Additive Manufacturing of Medical Implants: A Review

Additive Manufacturing of Medical Implants: A Review E. Gordon Wayne State University College of Engineering Abstract Additive manufacturing has numerous applications and is gaining interest in the biomedical field. The quality of additively manufactured parts is constantly improving, which contributes to their increased use for medical implants in patients. This paper reviews the literature on surgical additive manufacturing applications used on patients, with a focus on the customization of 3D printed implants and the ability to incorporate scaffolds on the implant surface. Scholarly literature databases were used to find general information on the focus topics, as well as case studies of surgical applications of additive manufacturing implants in rodents and humans. The advantages of additive manufacturing medical implants include improved medical outcome, cost effectiveness, and reduced surgery time, as well as customization and incorporated scaffold. Overall, the most effective type of additive manufacturing for the medical implant application is electron beam melting using Ti-6Al-4V because it can produce a high quality, high purity biocompatible implant that has the required mechanical properties. Keywords: Additive Manufacturing, Customized implants, Scaffold, 3D Printing, Ti-6Al-4V Introduction In recent years, additive manufacturing technologies have improved significantly, thus expanding the fields and applications for which they can be used. These 3D printing technologies create physical models from digital models without the need for tool and die and process planning. Additive manufacturing can fabricate prototypes of complex shapes in a variety of materials such as metals, polymers, and nylon. Metal components, in particular, can be used for practical applications such as medical implants: devices manufactured to replace or support a biological structure. The biocompatibility of these metallic devices must be considered, creating rigorous requirements for the material selection and final material properties of the structure. Studies have shown that additive manufacturing successfully produces implants with biocompatible materials that meet the structural requirements [1-6]. 3D printing medical implants can provide many benefits such as the customization and personalization of the implants, cost-effectiveness, increased productivity, and the ability to incorporate scaffold. Using custom made implants, fixtures and surgical tools can help decrease surgery time and patient recovery time, while increasing the likelihood of a successful surgery [7]. Another benefit is the cost efficiency of 3D printing medial implants. Traditional manufacturing methods are cheaper for large quantities, but are more expensive for personalized designs and small production runs [8, 9]. 3D printing is especially cost effective for small-sized implants like spinal or dental implants. 3D printing is also faster than traditional manufacturing if a custom implant needs to be made; traditional methods require milling, forging, and a long delivery time while 3D printing may only take about a day [1]. Another notable benefit of additive manufacturing is the ability to share data files of designs. Files saved as an .STL can be downloaded and printed anywhere in the world. The National Institutes of Health established a 3D Print Exchange to promote open-source sharing of 3D print files for medical models [7]. The most significant benefits for the biomedical industry, however, are the ability to manufacture biocompatible materials, customize implants, and incorporate a porous scaffold surface. Types of Additive Manufacturing The additive manufacturing approach uses computer software to slice a complex 3D model into layers of 2D cross-sections with a minute thickness. The layers are then printed layer by layer depending on the particular method chosen for the application. There are dozens of types of additive manufacturing systems on the market, some of the most common being stereolithography (SLA), direct metal laser sintering (DMLS), selective laser sintering (SLS), selective laser melting (SLM), 3D printing (3DP), and electron beam melting (EBM). These systems are classified according to the form of the raw material, which can be powder, liquid, or solid form [8]. The two types of additive manufacturing that are most commonly used for medical implants are SLS and EBM. Figure 1: Process chain for SLM and EBM. The pre-processing before manufacturing includes 3D modeling, file preparation, and slicing of the 3D model into layers. Post-processing may include heat treatment and polishing of fabricated parts [10]. Selective Laser Sintering An SLS printer uses a powder form of material for printing objects. A laser fuses a single layer of powder by drawing the shape of the object according to the first 2D cross-section of the 3D model. Immediately, the