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The effect of radiosterilization on cytotoxicity of polyurethane film
Nuclear Instruments and Methods in Physics Research B 208 (2003) 215–219 www.elsevier.com/locate/nimb
The effect of radiosterilization on cytotoxicity of polyurethane film N. Sheikh
*
Gamma Irradiation Centre, Atomic Energy Organisation of Iran, P.O. Box 11365-3486, Tehran, Iran
Abstract Nowadays a sequence of tests for evaluation of sterilized biomaterial includes an initial set of tests in vitro, both biological (cell culture) and non-biological (mechanical tests). In this paper the cytotoxicity of a sterilized polyurethane film, in order to use as biomaterial, has been investigated. For this purpose NCO-terminated urethane prepolymer in medical quality was synthesized without ingredients beside monomers (polyethylene glycol/castor oil and toluene diisocyanate). The cured prepolymer films were prepared under ambient conditions due to the reaction of free NCOgroups of prepolymer with air moisture. The polyurethane films were sterilized by gamma-ray (25 kGy). The surface structure of sterilized polyurethane film was observed by SEM and compared to that of the unsterilized film. Also, the in vitro interaction of fibroblast cells and sterilized polyurethane film in culture medium containing serum was evaluated in comparison with control samples. Results showed no signs of cell toxicity. Ó 2003 Elsevier B.V. All rights reserved. PACS: 61.82.PV Keywords: Sterilization; Gamma-ray; Polyurethane film; SEM; Porosity; Cytotoxicity
1. Introduction A biomaterial is expected to be selected from untoxical and biocompatible materials [1]. Also, in the most cases it must be capable of withstanding radiosterilization [2]. As an effective and industrial method for medical products sterilization is the use of radiation [3]. This method is advantageous because it is economically feasible for large-scale terminal sterilization of products in sealed packages. Among synthetic materials, polyurethanes have been considered to be the most suitable material in
*
Tel.: +98-21-800-4065; fax: +98-21-800-9054. E-mail address: [email protected] (N. Sheikh).
various biomedical applications, which is connected to their biocompatibility, biodegradability and controlled microstructure and properties [4]. However, in the case of materials for biomedical applications in addition to the evaluation of their physico-mechanical properties, risks caused by the emission of harmful substances escaping from the material due to sterilization procedures have to be kept at a minimum. In other words, it is important to know about the biological safety of a sterilized biomaterial. So, toxicity tests have to be conducted with the final sterilized product. In our laboratory, attempts have been made during several past years to find structure–properties relationships of polyurethanes in order to obtain a suitable urethane biomaterial. The synthesis
0168-583X/03/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-583X(03)01107-8
216
N. Sheikh / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 215–219
was performed on the basis of prepolymer method by using of different monomers. It was found that the use of a mixture of polyethylene glycol (PEG) and castor oil together with toluenediisocyanate (TDI) as polyol and diisocyanate components, gives the possibility of optimizing the required properties of the product such as fluidability in the liquid prepolymer state, and also hydrophilicity, flexibility and mechanical strength in a water-cured prepolymer film. This article focuses on the evaluation of cytocompatibility of the gamma-sterilized polyurethane film which contains selected chemical composition.
polyol mixture under an atmosphere of dry N2 . The reaction conditions (temperature and time) were controlled so that a clear yellowish liquid prepolymer was obtained. As the medical end use is intended, a purification process by thin layer evaporation method was carried out on the reactive prepolymer product to remove the unreacted TDI [5]. This method can be performed easily due to the high vapour pressure and volatility of TDI.
2. Experimental 2.1. Chemicals Polyethylene glycol (PEG 1000, Sigma) and castor oil (Aldrich) were used as polyol and dried at 80 °C under vacuum. 2,4 TDI supplied by Riedelde Haen Co. was used as received. 2.2. In vitro cell system
2.4. Film preparation
Mouse C34 /connective tissue (L929) was obtained from the National Cell Bank, Pasteur Institute of Iran. The cells were maintained in growth media RPMI-1640 and supplemented with 10% fetal calf serum (PCS), 100 U ml 1 penicillin and 100 lg ml 1 streptomycin (Gibco BRL Laboratories). A routine subculture method was used to maintain the cell line. The cells were incubated in a humidified atmosphere with 5% CO2 at a temperature of 37 °C. After incubation of one week the monolayer was then harvested by trypsinization. A cell suspension of 4 105 cells ml 1 was prepared before seeding.
