1. Introductıon to sterılızatıon
Sterilization is an absolute term meaning the destruction of all life (Joslyn, 1991). Sterilization generally alters or damages the type of materials subjected to it. Sterilization can be classified as physical or chemical, and these are either liquid, gaseous, or electromagnetic radiation. However, there are no sharp distinctions in between them.
In August 2002, FDA (Food and Drug Administration) released a guide concerning the sterilization of biomaterials. Sterilization methods are divided into two; traditional, and non-traditional methods. Traditional methods are those recognized by the FDA. And non-traditional methods are those that are outside the scope of specific CDRH (Center for Devices and Radiological Health)-recognized standards are non-traditional. A new method of sterilization remains a non-traditional method unless: a) the specific sterilization method is incorporated into a new or existing voluntary consensus standard formally recognized by the Agency or b) CDRH evaluates the validation data for the method of sterilization as part of a quality system evaluation and finds it satisfactory for specified types of devices.
a. Traditional Methods of Sterilization :
· Dry heat sterilization
· Moist heat sterilization
· Ethylene Oxide (EO) with devices placed in a fixed chamber
· Radiation (gamma and electron beam)
· Liquid chemical sterilants for sterilizing single-use devices incorporating materials of animal origin
b. Non-Traditional Methods of Sterilization:
· EO not using a fixed chamber, e.g., EO injection into a porous polymer bag. Terms used for this process include:
· "bag method"
· "diffusion method"
· "sterilization pouch"
· "injection method"
· Chlorine dioxide
· Ultraviolet light
· Combined vapor and gas plasma
· Vapor systems (e.g., peroxide or peracetic acid)
· Filtration methods
· Limited use of a liquid peracetic acid system in endoscopy and with metal instruments.
1.1. Dry heat
The action of dry heat on objects is that of conduction. The heat is absorbed by the exterior surfaces of an article, eventually heating the interior, but the factor of moisture is lacking. The difference in moisture contents is responsible from different temperatures required to sterilize materials. It is generally accepted that 1 hour at 180 0C or 2 hours at 160 0C is necessary to dry sterilize the materials.
The principal advantage of dry heat sterilization is its penetrating power. It penetrates materials that are not permeable to steam, i.e. oil, powders, petrolatum etc. It is also not as corrosive as steam for metals and sharp instruments, and it does not erode ground glass surfaces.
There are also disadvantages of dry heat sterilization, these include:
· Slow heating: diffusion and penetration of heat are slow because the heat transfer medium is poor.
· Long time: long exposure is required to kill microorganisms.
· High temperatures: extreme temperatures may be harmful to materials.
· Tendency to stratify: a severe tendency to stratify over a considerable range of temperature must be overcome.
1.2. Moist heat
Sterilization by moist heat is done by saturated steam under pressure. Sterilization depends on five parameters; vacuum, pressure, temperature, relative humidity, and time. The advantages of this method are those:
· Sterilization is achieved in a short time (15 min at 121 0C, at 1 atm pressure), even the most resistant form of life, the spores of Bacillus stearothermophilus is destructed,
· It rapidly heats and penetrates textiles or fabrics,
· There is no toxic residue left on the material, and
· It is the most economic sterilization method among others.
The method also has disadvantages, these include:
· Incomplete air elimination from the sterilizer decreases temperature and prevents adequate sterilization.
· Sterilization of anhydrous oils, greases, and powders cannot be done.
1.3. Ethylene oxıde (eo)
Ethylene oxide was first discovered and described by Wurtz in 1859 (Perkins, 1969). It was used as a fumigant and pesticide at these times. Later it was used as a sterilizing agent. This compound inactivates all organisms without exerting detrimental effects resulting from dry heat or steam sterilization.
EO is an epoxy compound, it is the simplest cyclic ether. The configuration is as follows:
CH2 CH2
CH2
The action of EO towards microorganisms is through alkylation of sulfhydryl, amino, carboxyl, phenolic and hydroxyl groups of molecules. EO primarily shows its action on alkylation adenine and guanine residues.