build platform is lowered by the defined layer thickness and another layer of powder is rolled across [10]. The process repeats, fusing each layer one at a time to form the object. SLS can be used with metal, ceramic, and plastic powders. The precision of the laser and the diameter of the powder determines the degree of detail of the final object, so it is possible to create detailed structures with an SLS printer [11]. Figure 2: Schematic of SLS system. The key components of SLM include the laser system (a fiber laser, F-theta and galvanometer used to control the laser beam movement) and the mechanical system (movable build platform and powder roller) [10]. Electron Beam Melting An EBM printer uses a powder form of material for printing objects, similar to SLS. However, while SLS uses a laser to fuse each layer of the powder, EBM uses an electron beam. This energy is delivered through an electric circuit between a tungsten filament inside of the electron gun and the build platform [10]. An electric current heats the filament to emit a beam of electrons [1]. Electric energy is transformed to heat energy which melts the powder on the build platform. The process continues similarly to SLS, where powder is spread across the platform in a thin layer, the cross-section of the object is melted, and then the build platform lowers by the layer thickness. A key element of EBM is that the build chamber is kept under vacuum, which allows the object to be maintain great detail (70-100ÃŽÂ ¼m) [1]. Figure 3: Schematic of EBM system. The key components of EBM include an electron beam system (electron gun assembly, electron beam focusing lens and deflection coils used to control the electron beam) and the mechanical system (movable powder rake and fixed powder cassettes) [10]. Materials of Medical Implants The most common metals used for surgical implants are stainless steel 316L (ASTM F138), Cobalt based alloys (ASTM F75 and ASTM F799) and titanium alloy Ti-6Al-4V (ASTM F67 and F136) [12, 13]. However, these metals have disadvantages such as the potential release of toxic ions and particles due to corrosion that cause inflammation and allergic reactions, affecting biocompatibility [14]. Also, the materials that have an elastic modulus that is not similar to natural bone stimulate new bone growth poorly [12]. Despite this, the low Youngs modulus, high strength, and nonlinear elasticity of titanium-based alloys make it the least harmful choice [3]. The most commonly used titanium alloy is Ti-6Al-4V (Ti64) because it also has a better resistance to corrosion compared to stainless steels and cobalt-based alloys [15]. Additive manufacturing has also been done using Tantalum. Tantalum is biocompatible, hard, ductile, and chemically resistant, but it is expensive and difficult to machine [6] . Titanium based alloys are superior, thus Ti-6Al-V4 is the best material for additive manufacturing medical implants. Material Youngs modulus (GPa) Ultimate tensile strength (MPa) Yield strength (MPa) Elongation (%) TiTa 75.77  ± 4.04 924.64  ± 9.06 882.77  ± 19.60 11.72  ± 1.13 Ti6Al4V 131.51  ± 16.40 1165.69  ± 107.25 1055.59  ± 63.63 6.10  ± 2.57 cpTi 111.59  ± 2.65 703.05  ± 16.22 619.57  ± 20.25 5.19  ± 0.32 Table 1: Tensile properties of SLS produced TiTa, Ti6Al4V and commercially pure titanium samples (n = 5) [16]. Customized Implants Additive manufacturing allows for the design and fabrication of customized prosthetic implants that are created to meet the specific needs of a patient, such as the size, shape, and mechanical properties of the implant. Additive Manufacturing reduces design time as well as manufacturing time because the implant pattern is computer generated with CT and MRI scans, thus removing the need for a physical model [8]. The ability to produce custom implants quickly solves a common problem with orthopedics where standard implants do not always fit the needs of certain patients. Previously, surgeons had to manually modify implants to make them fit the patient [7]. These techniques can be used by professionals in a variety of specialties such as neurosurgery, orthopedics, craniofacial and plastic surgery, oncology, and implant dentistry [8]. One example of an application in which a customized implant is required is craniofacial reconstruction. Craniofacial abnormalities are a diverse group of congenital defects that affect a large number of people and can be acquired at birth or due to injuries or tumors [8]. Standard cranial implants rarely fit a patient precisely because skulls have irregular shapes [7]. The custom implant can be created by using a CT scan to create a 3D virtual model of the patients skull. Then the model can be used with CAD software to design an implant that would perfectly fit the patient [8]. Using custom implants has shown to improve the morphology for large and complex-shaped cranial abnormalities, and some researchers have observed a greater improvement in