For the preparation of the urethane film, the liquid purified prepolymer was cast onto the surface of a glass plate and then left to be cured under ambient conditions.
(The reaction between a H2O molecule and 2(NCO) groups of prepolyner chains)
2.3. Urethane synthesis The urethane prepolymer was obtained by the reaction between a mixture of PEG and castor oil (50/50, w/w) with TDI. The amount of isocyanate and hydroxyl containing materials were adjusted so that a ratio of 2 was obtained for NCO to OH groups. Synthesis was carried out on the basis of prepolymer method. TDI was slowly added to
A combination of above reactions is occurred due to the simultaneous presence of PEG (diol) and
N. Sheikh / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 215–219
castor oil (triol) in the reaction system. Finally, prepolymer chains completely react with an equivalent amount of water, lead to the formation of crosslinked poly(urea)urethane. 2.5. Sterilization The cured urethane film (PU) and control films, latex and tissue culture polystyrene (TCPS),with 0.15 mm thickness, were punched into discs of 15 mm diameter and then sterilized (25 kGy). Sterilization was carried out using gamma rays from 60 Co source, in a Gammacell-220 with a dose rate of 3.7 kGy/h, in air at room temperature. The absorbed dose was measured by the Frick dosimeter.
217
work reactive urethane prepolymer was prepared without ingredients beside monomers and purified in order to obtain medical purity. Also, it is found that controlled porosity of biomaterials is important in numbers of biomedical applications [7]. In the case of urethane prepolymer, the curing reaction rate of prepolymer with water which is clearly dependent on the type of used polyol and diisocyanate, amount of moisture, accelarator and thickness of prepolymer layer, has influence on porosity of cured film. On the other hand, sterilization process may affect on the film porosity. So, the physical form of the sterilized urethane surface was compared with that of the unsterilized sample using SEM. It can be observed from Fig. 1(a) and (b) that radiation sterilization
2.6. Surface structure The surface porosity of gamma sterilized and unsterilized urethane films were observed with a SEM (Philips-XL30) after sputter gold coating of samples. 2.7. Cytocompatibility assay The cytocompatibility of the urethane film was evaluated by in vitro cell culture test in comparison with controls. For this purpose gamma sterilized polymeric discs were individually placed into a multiwell plate (Nunc, Denmark). A volume of 5 ml cell suspension was seeded into each well. These cultures were placed in a CO2 controlled incubator at 37 °C for 5 days and then media were decanted. The samples were washed with phosphate buffer saline solution, their attached cells were fixed by ethanol solution and stained with crystal violet or giemsa solution as staining agents. All air-dried samples were examined by light microscopy (Hund-H500).
3. Results and discussion As it is reported that small molecular weight additives may leach out from biomaterial and decrease the biocompatibility [6] therefore, in this
Fig. 1. SEM micrographs of the surface of polyurethane film (mag. 2000 ): (a) before sterilization and (b) after sterilization.
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N. Sheikh / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 215–219
has no significant effect on the number and size of pores of surface film. The preliminary in vitro test to evaluate the biocompatibility of the material is cytotoxicity test. This test indicates cellular behaviour consisting of cell adhesion, morphology and growth
pattern, which results from complex processes with biological and non-biological mechanisms. These processes are not only linked to the release of toxic products but also to the chemical nature and surface properties of the substrate including; surface tension, roughness, hydrophilicity, elasticity and electrical charge. The sterilized urethane film was also evaluated by a cytotoxicity test. Results obtained from in vitro cell culture are presented in Fig. 2(a)–(c). The positive control (Latex) is a toxic material as only few detached cells are observed on its surface (Fig. 2(a)). This behaviour is due to the presence of toxic vulcanizing ingredients in its compound. The negative control (TCPS) supports cell adhesion and growth (Fig. 2(b)) because it has no toxicity moreover, its surface properties is probably desirable for cell attachment and cell proliferation. In the case of polyurethane film (Fig. 2(c)) similar behaviour has been shown to the TCPS substrate. The affinity of the cells towards the polyurethane surface is related to its high surface energy resulting from the presence of the polar chemical groups on the surface of the polyurethane film. Also, polyurethane surface supports a significant degree of cell spreading due to the presence of the hard segments, which are distributed throughout the soft domains. This microstructure leads to high elasticity for the polyurethane film so it can tolerate the forces imposed by the cells for being stretched. The mentioned cell response is a sign of cytocompatibility of the sterilized urethane substrate.