The parameters of EO sterilization are time, temperature, ethylene oxide concentration and moisture. As an example, in sterilization of polyurethane materials, Lucas et.al., (2003) used 10% EO at 54 °C, at 65% RH (relative humidity) for 130 min, followed by 12 h aeration at 132 °C.
Telescopic instruments (bronchoscopes, endoscopes, pharyngoscopes), catheters, syringes, and surgical gloves can be sterilized with ethlylene oxide (Perkins, 1969).
Although applicable to a wide variety of materials, ethylene oxide has many disadvantages. The vapor of ethylene oxide is flammable and explosive and should be handled carefully. It has mutagenicity and carcinogenicity, and for this reason long aeration times are required to eliminate residual ethylene oxide gas (or at least to drop to tolerable levels).
1.4. radıatıon
Radiation may be classified into two groups:
- electromagnetic radiation, and
- particle radiation.
Electromagnetic radiation includes microwave, UV, gamma rays, x-rays, and electrons. Gamma rays, x-rays, and electrons are called as ionizing radiation, because they show their effect by transferring the energy of a photon into characteristic ionizations in or near a biological target (activated molecules and free radical production). Non-ionizing radiation is mainly UV light.
1.4.1. Ionizing radiation
Gamma radiation is emitted by radioactive cobalt. It is the result of a transition of an atomic nucleus from an excited state to a ground state. In X-ray, emission occurs by transition of an electron from an outer shell to a vacancy further within an inner shell, and is produced by bombarding a heavy metal target with fast electrons in an accelerator (Silverman, 1991). High-energy electron beams are produced by acceleration of electrons to high energies.
Gamma radiation is used in sterilization of medical products not only to sterilize materials, but it also confers decreased immunogenicity to implants. At the same time, however, a decrease in mechanical strength is also accomplished.
The primary cellular target of gamma radiation is DNA molecule. There are also indirect effects caused by the diffusion of radicals produced in the adjacent volume. The materials that can be sterilized with gamma rays include cartilage, tendons, skin, heart valve, drugs, vaccines etc. In sterilization of medical devices, the general choice of application dose is 2.5 Mrad.
1.4.2. Non-ionizing radiation
The non-ionizing radiation used in sterilization of biomaterials is UV-radiation. The wavelength of UV light is greater than 1 nm, while the wavelength of ionizing radiation has shorter wavelength. Thus, the energy of the UV light is much less than that of ionizing radiation, and it has less penetration power.
The UV spectrum ranges from 15nm to the borders of visible light; however, the region between 220-300nm is interested in sterilization. The primary action of UV on target cells is the formation of thymine dimers. The dimers cause increased mutation rate, and increased rate of chromosomal aberrations.
As UV light has low penetration power, it can be used against airborne microorganisms, or to the surfaces that are accessible to UV light. They are commonly used to sterilize air and material surfaces in surgical operation rooms.
1.5. lıquıd sterılants
Liquid sterilants include glutaraldehyde, hydrogen peroxide, peracetic acid and formaldehyde. The most commonly used liquid sterilant is glutaraldehyde (1,5-pentadial). The structure is as follows:
Glutaraldehyde is a powerful biocidal agent. It is used where moist heat, dry heat, or other types of sterilization methods cannot be used on the material. It is used in concentrations between 0.5-2% depending on the material and purpose. This chemical is able to kill spores, mycobacteria and viruses. The main advantage of this agent is that it is able to show its activity in the presence of organic material (Scott and Gorman, 1991). This is important because, for the other sterilization methods, a careful cleaning step should be performed, otherwise the sterilization would be of no value. Another advantage is that this chemical is not corrosive to metals, rubbers, and lenses, and can be used to sterilize urologic, laparoscopic instruments, and anethesic equipment.