neurological functions than after similar surgeries using traditionally manufactured implants [17, 18]. Figure 4: Skull model and customized implant for craniofacial reconstruction surgery [8]. Scaffold Additive manufacturing medical implants allows the porosity of the surface to be designed, controlled, and interconnected, which provides better bone growth into implants, thus decreasing the chances of the body rejecting the implant. Additionally, the rough surface quality of 3D printed implants enhances bone-implant fixation [1]. Without scaffold, there is a risk of bone weakening and bone loss around the implant, which is a consequence of stress shielding due to high stiffness of materials [19]. The probability of this problem occurring is lessened when bone can grow into a porous surface of the implant [19]. Cellular lattice structures are classified by stochastic and non-stochastic geometries. The pores in stochastic structures have random variations in size and shape, while the pores in non-stochastic structures have repeating patterns of particular shapes and sizes [10]. The main challenge in additively manufacturing scaffolds is the difficulty to remove the loose powder from within the pores, but an advantage is that additive manufacturing technology allows for the manufacturing of different types of scaffolds if a design requires it; different regions of the implant could have different porosities [1, 10]. The procedure used to achieve the porous areas with traditional manufacturing methods includes coating a smooth surface with other materials such as plasma-sprayed titanium or a titanium wire mesh; however, combining different metals increases the risk of the body rejecting the implant. Additive manufacturing allows the smooth and porous surfaces to be fabricated with the same material, thus decreasing that risk. A variety of additive manufacturing techniques can be used to create the lattice structure, but scaffold can be fabricated by SLS or EBM without the need for support structures, thus making it the most effective method [5]. Figure 5: Acetabular cup with designedFigure 6: (a) Porous femoral stem on the building porous surface [10].platform, (b) post-processed femoral stem [5]. Conclusion There are many advantages to using additive manufacturing to fabricate surgical implants. These benefits include improved medical outcome, cost effectiveness, reduced surgery time, as well as customization and scaffold. Overall, the most effective type of additive manufacturing for the medical implant application is Electron Beam Melting because it can produce a high quality, high purity biocompatible implant that has the required mechanical properties. The recommended metal to use for most implants is the titanium-based alloy Ti-6Al-4V because of its low Youngs modulus, high strength, nonlinear elasticity, and corrosion resistance. Overall, additive manufacturing is an excellent production method for medical implants because it allows surgeons to customize implants and scaffold to the specific needs of the patient. 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Gaviria, Neurocognitive Assessment Before and after Cranioplasty. Acta Neurochirurgica, 2002. 144(10): p. 1033-1040. 19.Shah, F.A., et al., Long-term osseointegration of 3D printed CoCr constructs with an interconnected open-pore architecture prepared by electron beam melting. Acta Biomaterialia, 2016. 36: p. 296-309. Bacillus Thuringiensis: Distribution and Habitat Bacillus Thuringiensis: Distribution and Habitat LITERATURE REVIEW For several decades since its discovery, formulations of Bacillus thuringiensis (B. t.) have been seen as the ideal means of controlling Lepidoteran pests in agriculture because of the many attributes that differentiate this microbial insecticide from the synthetic chemical formulations. No toxicity to mammals, environmental friendliness, apparent immunity to the pesticide resistance phenomenon (no longer true), good integration with other pest control methods and the possibility of being mass produced at farm level at low cost, all made B. thuringiensis the much-needed tool for IPM programmes in developing countries. Research of almost 85 years reveals that Bacillus spp., especially B. thuringiensis and Bacillus sphaericus are the most potent biopesticides (Boucias Pendland, 1998). B. thuringiensis is a species of bacteria that has insecticidal properties that affects a specific range of insect orders. There are at least 34 subspecies of  B. thuringiensis (also called serotypes o r varieties) and possibly over 800 strain isolates (Swadener, 1994). B. thuringiensis accounts for about 5-8% of Bacillus spp. population in the environment (Hastowo et al., 1992). Till date more than 130 species of lepidopteran, dipteran and coleopteran insects are found to be controlled by  B. thuringiensis (Dean, 1984). Historical Background of B. thuringiensis B. thuringiensis are interesting and important