4. Conclusion
Fig. 2. Light photomicrographs (mag. 200 ), fibroblast cells behaviour on the surface of the polymeric films after 5 days incubation: (a) Latex (b) TCPS (c) PU.
The biological safety of the sterilized polyurethane film towards gamma irradiation was evaluated. According to the results obtained from toxicity test gamma irradiation in a dose of 25 kGy performed at polyurethane film do not have a relevant influence on cell toxicity. So, this gammasterilized polyurethane film with specially chemical composition and also without additives beside monomers which supports cell attachment and growth is not cytotoxic.
N. Sheikh / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 215–219
References [1] M. Donkerwolcke, F. Burny, D. Muster, Biomaterials 19 (1998) 1461. [2] P. Klemchuk, Radiat. Phys. Chem. 41 (1–2) (1993) 165. [3] Plastics design library, The effect of sterilization methods on plastics and elastomers, New York, 1994, p. IV.
219
[4] S. Corneillie, P. Lan, E. Schacht, M. Davies, A. Shared, Polym. Int. 46 (1998) 251. [5] N. Sheikh, A.A. Katbab, H. Mirzadeh, Int. J. Adhesion Adhesives 20 (2000) 299. [6] J. Autian, Artif. Organs 1 (1977) 53. [7] M.D. Laleh, S.L. Cooper, in: Polyurethane in Medicine, CRC Press Inc., Florida, 1986, p. 103.
The effect of radiosterilization on cytotoxicity of polyurethane film N. Sheikh
*
Gamma Irradiation Centre, Atomic Energy Organisation of Iran, P.O. Box 11365-3486, Tehran, Iran
Abstract Nowadays a sequence of tests for evaluation of sterilized biomaterial includes an initial set of tests in vitro, both biological (cell culture) and non-biological (mechanical tests). In this paper the cytotoxicity of a sterilized polyurethane film, in order to use as biomaterial, has been investigated. For this purpose NCO-terminated urethane prepolymer in medical quality was synthesized without ingredients beside monomers (polyethylene glycol/castor oil and toluene diisocyanate). The cured prepolymer films were prepared under ambient conditions due to the reaction of free NCOgroups of prepolymer with air moisture. The polyurethane films were sterilized by gamma-ray (25 kGy). The surface structure of sterilized polyurethane film was observed by SEM and compared to that of the unsterilized film. Also, the in vitro interaction of fibroblast cells and sterilized polyurethane film in culture medium containing serum was evaluated in comparison with control samples. Results showed no signs of cell toxicity. Ó 2003 Elsevier B.V. All rights reserved. PACS: 61.82.PV Keywords: Sterilization; Gamma-ray; Polyurethane film; SEM; Porosity; Cytotoxicity
1. Introduction A biomaterial is expected to be selected from untoxical and biocompatible materials [1]. Also, in the most cases it must be capable of withstanding radiosterilization [2]. As an effective and industrial method for medical products sterilization is the use of radiation [3]. This method is advantageous because it is economically feasible for large-scale terminal sterilization of products in sealed packages. Among synthetic materials, polyurethanes have been considered to be the most suitable material in
*
Tel.: +98-21-800-4065; fax: +98-21-800-9054. E-mail address: [email protected] (N. Sheikh).