1.6. gas plasma
Menashi was the first to use a corona discharge to sterilize the surface of materials (Silverman, 1991). Nowadays a gas plasma sterilizing apparatus consists of a sterilization chamber with vacuum, a radio frequency generator, an impedance matching network, and gauges for admitting controlled amounts of gases. There are carrier gases used in the gas plasma, these include nitrogen, oxygen, argon etc. The efficiency of the method is increased if aldehydes, nitrous oxides are added to gas mixture.
Two sterilizers using plasma technology are available commercially, namely Sterrad™ (Advanced Sterilization Products, Johnson and Johnson), and Plazlyte™ (AbTox). Sterrad™ uses hydrogen peroxide with RF generator, and Plazlyte™ uses peracetic acid gas plasma with RF generator. Both systems allow fast low-temperature reprocessing of medical instruments.
The advantages of gas plasma are:
· no residue is left on the material, and
· no toxic chemicals are involved in sterilization process.
According to manufacturers of these devices, gas plasma has wide materials compatibility (materials like catheters, scissors, surgical tubing, etc.), however, there is not enough research revealing the effects of gas plasma on the materials.
1.7. fıltratıon
The use of filters dates back to Egyptians. They used fabrics to strain grape juice, and at those times Carthage was famous for the clarity of its wines. In modern times Pasteur and Chamberlain were the first to use filters to remove bacteria from solution (Levy and Leahy, 1991).
Filtration is done through passage of a liquid or gas through a screenlike material with pores small enough to retain microorganisms. For this purpose, membrane filters (0.22-0.45µm pore size) are used. For use in hospital operating rooms HEPA filters (0.30µm pore size) are convenient to filter microorganisms.
Filtration is done where labile solutions need to be sterilized (those that cannot be treated with heat sterilization). These include antibiotics, ophtalmic solutions, intravenous solutions, vaccines, tissue culture medium, etc.
2. Influence of lamella features of UHMWPE on its physical and uniaxial tensile properties. I. Effect of sterilization method in uncrosslinked and unaged materials
Mushra et al., (2003) Bio-Medical Materials and Engineering
2.1. ıntroduction to Ultra high molecular weight polyethylene (uhmwpe) and methods used in sterilization of UHMWPE
UHMWPE is a thermoplastic polymer, composed of is ethylene monomers. Its molecular formula is (C2H4)n, and it has a molecular weight in the range of 3000000-6000000 D. The structure of UHMWPE is shown in Figure 1.
Figure 1. Chemical structure of UHMWPE
(www.centerpulseorthopedics.com/durasul/polyethylene)
UHMWPE is the most commonly used bearing material in total joint replacement (Figures 2 and 3). The most widely accepted implant configuration includes a metal component articulating against a polymeric component fabricated from ultra-high molecular weight polyethylene (UHMWPE). Its excellent combination of biocompatibility, structural strength, and wear resistance makes it the most common material (Shen and McKellop, 2002). UHMWPE consists of extremely long molecular chains, which makes it an excellent 
abrasion-resistant material.
Figure 2. Figure of acetabular cups made up of UHMWPE used in total hip joints
(www.tu-berlin.de/fbb/polymer/uhmwpe.html)
Figure 3. Figure showing X-Ray of a normal hip joint (© 2003 Herbert D. Huddleston, M.D.)
2.1. 1. sterilization of UHMWPE
Most frequent fixation problems in total hip prosthesis are related to (1) infection, (2) wear and wear particulate, (3) migration and failure of implants, and (4) loosening of which the “long-term loosening” of the implant is especially important (Park, 2000).
Sterilization of UHMWPE can disturb the chemical stability of this molecule and result in the abovementioned problems (Reno et. al., 2003).
There exist three methods commonly used by companies in USA in sterilization of UHMWPE (Kurtz et al., 1999). These are:
· Gamma radiation,
· Ethylene oxide gas sterilization, and
· Peracetic acid gas plasma.
The most common methods are gamma radiation and ethylene oxide gas sterilization, and there has not been a consensus on which method is better than the other.