bacteria used in the biological control of insect pest which form toxic crystal proteins at the time of sporulation. Perhaps the most well known and widely used biopesticide comes from B. thuringiensis, a bacterium that produces insecticidal proteins during its sporulation. This common soil bacterium, most abundantly found in grain dust from soil and other grain storage facilities, was discovered first in Japan in 1901 by Ishawata and then in 1911 in Germany by Berliner (Baum et al., 1999). It was subsequently found that thousands of strains of B. thuringiensis exist (Lereclus, 1993). The bacterium was isolated from diseased larvae of Anagasta kuehniella, and this finding led to the establishment of B. thuringiensis as microbial insecticide. The first record of its application to control insects was in Hungary at the end of 1920, and in Yugoslavia at the beginning of 1930s, it was applied to control the European corn borer (Lords, 2005). Sporine which was the first commercial product of B. thuringiensis was available in 1938 in France (Waiser, 1986) for the control of flour moth (Jacobs, 1951). Unfortunately, the product was used only for a very short time, due to World War II (Nester et al., 2002). Formation of transgenic plant was also observed. The first reports of insertion of genes encoding for B. thuringiensis delta-endotoxins into plants came in 1987 and the first transgenic plants to express B. thuringiensis toxins were tobacco and tomato plants (van Frankenhuyzen, 1993). In 1957 pacific yeast products commercialized the first strain on B. thuringiensis, named as Thuricide due to the increasing concern of biopesticide over the use of chemical insecticides. B. thuringiensis is a gram-positive spore-forming bacterium that produces crystalline proteins called deltaendotoxins during its stationary phase of growth (Schnepf et al., 1998). The crystal is released to the environment after analysis of the cell wall at the end of sporulation, and it can account for 20 to 30% of the dry weight of the sporulated cells (Schnepf et al., 1998) Distribution Habitat of B. thuringiensis This bacterium is distributed worldwide (Martin Travers, 1989). The soil has been described as its main habitat; however it has also been isolated from foliage, water, storage grains, and dead insects, etc (Iriarte Caballero, 2001). Isolation of strains from dead insects has been the main source for commercially used varieties, which include kurstaki, isolated from A. kuehniella; israelensis, isolated from mosquitoes, and tenebrionis, isolated from Tenebrio monitor larvae (Ninfa Rosas, 2009; Iriarte Caballero, 2001).. The spores of B. thuringiensis persist in soil, and vegetative growth occurs when nutrients are available (DeLucca et al., 1981; Akiba, 1986; Ohba Aizawa, 1986; Travers et al., 1987; Martin Travers, 1989). DeLucca et al., (1981) found that B. thuringiensis represented between 0.5% and 0.005% of all Bacillus species isolated from soil samples in the USA. Martin Travers (1989) recovered B. thuringiensis from soils globally. Meadows (1993) isolated B. thuringiensis from 785 of 1115 soil samples, and the percentage of samples that contained  B. thuringiensis ranged from 56% in New Zealand to 94% in samples from Asia and central and southern Africa. Ohba Aizawa (1986) isolated B. thuringiensis from 136 out of 189 soil samples in Japan. There are several theories on the ecological niche filled by B. thuringiensis. Unlike most insect pathogenic microbes, B. thuringiensis generally recycle poorly and rarely cause natural epizootics in insects, leading to speculation that B. thuringiensis is essentially a soil micro-organism that possesses incidental insecticidal activity (Martin Travers 1989). Evidence to support this view is that B. thuringiensis are commonly reported in the environment independent of insects and there is a lack of association between occurrence and insect activity (van Frankenhuyzen 1993). Meadows (1993) suggested four possible explanations for the presence of B. thuringiensis in soil: 1) rarely grows in soil but is deposited there by insects; 2) may be infective to soil-dwelling insects (as yet undiscovered); 3) may grow in soil when nutrients are available; and 4) an affinity with B. cereus. B. thuringiensis has been found extensively in the phylloplane. Numerous  B. thuringiensis subspecies have been recovered from coniferous trees, deciduous trees and vegetables, as well as from other herbs (Smith Couche, 1991; Damgaard et al., 1997). B. thuringiensis deposited on the upper side of leaves (exposed to the sun) may remain effective for only 1-2 days, but B. thuringiensis on the underside of leaves (i.e. protected from the sun) may remain active for 7-10 days (Swadner, 1994). B. thuringiensis kurstaki has been recovered from rivers and public water distribution systems after an aerial application of Thuricide 16B (Ohana, 1987). Crystal Composition and Morphology The existence of parasporal