern Clay Products Inc. D. USA for providing Cloisite®30B. and thermal stability of PP were increased in a presence of OMMT. 1083-1093. pp. Sci. J. J. Part A. and Chemical Innovation Co. Lee and D. B. 1177-1191. for supplying Fusabond®P MZ 109D.  S. McGenity and J. J. 111 (2009). Appl. 117 (2010). R. De Kee. Nayak. 116 (2010). M. for supplying P700J. XRD patterns showed the intercalated nanostructure for PP/OMMT composite and presented the exfoliated nanostructure for PP/Si/OMMT composite. Appl. P. 118 (2010). Polym. Sinha Ray and M. Appl. the mechanical properties and flame retardancy of PP/Si composite were improved by incorporating the OMMT. Ishak.
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