Abstract

1. Introduction

Biomaterials are substances of natural or synthetic origin used to replace human body tissues, organs or functions temporarily or permanently¹1 Ramakrishna S, Mayer J, Wintermantel E, Leong KW. Biomedical applications of polymer-composite materials: a review. Composites Science and Technology. 2001;61(9):1189-1224.. However, biomaterials must comply with basic requirements for use on living beings, such as: biocompatibility, biofunctionality and sterilization, without compromising their chemical structure²2 Ratner BD, Hoffmann AS, Schoen FJ, Lemons JE. Biomaterials Science: An Introduction to Materials in Medicine. 3rd Ed. New York: Academic Press; 2013.^,33 Hench LL. Biomaterials: a forecast for the future. Biomaterials. 1998;19(16):1419-1423.. Sterilization is a crucial procedure in disinfecting biomaterials to be used on living beings, because it would eliminate microorganisms, prevent the spread of diseases and avoid inflammatory reactions triggered by the body defense mechanism. One of the most frequent methods of polymeric biomaterial sterilization is the use of ethylene oxide because of its excellent antimicrobial properties⁴4 Rutala WA, Gergen MF, Weber DJ. Comparative evaluation of the sporicidal activity of new low-temperature sterilization technologies: Ethylene oxide, 2 plasma sterilization systems, and liquid peracetic acid. American Journal of Infection Control. 1998;26(4):393-398.

5 Alfa MJ, DeGagne P, Olson N, Hizon R. Comparison of liquid chemical sterilization with peracetic acid and ethylene oxide sterilization for long narrow lumens. American Journal of Infection Control. 1998;26(5):469-477.^-66 Morejón-Alonso LM, Carrodeguas RG, García-Menocal JAD, Pérez JAA, Manent SM. Effect of sterilization on the properties of CDHA-OCP-ß-TCP biomaterial. Materials Research. 2007;10(1):15-20.. However, the method has the disadvantage of possibly causing harmful side effects, from skin irritation to damage to the gastrointestinal and central nervous system tissues. At a high level of exposure, side effects may lead to neurological and cognitive dysfunctions, polyneuropathic disability, hematological disorders, increased risk of spontaneous abortions and various types of cancerous growth (in the event of chronic exposure)⁷7 Rutala WA, Weber DJ. Clinical effectiveness of low-temperature sterilization technologies. Infection Control and Hospital Epidemiology. 1998;19(10):798-804..

Other common sterilization techniques include dry heat sterilization, steam sterilization, sterilization by radiation, besides sterilization by peracetic acid and glutaraldehyde. Heat and high humidity are avoided because these conditions cause degradation of a variety of biodegradable polymers⁸8 Mainil-Varlet P, Rahn S, Gogolewski S. Long-term in vivo degradation and bone reaction to various polylactides: 1. One-year results. Biomaterials. 1997;18(3):257-266.

9 Hooper KA, Cox JD, Kohn J. Comparison of the effect of ethylene oxide and γ-irradiation on selected tyrosine-derived polycarbonates and poly(L-lactic acid). Journal of Applied Polymer Science. 1997;63(11):1499-1510.^-1010 Holy CE, Cheng C, Davies JE, Shoichet MS. Optimizing the sterilization of PLGA scaffolds for use in tissue engineering. Biomaterials. 2001;22(1):25-31.. After applying the ethylene oxide sterilization method, oxide residual should be checked according to the ANSI/AAMI/ISO 10993-7¹¹11 Association for the Advancement of Medical Instrumentation. Biological evaluation of medical devices - Part 7: Ethylene oxide sterilization residual: ISO 10993-7:1995.2001 Arlington: Association for the Advancement of Medical Instrumentation; 2001. Standard, so that the samples can be used as biomaterials without causing damage.