inclusions in B. thuringiensis was first noted in 1915 (Berliner, 1915), but their protein composition was not delineated until the 1950s (Angus, 1954). Hannay (1953) detected the crystalline fine structure that is a property of most of the parasporal inclusions. B. thuringiensis subspecies can synthesize more than one inclusion, which may contain different ICPs (Hannay, 1953). Depending on their ICP composition, the crystals have various forms (bipyramidal, cuboidal, flat rhomboid, or a composite with two or more crystal types) (Bulla et al., 1977; Hà ¶fte Whiteley, 1989). A partial correlation between crystal morphology, ICP composition, and bioactivity against target insects has been established (Bulla et al., 1977; Hà ¶fte Whiteley, 1989; Lynch Baumann, 1985). Classification of B. thuringiensis subspecies The classification of B. thuringiensis subspecies based on the serological analysis of the flagella (H) antigens was introduced in the early 1960s (de Barjac Bonnefoi, 1962). This classification by serotype has been supplemented by morphological and biochemical criteria (de Barjac, 1981). Until 1977, only 13 B. thuringiensis subspecies had been described, and at that time all subspecies were toxic to Lepidopteran larvae only. The discovery of other subspecies toxic to Diptera (Goldberg Margalit, 1977) and Coleoptera (Krieg et al., 1983) enlarged the host range and markedly increased the number of subspecies. Up to the end of 1998, over 67 subspecies based on flagellar H-serovars had been identified. Genetics of ICP In the early 1980s, it was established that most genes coding for the ICPs reside on large transmissible plasmids, of which most are readily exchanged between strains by conjugation (Gonzà ¡lez Carlton, 1980; Gonzà ¡lez et al., 1981). Since these initial studies, numerous ICP genes have been cloned, sequenced and used to construct  B. thuringiensis strains with novel insecticidal spectra (Hà ¶fte Whiteley, 1989). The currently known crystal (cry) gene types encode ICPs that are specific to either Lepidoptera (cryI), Diptera and Lepidoptera (cryII), Coleoptera (cryIII), Diptera (cryIV), or Coleoptera and Lepidoptera (cryV) (Hà ¶fte Whiteley, 1989). All ICPs described to date attack the insect gut upon ingestion. To date, each of the proteolytically activated ICP molecules with insecticidal activity has a variable C-terminal domain, which is responsible for receptrecognition (host susceptibility), and a conserved  N-terminal domain, which induces pore formation (toxicity) (Li et al., 1991). Most naturally occurring B. thuringiensis strains contain ICPs active against a single order of insects. However, conjugative transfer between B. thuringiensis strains or related species can occur, resulting in new strains with various plasmid contents (Gonzà ¡lez Carlton, 1980). Thus the mobility of the cry genes and the exchange of plasmids may explain the diverse and complex activity spectra observed in B. thuringiensis (Gonzà ¡lez Carlton, 1980; Gonzà ¡lez et al., 1981; Gonzà ¡lez et al., 1982; Reddy et al., 1987; Jarrett Stephenson, 1990). New B. thuringiensis strains have been developed by conjugation that is toxic to two insect orders. Nutritional status of B. thuringiensis Since sporulation and germination in bacilli are dependent on the nutritional status of the organism (Hardwick Foster, 1952), a study of the nutritional requirement of  B. thuringiensis var. thuringiensis is important for delineating the control mechanisms which regulate spore and parasporal crystal formation. Certain amino acids support growth, sporulation and crystal formation of B. thuringiensis var. thuringiensis, while others inhibit the growth (Singer et al., 1966; Singer Rogoff, 1968; Bulla et al., 1975; Nickerson Bulla, 1975; Rajalakshmi Shethna, 1977). A lower concentration of cystine (Nickerson Bulla, 1975) or cysteine (Rajalakshmi Shethna, 1977) promotes growth, sporulation and crystal formation in Î’. thuringiensis, while at a higher concentration of cys/cysSH, only the vegetative growth was observed, (Rajalakshmi Shethna, 1977). Classification of B. thuringiensis The classification of B. thuringiensis subspecies based on the serological analysis of the flagella (H) antigens was introduced in the early 1960s (de Barjac Bonnefoi, 1962). This classification by serotype has been supplemented by morphological and biochemical criteria (de Barjac, 1981). Many strains of B. thuringiensis have been isolated and classified within more than 20 different varieties by serological techniques. On the basis of their potency for insect these varieties have been grouped into five pathotypes: Lepidopteran-Specific (e.g. B. thuringiensis .var Kurstaki) Dipteran-Specific (e.g. B. thuringiensis . var israelensis) Coleopteran-Specific (e.g. B. thuringiensis .var. tenebrionis) Those active against Lepidoptera and Dipter(e.g. B. thuringiensis . var. aizawai) Those with no toxicity recorded in insects (e.g. B. thuringiensis . var. Dakota) Mode of Action The ICP structure and function have been reviewed in detail by Schnepf et al., (1998). Binding of the ICP to putative receptors is a major determinant of ICP specificity and the formation of pores in the midgut epithelial cells is a major mechanism of toxicity (Van Frankenhuyzen, 1993). After ingestion of B. thuringiensis by insect the crystal is dissolved in the insects alkaline gut. Then the digestive enzymes that are present in insects body break down the crystal structure and activate B. thuringiensiss insecticidal component, called the delta-endotoxin (Swadner, 1994). The delta-endotoxin binds to the cells lining the midgut membrane and creates pores in the membrane, upsetting the guts ion balance. The insect soon stops feeding and starves to death (Gill et al., 1992). Target Organisms In the past decades, B. thuringiensis Cry toxins were classified according to the target pest they attacked (Hofte Whiteley, 1998); however, due to the dual toxic activity exhibited by some cry genes and the inconsistencies in the original classification proposed by Hà ¶fte and Whiteley(1998), Crickmore et al., (1998) proposed a revision of the nomenclature for insecticidal crystal proteins, based on the ability of a crystal protein to exhibit some experimentally verifiable toxic effect in a target organism (Crickmore et al., 1998; Hà ¶fte Whiteley, 1998). The diversity of B. thuringiensis is demonstrated in the almost 70 serotypes and the 92 subspecies described to date (Galan-Wong et al., 2006). It is well known that many insects are susceptible to the toxic activity of  B. thuringiensis; among them, lepidopterans have been exceptionally well studied, and many toxins have shown activity against them (Jarret Stephens., 1990; Sefinejad et al., 2008). Order Lepidoptera encompasses the majority of susceptible species belonging to agriculturally important families such as Cossidae, Gelechiidae, Lymantriidae, Noctuidae, Pieridae, Pyralidae, Thaumetopoetidae, Tortricidae, and Yponomeutidae (Iriarte Caballero, 2001). General patterns of use: Commercial applications of B. thuringiensis have been directed mainly against lepidopteran pests of agricultural and forest crops; however, in recent years strains active against coleopteran pests have also been marketed (Tomlin, 1997). Strains of B. thuringiensis kurstaki active against dipteran vectors of parasitic disease organisms have been used in public health programmes (Tomlin, 1997). Applications in agriculture and forestry Commercial use of B. thuringiensis on agricultural and forest crops dates back nearly  30 years, when it became available in France (Van Frankenhuyzen, 1993). Use of  B. thuringiensis has increased greatly in recent years and the number of companies with a commercial interest in B. thuringiensis products has increased from four in 1980 to at least 18 (Van Frankenhuyzen, 1993). Several commercial B. thuringiensis products with B. thuringiensis aizawai, B. thuringiensis kuehniella or B. thuringiensis tenebrionise have been applied to crops using conventional spraying technology. Various formulations have been used on major crops such as cotton, maize, soybeans, potatoes, tomatoes, various crop trees and stored grains. Formulations have ranged from ultralow-volume oil to high-volume, wettable powder and aqueous suspensions (Tomlin, 1997). In the main, naturally occurring B. thuringiensis strains have been used, but transgenic microorganisms expressing B. thuringiensis toxins have been developed by conjugation and by genetic manipulation, and in some cases, these have reached the commercial market (Carlton et al., 1990). These modified organisms have been developed in order to increase host range, prolong field activity or improve delivery of toxins to target organisms. For example, the coleopteran-active cryIIIA gene has been transferred to a lepidopteran-active B. thuringiensis kuehniella (Carlton et al., 1 990). A plasmid bearing an ICP gene has been transferred from B. thuringiensis to a non-pathogenic leaf-colonizing isolate of Pseudomonas fluorescens; fixation of the transgenic cells produces ICP contained within a membrane which prolongs persistence (Gelernter, 1990). Applications in vector control B. thuringiensis Kurstaki has been used to control both mosquitos and blackflies in large-scale programmes (Lacey et al., 1982; Chilcott et al., 1983; Car, 1984; Car de Moor, 1984; Cibulsky Fusco, 1987; Becker Margalit, 1993; Bernhard Utz, 1993). For example, in Germany 23 tonnes of B. thuringiensis Kurstaki wettable powder and 19 000 litres of liquid concentrate were used to control mosquitos (Anopheles and Culex species) between 1981 and 1991 in the Upper Rhine Valley (Becker Margalit, 1993). In China, approximately 10 tonnes of B. thuringiensis Kurstaki have been used in recent years to control the malarial vector, Anopheles sinensis. Resistance of Insect Populations A number of insect