various biomedical applications, which is connected to their biocompatibility, biodegradability and controlled microstructure and properties [4]. However, in the case of materials for biomedical applications in addition to the evaluation of their physico-mechanical properties, risks caused by the emission of harmful substances escaping from the material due to sterilization procedures have to be kept at a minimum. In other words, it is important to know about the biological safety of a sterilized biomaterial. So, toxicity tests have to be conducted with the final sterilized product. In our laboratory, attempts have been made during several past years to find structure–properties relationships of polyurethanes in order to obtain a suitable urethane biomaterial. The synthesis
0168-583X/03/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-583X(03)01107-8
216
N. Sheikh / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 215–219
was performed on the basis of prepolymer method by using of different monomers. It was found that the use of a mixture of polyethylene glycol (PEG) and castor oil together with toluenediisocyanate (TDI) as polyol and diisocyanate components, gives the possibility of optimizing the required properties of the product such as fluidability in the liquid prepolymer state, and also hydrophilicity, flexibility and mechanical strength in a water-cured prepolymer film. This article focuses on the evaluation of cytocompatibility of the gamma-sterilized polyurethane film which contains selected chemical composition.
polyol mixture under an atmosphere of dry N2 . The reaction conditions (temperature and time) were controlled so that a clear yellowish liquid prepolymer was obtained. As the medical end use is intended, a purification process by thin layer evaporation method was carried out on the reactive prepolymer product to remove the unreacted TDI [5]. This method can be performed easily due to the high vapour pressure and volatility of TDI.
2. Experimental 2.1. Chemicals Polyethylene glycol (PEG 1000, Sigma) and castor oil (Aldrich) were used as polyol and dried at 80 °C under vacuum. 2,4 TDI supplied by Riedelde Haen Co. was used as received. 2.2. In vitro cell system
2.4. Film preparation
Mouse C34 /connective tissue (L929) was obtained from the National Cell Bank, Pasteur Institute of Iran. The cells were maintained in growth media RPMI-1640 and supplemented with 10% fetal calf serum (PCS), 100 U ml 1 penicillin and 100 lg ml 1 streptomycin (Gibco BRL Laboratories). A routine subculture method was used to maintain the cell line. The cells were incubated in a humidified atmosphere with 5% CO2 at a temperature of 37 °C. After incubation of one week the monolayer was then harvested by trypsinization. A cell suspension of 4 105 cells ml 1 was prepared before seeding.
For the preparation of the urethane film, the liquid purified prepolymer was cast onto the surface of a glass plate and then left to be cured under ambient conditions.
(The reaction between a H2O molecule and 2(NCO) groups of prepolyner chains)
2.3. Urethane synthesis The urethane prepolymer was obtained by the reaction between a mixture of PEG and castor oil (50/50, w/w) with TDI. The amount of isocyanate and hydroxyl containing materials were adjusted so that a ratio of 2 was obtained for NCO to OH groups. Synthesis was carried out on the basis of prepolymer method. TDI was slowly added to
A combination of above reactions is occurred due to the simultaneous presence of PEG (diol) and
N. Sheikh / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 215–219
castor oil (triol) in the reaction system. Finally, prepolymer chains completely react with an equivalent amount of water, lead to the formation of crosslinked poly(urea)urethane. 2.5. Sterilization The cured urethane film (PU) and control films, latex and tissue culture polystyrene (TCPS),with 0.15 mm thickness, were punched into discs of 15 mm diameter and then sterilized (25 kGy). Sterilization was carried out using gamma rays from 60 Co source, in a Gammacell-220 with a dose rate of 3.7 kGy/h, in air at room temperature. The absorbed dose was measured by the Frick dosimeter.