The literature has reported that poly (ether ether ketone) (PEEK) shows excellent mechanical and chemical resistance¹²12 Gao X, Wang R, Zhang A. Synthesis of Poly(ether ether ketone)s containing tertiary amine. Materials Letters. 2007;61(17):3647-3651.^,1313 Xi D, Huang W, Li Z, Lu J, Chen X, Zhou Z. Fullerene-induced novel spherulitic structures of poly (ether ether ketone) at high pressure. Materials Letters. 2012;81:189-192. as well as physical properties at high temperatures¹⁴14 Vasconcelos GC, Mazur RL, Ribeiro B, Botelho EC, Costa ML. Evaluation of decomposition kinetics of poly (ether-ether-ketone) by thermogravimetric analysis. Materials Research. 2014;17(1):227-235., it is inert and biocompatible, including its composites, that show improved mechanical properties¹⁵15 Toth JM, Wang M, Estes BT, Scifert JL, Seim III HB, Turner AS. Polyetheretherketone as a biomaterial for spinal applications. Biomaterials. 2006;27(3):324-334.^,1616 Kurtz SM, Devine JN. PEEK biomaterials in trauma, orthopedic and spinal implants. Biomaterials. 2007;28(32):4845-4869.. In this context, this study aims at performing sterilization of PEEK samples, for both medical and commercial grades using ethylene oxide and monitoring the sterilized parts for residues of ethylene oxide by means of thermogravimetry (TGA).

2. Experimental procedure

Two different types of PEEK were selected for the study of ethylene oxide sterilization. PEEK commercial and medical degree, differentiating the purity of the monomers and their synthesis by the polymerization process. Commercial poly(ether ether ketone (PEEKc) was supplied by Autotravi Plásticos (Caxias do Sul -RS, Brazil) cylindrical parts with as 4 cm diameter and 10 cm length. Medical grade PEEK (PEEKm) is manufactured by Optima of InvibioLtd. (Holland), cylindrical parts with a 6.4 mm diameter and 100 cm length.

The cylindrical PEEKc were machined to manufacture the specimens by mechanical processing in a Dyna DM 4500 vertical Machining Centre (USA) resulting in nine smaller tubes of 2 mm internal diameter, 3 mm external diameter and 20 mm length.

For cylindrical PEEKm were machined, from a 6.4 mm diameter solid cylinder tubes of the same diameters and lengths as those of PEEKc were obtained.Formachining, Blasocut BC20 oil was used. The cylindrical parts were submitted to compressed air jets to remove any residues remaining from the machining process.

Hygienization of PEEKc and PEEKm samples was carried out by a first rinse with double filtered water, followed by drying on sterilized compressed air and then sonication with the aid of enzymatic compounds for better release of possible residues. The advanced proteolytic action enzymatic detergent employed in this stage is Endozime^® AW Plus, based on amylase, lipase, protease and carbohydrase, developed for full removal of all organic materials andhaving corrosion inhibition properties. Its composition includes isopropyl alcohol, and the cited group of enzymes amylase, protease, lipase, carbohydrase, ethoxylated nonylphenol nonionic detergent, dye, fragrance and water. Standard sterilization techniques for using ethylene oxide procedures were in agreement with the instructions of Interministerial Ordinance Nº. 482 of 16 April 1999¹⁷17 Brazil. ANVISA. Interministerial Order Nº. 482/2010, Brasília: ANVISA; 2010..

Polymers were characterized by differential scanning calorimetry (DSC) on a DSC Shimadzu DSC-50 (Japan), at heating and cooling rates of 10ºC‧min^-1 and N₂flow circulation of 50 mL‧min^-1 in an oven at temperatures from 23 to 420ºC. The crystallinity index (Xc) was calculated based on the fusion enthalpy of PEEK readings of DSC (ΔH_m) and fusion enthalpy of theoretically 100% crystalline polymer (ΔHº_m = 130 J‧g^-1)¹⁸18 Díez-Pascual AM, Naffakh M, Gómez MA, Marco C, Ellis G, Martínez MT, et al. Development and characterization of PEEK/carbon nanotube composites. Carbon. 2009;47(13):3079-3090. using the equation:

Thermal degradation of the polymers was measured by thermogravimetric analysis (TGA) run on a Shimadzu TGA-50 (Japan) instrument N₂ inert atmosphere (flow of 50 mL‧min^-1) at temperatures from 23 to 900ºC.

In this study this technique was chosen in order to monitor residual ethylene oxide residues in samples submitted to hygienization and sterilization at temperatures ranging from 100 to 500ºC, and to assess the residual ethylene oxide degradation of the samples (at 450 - 550ºC) and at temperatures prior to the event of PEEK degradation (560ºC). The uncertainty of the thermal gravimetric technique was 0.01 mg, obtained by analysis of measurement uncertainty, for this purpose it being considered: scale accuracy, standard weight and calcium oxalate analytical grade standard deviation.