2.1.1.1. Gamma radiation of UHMWPE
Gamma radiation is the most widespread sterilization method for UHMWPE. In the presence of oxygen gamma irradiation results in promoted chain scission, which in turn result in lowering of molecular weight, increased density, stiffness, and brittleness, and reduces the fracture strength (Medel et.al., 2004, Kurtz et.al., 1999). For this reason radiation is preferably done in low oxygen by either sealing in a vacuum, in a gas (nitrogen or argon), or with a chemical scavenger of ethylene oxide.
Apart from the sterilization of UHMWPE, gamma radiation is also used in cross-linking of UHMWPE (Muratoğlu and Harris, 2001). If little oxygen is present, the free radicals generated during radiation sterilization can form crosslinks between the carbon atoms, and crosslinking has been shown to markedly improve the wear resistance of polyethylene acetabular cups in laboratory wear simulators and in clinical studies.
Benson (2002), studied gamma radiation on UHMWPE and has stated that gamma-radiated samples show increased elasticity and hardness over untreated samples, which implies that, chemical or physical modifications occurred on the surface layer.
Affatato et.al., (2002a), states that the mechanical integrity and toughness of UHMWPE is preserved as long as the long molecular chains of polyethylene are folded into dispersed crystalline lamellae connected by tie molecules within an amorphous matrix.
2.1.1.2. Ethylene oxide gas sterilization of UHMWPE
UHMWPE is a good candidate for sterilization by ethylene oxide, because it has no constituents that will react with or bind to the toxic gas. As true with other materials, ethylene oxide gas is toxic, so it should be performed carefully.
In a review by Kurtz et.al., ethylene oxide was favored over gamma radiation. It was said that ethylene oxide sterilized components show significantly less surface damage and delamination than gamma radiated samples.
However, in another study comparing the gamma radiation and ethylene oxide sterilization methods, Affatato et.al., (2002b) concluded that ethylene oxide sterilized specimens wore 1.13 times faster than the gamma radiated specimens. In the second part of this study (Affatato et.al., 2003), however, they have shown that while ethylene oxide sterilized compounds show highest weight loss, gamma radiated compounds had more crystallinity (and hence more chain scission).
2.1.1.3. Gas plasma sterilization
This method is a relatively new commercial sterilization method, when compared to ethylene oxide or gamma radiation. It has the advantage of leaving no toxic residues or by products on the material. Lerouge et al., (2002) studied effects of gas plasma on different polymers including polyethylene tubing, and they found that surface characteristics have been changed more than that of EO sterilization.
2.2. aim of the study
In our paper of interest, the purpose of the study is to determine the extent to which sterilization methods can change the polymer’s (UHMWPE) tensile toughness (U), degree of crystallinity (%C), and melting temperature (Tm). It was also studied that if the change in the thickness and alignment of its lamellae is dependent on these three factors.
2.3. materials and methods
The UHMWPE specimens used in the experiment were uncrosslinked.
Five sets of specimens were prepared (five specimens per set), each having been sterilized as: none (CONTROL); packaged in air and then gamma irradiated in air [gamma-AIR]; packaged in a nitrogen-gas-filled pouch and then gamma irradiated in air [gamma-N2]; using ethylene oxide gas [EO]; and using the gas plasma [GP]. The specimens were tested as sterilized, with no aging of any type.
The tension test were uniaxial and performed using a servohydraulic universal materials testing machine.
A differential scanning calorimeter was used to determine %C and Tm.
To measure lamella thickness and alignment TEM (Transmission Electron Microscopy) was used. Atomic Force Microscopy (AFM) was used to obtain high-resolution three-dimensional relief maps of the surface, from which the lamella thickness was determined.
Test of significance between the mean values for any pair of test specimens was conducted using the paired Student’s t-test (p < 0.05).
2.4. results and discussion
The results of the tensile toughness (U), % crystallinity (C), and melting temperature (TM) are shown in Table 1.