populations of several different species with different levels of resistance to B. thuringiensis have been obtained by laboratory selection experiments during the last 15 years (Schnepf et al., 1998). The species include Plodia interpunctella, Cadra cautella, Leptinotarsa decemlineata, Chrysomela scripta, Tricholplusia ni, Spodoptera littoralis, Spodoptera exigua, Heliothis virescens, Ostrinia nubilalis and Culex quinquefasciatus (Schnepf et al., 1998). The Indian meal moth, a pest of grain storage areas, was the first insect to develop resistance to B. thuringiensis. Kurstaki (Swadner, 1994). Resistance progresses more quickly in laboratory experiments than under field conditions due to higher selection pressure in the laboratory (Tabashnik, 1991). No indications of insect resistance to B .thuringiensis were observed in the field, until the development of resistance was ob-served in the diamondback moth in crops where B. thuringiensis had been used repeatedly. Since then, resistance has been observed in the laboratory in the tobacco budworm, the Colorado potato beetle and other insect species (McGaughey, 1992) B. thuringiensiss Ecological Impacts Some of the most serious concerns about widespread use of B. thuringiensis as a pest control technique come from the effects it can have on animals other than the pest targeted for control. All B. thuringiensis products can kill organisms other than their intended targets. In turn, the animals that depend on these organisms for food are also impacted (Swadner, 1994). Effect on Beneficial insects: Many insects are not pests, and any pest management technique needs to be especially concerned about those that are called beneficials, the insects that feed or prey on pest species (Swadner, 1994). B. thuringiensis has impacts on a number of beneficial species. For example, studies of a wasp that is a parasite of the meal moth (Plodia interpunctella) found that treatment with B. thuringiensis reduced the number of eggs produced by the parasitic wasp, and the percentage of those eggs that hatched (Salama, 1993). Production and hatchability of eggs of a predatory bug were also decreased (Salama, 1991). Other insects: Many insects that do not have as directly beneficial importance to agriculture are important in the function and structure of ecosystems. A variety of studies have shown that B. thuringiensis applications can disturb insect communities (Swadner, 1994). Research following large-scale B. thuringiensis applications to kill gypsy moth larvae in Lane County, Oregon, found that the number of oak-feeding caterpillar species was reduced for three years following spraying, and the number of caterpillars was reduced for two years (Miller, 1990). Birds: Because many birds feed on the caterpillars and other insects affected by B. thuringiensis applications, it is not surprising that impacts of B. thuringiensis spraying on birds have been documented (Swadner, 1994). In New Hampshire, when B. thuringiensis-treatment reduced caterpillar abundance, black-throated blue warblers made fewer nesting attempts and also brought fewer caterpillars to their nestlings (Rodenhouse, 1992). Effects on Humans Eight human volunteers ingested 1 gram of a B. thuringiensis kuehniella formulation  (3 ÃÆ'- 109 spores/g of powder) daily for 5 days. Of the eight volunteers, five also inhaled 100 mg of the B. thuringiensis kuehniella powder daily for five days. Comprehensive medical examinations immediately before, after, and 4 to 5 weeks later failed to demonstrate any adverse health effects, and all the blood chemistry and urinalysis tests were negative (Fisher Rosner, 1959). Pivovarov et al., (1977) reported that ingestion of foods contaminated with  B. thuringiensis gastroenteitis at concentrations of 105 to 109 cells/g caused nausea, vomiting, diarrhoea and tenesmus, colic-like pains in the abdomen, and fever in three of the four volunteers studied. The toxicity of the B. thuringiensis gastroenteritis strain may have been due to beta-exotoxin (Ray, 1990). In a purified form, some of the proteins produced by B. thuringiensis are acutely toxic to mammals. However, in their natural form, acute toxicity of commonly-used  B. thuringiensis varieties is limited to caterpillars, mosquito larvae, and beetle larvae (Swadner, 1994). Special Concerns about B. thuringiensis Toxicity The earliest tests done regarding B. thuringiensiss toxicity were conducted using B. thuringiensis var. thuringiensis, a B. thuringiensis strain known to contain a second toxin called beta-exotoxin (Swadner, 1994). The beta-exotoxin is toxic to vertebrates, with an LD 50 (median lethal dose; the dose that kills 50 percent of a population of test animals) of 13-18 milligrams per kilogram of body weight (mg/kg) in mice when injected into the abdomen. An oral dose of 200 mg/kg per day killed mice after eight days (swadner, 1994) Beta-exotoxin also causes genetic damage to human blood cells (Meretoja, 1977).