Different heating rates were proposed for the TGA analysis: 5, 10 and 20ºC‧min^-1, to evaluate the residual ethylene oxide degradation after simulation of different processes and conclusion of the sterilization. Chemical analysis was performed by Fourier Transform Infrared Spectroscopy (FTIR) (USA), on a Thermo Nicolet, model iS10 device, band range from 4000 to 600 cm^-1, using 32 scans at 4 cm^-1 resolution, utilizing the technique of Attenuated Total Reflectance (ATR) with diamond crystal at one reflection for PEEK and NaCl oil cell.

3. Results and discussion

For sterilization, PEEKc and PEEKm samples were submitted to three different process stages: machining, hygienization and sonication.

Figure 1 illustrates the %weight loss obtained by TGA for the different process stages of the study.

During machining, there was a percentage of weight loss between the temperatures of 100 and 500ºC attributed to the oil used in cutting associated with sample humidity. This amount of oil shows significant loss after hygienization. Oil removal from the PEEKc sample can be confirmed by the TGA of oil, Figure 2, and FTIR, as shown in Figure 3.

Analysis of the cutting oil thermogravimetric curve as illustrated in Figure 2 points out that the highest degradation range which corresponds to 99.7% weight loss could be found between 314ºC and 352ºC. When the influence of cutting oil degradation is evaluated on the PEEKc and PEEKm TGA curves, practically no significant loss changes at this temperature range could be found, which indicates that no significant amounts of cutting oil were lost, even after the machining process stage.

By analyzing the mineral oil absorption bands of Figure 3 (B), one can see bands at 2954.5, 2922.7 and 2852.8 cm^-1 from CH₂ and CH₃ groups stretching, also bands at 1376.9 and 1463.2 cm^-1 related to angular deformations of the same groups and at 721.7 cm^-1 for rock of grouping (CH₂)₄ chain indicating an essentially aliphatic mineral oil. In seeking traces of these bands in PEEKc and PEEKm spectra, according to Figure 3 (A) these were not seen. The choice of checking the success of the chosen approach usingthe ATR technique is due to the fact that superficial contamination is evident since penetration of the IR beam into the sample is approximately 5 µm, so this technique is sufficiently adequate for contamination observations¹⁹19 Pikes Technologies. ATR - Theory and Applications - Application Note. PIKES Technologies. Madison, WI, 2011. Available from: <http://www.piketech.com/files/pdfs/ATRAN611.pdf>. Access in: 07/03/2015.
http://www.piketech.com/files/pdfs/ATRAN...

20 Pikes Technologies. Analysis of Polymers by ATR/FT-IR Spectroscopy. PIKES Technologies. Madison, WI, 2013. Available from: <http://www.piketech.com/files/pdfs/Analysis_of_Polymers_ATR_FTIR_AN.pdf>. Access in: 07/03/2015.
http://www.piketech.com/files/pdfs/Analy... ^-2121 Soares IP, Russo RMDO, Prates RGD, Augusti R, Fortes ICP, Pasa VMD. Performance evaluation of techniques ESI-MS and ATR / FTIR in determination of BX adulteration with of kerosene and residual oil. Química Nova. 2011;34(8):1439-1442..

The lowest weight loss of all processes was found for the hygienization stage performed by cleaning samples with enzymatic detergents. Hygienization obtained by cleaning could be observed by color alteration detected through optical microscopy (OM) during the PEEKc samples hygienization process, with gradual removal of dirt (Figure 4) with initial 50x magnification.

Table 1 shows the weight loss (in mg) by TGA after samples machining, hygienization and sterilization process stages.

For the sterilized samples, weight loss percentage was found through all the process stages, as being 1.3% for PEEKc and 1.2% for PEEKm at a temperature range between 100 and 500ºC. These temperatures are lower than the PEEK degradation temperatures (± 560ºC)²²22 Celso F, Mauler RS, Gomes AS. Thermal properties of Speek-based polymeric films containing benzimidazole derivatives and fosfotungstic acid. Polímeros. 2008;18(2):178-186., however they are within the temperature range of ethylene oxide degradation (450 - 550ºC), to which the loss is attributed. It should be emphasized that care must be taken in removing humidity at the hygienization and sterilization stages prior to carrying TGA analysis. From Table 1 it is clear that the analysis results for weight loss are higher for PEEKc than for PEEKm, since the processes for obtaining commercial and medical grade polymers are different.