Table 1. Means of mechanical and physical properties and lamella parameters of the five study sets (U: tensile toughness, % C: degree of crystallinity, Tm: melting temperature, WT: lamellar thickness, WA: lamellar thickness after sterilization, f: lamellar alignment)
Set |
U
(MJm-3) |
%C |
Tm
(0C) |
WT
(nm) |
WA
(nm) |
f
(o) |
Control |
102 |
72.9 |
130 |
44.4 |
23.0 |
28.6 |
Gamma-air |
165 |
73.2 |
144.4 |
36.8 |
28.0 |
24.7 |
Gamma-N2 |
114 |
82.9 |
142.3 |
44.2 |
23.0 |
36.3 |
EO |
127 |
68.9 |
138.0 |
47.0 |
21.0 |
33.8 |
GP |
114 |
79.8 |
137.1 |
41.3 |
19.0 |
33.7 |
From the table it is seen that for the values U, % C, and Tm, there is a marked difference between the gamma-air and the others.
The results of the TEM are shown in Figure 4. The mean values obtained from TEM measurements for the lamella thickness are shown in the Table 1 as WT . It was stated that the mean values of each set are not statistically significant.
The distribution of lamella thickness is shown in Figure 5. It is in the range of 30-60 nm, which is in accordance with the literature. It is observed that different sterilization methods show a similar distribution of lamella thickness, all are in the range of 30-60 nm.
Figure 5. Summary of overlapped histograms of lamella thickness distributions, as obtained using TEM.
The results of the lamellar alignment were obtained from both TEM and AFM. In Figure 6, the distibution of lamellar arrangement are shown. In Figure 7, the photos taken by AFM show the lamellar arrangement and orientation after uniaxial stress is applied (from top to bottom).
Figure 6. Histograms showing the distribution of lamellar alignment (f).
The histograms show that there is distinct alignment at a certain range, indicating the stress application direction. A value called FWHM (full width at half-max) was defined from the histograms, which is an indication of the degree of lamellar arrangement. Smaller FWHM is an indication of increased lamella alignment. Compared to control specimens, the FWHM of the gamma-air is smaller than the control, while it is larger for other four. Thus, the least lamellar arrangement is seen for gamma-N2, then comes GP, control, EO, and gamma-air is the most lamellar arranged specimen.
Figure 7: AFM of specimens.
In Figure 7, it is seen that the arrangement and orientation of the specimens are in the direction of tensile loading. The lamella is arranged from top to bottom. In the sterilized specimens, lamellar thickness (WA) is shorter than the control specimen, also shown in Table 1. But it also is in the accepted range (Mishra et.al.).
2.5. conclusion
The changes in the tensile toughness (U), degree of crystallinity (%C), and melting temperature (Tm) of an uncrosslinked and unaged UHMWPE is brought about as result of a change in the sterilization method used.
From the results of TEM and AFM, and that of the Table 1, it is seen that lamellar thickness does not change significantly, so it is probably lamellar alignment that changed the tensile toughness (U), degree of crystallinity (%C), and melting temperature (Tm) properties.
It is concluded from changes in tensile toughness (U), degree of crystallinity (%C), and melting temperature (Tm), that gamma radiation in air severely degrades the material and, thus, changes the in vivo performance of bearing components fabricated from it.
2.6. comments
In the paper nothing was said about the effect of different types of sterilization on the wear rate of the material, simulatory tests could have been performed to determine weight loss which is an indication of material loosening, an important factor in failure of hip implants.
Gamma-radiated (air) sample showed the worse performance in terms of tensile strength, crystallinity and melting point. These are characteristic of gamma-radiation, resulting from oxidation, and subsequent chain scission. To prevent oxidation, incorporation of antioxidants into the biomaterial can be done.
2.7. references
1. Affatato S., B. Bordini, C. Fagnano, P. Taddei, A. Tinti, A. Toni (2002a) Effects of the sterilisation method on the wear of UHMWPE acetabular cups tested in a hip joint simulator. Biomaterials 23: 1439–1446
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