217
work reactive urethane prepolymer was prepared without ingredients beside monomers and purified in order to obtain medical purity. Also, it is found that controlled porosity of biomaterials is important in numbers of biomedical applications [7]. In the case of urethane prepolymer, the curing reaction rate of prepolymer with water which is clearly dependent on the type of used polyol and diisocyanate, amount of moisture, accelarator and thickness of prepolymer layer, has influence on porosity of cured film. On the other hand, sterilization process may affect on the film porosity. So, the physical form of the sterilized urethane surface was compared with that of the unsterilized sample using SEM. It can be observed from Fig. 1(a) and (b) that radiation sterilization
2.6. Surface structure The surface porosity of gamma sterilized and unsterilized urethane films were observed with a SEM (Philips-XL30) after sputter gold coating of samples. 2.7. Cytocompatibility assay The cytocompatibility of the urethane film was evaluated by in vitro cell culture test in comparison with controls. For this purpose gamma sterilized polymeric discs were individually placed into a multiwell plate (Nunc, Denmark). A volume of 5 ml cell suspension was seeded into each well. These cultures were placed in a CO2 controlled incubator at 37 °C for 5 days and then media were decanted. The samples were washed with phosphate buffer saline solution, their attached cells were fixed by ethanol solution and stained with crystal violet or giemsa solution as staining agents. All air-dried samples were examined by light microscopy (Hund-H500).
3. Results and discussion As it is reported that small molecular weight additives may leach out from biomaterial and decrease the biocompatibility [6] therefore, in this
Fig. 1. SEM micrographs of the surface of polyurethane film (mag. 2000 ): (a) before sterilization and (b) after sterilization.
218
N. Sheikh / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 215–219
has no significant effect on the number and size of pores of surface film. The preliminary in vitro test to evaluate the biocompatibility of the material is cytotoxicity test. This test indicates cellular behaviour consisting of cell adhesion, morphology and growth
pattern, which results from complex processes with biological and non-biological mechanisms. These processes are not only linked to the release of toxic products but also to the chemical nature and surface properties of the substrate including; surface tension, roughness, hydrophilicity, elasticity and electrical charge. The sterilized urethane film was also evaluated by a cytotoxicity test. Results obtained from in vitro cell culture are presented in Fig. 2(a)–(c). The positive control (Latex) is a toxic material as only few detached cells are observed on its surface (Fig. 2(a)). This behaviour is due to the presence of toxic vulcanizing ingredients in its compound. The negative control (TCPS) supports cell adhesion and growth (Fig. 2(b)) because it has no toxicity moreover, its surface properties is probably desirable for cell attachment and cell proliferation. In the case of polyurethane film (Fig. 2(c)) similar behaviour has been shown to the TCPS substrate. The affinity of the cells towards the polyurethane surface is related to its high surface energy resulting from the presence of the polar chemical groups on the surface of the polyurethane film. Also, polyurethane surface supports a significant degree of cell spreading due to the presence of the hard segments, which are distributed throughout the soft domains. This microstructure leads to high elasticity for the polyurethane film so it can tolerate the forces imposed by the cells for being stretched. The mentioned cell response is a sign of cytocompatibility of the sterilized urethane substrate.
4. Conclusion
Fig. 2. Light photomicrographs (mag. 200 ), fibroblast cells behaviour on the surface of the polymeric films after 5 days incubation: (a) Latex (b) TCPS (c) PU.
The biological safety of the sterilized polyurethane film towards gamma irradiation was evaluated. According to the results obtained from toxicity test gamma irradiation in a dose of 25 kGy performed at polyurethane film do not have a relevant influence on cell toxicity. So, this gammasterilized polyurethane film with specially chemical composition and also without additives beside monomers which supports cell attachment and growth is not cytotoxic.
N. Sheikh / Nucl. Instr. and Meth. in Phys. Res. B 208 (2003) 215–219
References [1] M. Donkerwolcke, F. Burny, D. Muster, Biomaterials 19 (1998) 1461. [2] P. Klemchuk, Radiat. Phys. Chem. 41 (1–2) (1993) 165. [3] Plastics design library, The effect of sterilization methods on plastics and elastomers, New York, 1994, p. IV.
219
[4] S. Corneillie, P. Lan, E. Schacht, M. Davies, A. Shared, Polym. Int. 46 (1998) 251. [5] N. Sheikh, A.A. Katbab, H. Mirzadeh, Int. J. Adhesion Adhesives 20 (2000) 299. [6] J. Autian, Artif. Organs 1 (1977) 53. [7] M.D. Laleh, S.L. Cooper, in: Polyurethane in Medicine, CRC Press Inc., Florida, 1986, p. 103.