The maximum amount of residual ethylene oxide among the polymer chains must obey the National Health Surveillance Agency ( Agência Nacional de Vigilância Sanitária) (ANVISA) regulation that establishes for small implants (10 g), similar to the mass of the samples of this study, a maximum limit of 250 ppm (0.0025 mg). Ethylene oxide remaining above this limit is not considered safe for living beings¹⁷17 Brazil. ANVISA. Interministerial Order Nº. 482/2010, Brasília: ANVISA; 2010.. In this study, it was observed that the remaining oxide in the sterilized, non-aerated hyperventilated samples was higher than that allowed by ANVISA and that the sensitivity of the proposed technique, TGA, was suitable for the monitoring of the sterilization process stages.

The crystallinity index of PEEK (Xc) calculated by DSC, is shown in Table 2.

The crystallinity index showed that samples crystallinity values after machining are similar, where PEEKm = 24.2% and PEEKc = 23.5%; both samples being semicrystalline, even though at low levels. After the various process stages effected for PEEKm sterilization it showed no changes (Xc = 24.2% after machining; Xc = 22.7% after hygienization and Xc = 23.8% after sterilization). This behavior was attributed to the purity of the starting monomers and the process for obtaining the products, which do not contain impurities influencing crystallization. For PEEKc, one can observe increased crystallinity after the proposed process stages (Xc = 23.5% to 42.4%). This increase can be attributed the impurities resulting from the polymerization process as well as any additives used, influencing in the crystallization²³23 Liu Q, Li C, Wei L, Shen M, Yao Y, Hu B, et al. The phase structure, chain diffusion motion and local reorientation motion: (13)C Solid-state NMR study on the highly-crystalline solid polymer electrolytes. Polymer. 2014;55(21):5454-5459..

For amorphous PEEK crystallization occurs at temperatures approaching Tg (~ 143ºC) but still far below the main crystalline melt transition at 335ºC. For industrial applications, which can expose PEEK to continuous temperatures of up to 250ºC, operating near or above Tg can induce in-service crystallization¹⁴14 Vasconcelos GC, Mazur RL, Ribeiro B, Botelho EC, Costa ML. Evaluation of decomposition kinetics of poly (ether-ether-ketone) by thermogravimetric analysis. Materials Research. 2014;17(1):227-235..

Ethylene oxide gas diffuses through the samples amorphous segments. For polymer materials, diffusion is generally higher throughthe amorphous than throughthe crystalline phase²³23 Liu Q, Li C, Wei L, Shen M, Yao Y, Hu B, et al. The phase structure, chain diffusion motion and local reorientation motion: (13)C Solid-state NMR study on the highly-crystalline solid polymer electrolytes. Polymer. 2014;55(21):5454-5459.. The amount of weight loss indicated by the TGA technique is that of the oxide residual since after biomaterials sterilization there is a period of aeration at a given temperature and time to ensure the removal of values compatible with the ANVISA regulations.

Considering the different process simulations carried outon PEEKc and PEEKm samples (Table 3) heating rate values observed at 10ºC‧min^-1 (by TGA) are lower when compared with other rates, but the results are similar to those displayed in Table 1.

Also highlighted are the results of Table 3 which show that the proposed drying, aeration and hyperventilation steps are efficient for the removal of residual ethylene oxide since weight loss Figures in mg (steps 5 and 6) are lower than those of the samples which were not submitted to these treatments. Hyperventilation and aeration times longer than those proposed in this study are however recommended since the values obtained so far are higher than those accepted by ANVISA, of up to 0.0025 mg for 10 g.TGA heating rate of 10ºC‧min^-1 was the one of higher coherence relative to the processes proposed in this study as well as the evidenced weight losses.

4. Conclusion

The chosen machining methodology enabled making PEEK cylindrical tubs of the required dimensions for the purpose of this study. Medical and commercial PEEK grades showed similar behaviors regarding sterilization processes due to similar crystallinity. Using thermogravimetry at a heating rate of 10ºC‧min^-1 it was possible to evaluate the residual ethylene oxide in the sterilized samples, these values being higher than those allowed by the Interministerial Ordinance Nº. 482 of 16 April 1999, in accordance with the thermogravimetric analyses carried out in this study.

5. Acknowledgments

The authors are grateful to UCS and CNPq.

6. References

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