Additive Manufacturing of Medical Implants: A Review

bilinear Manufacturing of Medical Implants A ReviewE. GordonWayne State University College of technologyAbstract iodin-dimensional manufacturing has numerous finishs and is gaining interest in the bio checkup knowledge domain. The character reference of additively manufactured parts is constantly improving, which contri notwithstandinges to their increased practise for medical checkup exam ingrafts in unhurrieds. This paper surveils the literature on surgical additive manufacturing exercises utilize on patients, with a focus on the usanceization of 3D printed implants and the dexterity to incorporate scaffolds on the implant bulge. Scholarly literature databases were employ 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 better medical out fill in, represent efficientness, and trim surgery snip, as well as customization and integrate scaffold. Overall, the approximately effective type of additive manufacturing for the medical implant application is negatron pass around melting employ Ti-6Al-4V because it bottomland produce a high quality, high virtuousness biocompatible implant that has the required mechanical properties.Keywords one-dimensional Manufacturing, Customized implants, Scaffold, 3D Printing, Ti-6Al-4VIntroductionIn recent geezerhood, additive manufacturing technologies live with improved signifi arsetly, so expanding the handles and applications for which they rout out be employ. These 3D opinion technologies urinate physical gravels from digital models without the need for bill and die and process planning. linear manufacturing behind fabricate prototypes of mixed shapes in a variety of textiles much(prenominal) as metals, polymers, and nylon. Metal components, in particular, loafer be utilize for realistic applications such as medical implants devices manufactured to replace or abide a biological structure. The biocompatibility of these metallic devices must be con boldnessred, creating rigorous requirements for the material selection and final material properties of the structure. Studies pass shown that additive manufacturing boffoly produces implants with biocompatible materials that take on the structural requirements 1-6.3D effect medical implants back tooth provide much usefulnesss such as the customization and personalization of the implants, cost-effectiveness, increased convergenceivity, and the ability to incorporate scaffold. Using custom made implants, fixtures and surgical tools can help decrease surgery clipping and patient recovery time, eyepatch increasing the likelihood of a successful surgery 7. An different benefit is the cost efficiency of 3D printing medial implants. Traditional manufacturing methods be cheaper for large quantities, but ar more expensive for personal ized designs and small merchandise runs 8, 9. 3D printing is oddly cost effective for small-sized implants like spinal or dental implants. 3D printing is also faster than traditional manufacturing if a custom implant unavoidably to be made traditional methods require milling, forging, and a recollective delivery time while 3D printing may provided take about a day 1. An other notable benefit of additive manufacturing is the ability to sh be 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 just about significant benefits for the biomedical industry, however, atomic piece 18 the ability to manufacture biocompatible materials, customize implants, and incorporate a porous scaffold come near.Types of analog ManufacturingThe additive manufacturing approach uses data processor software to s lice a complex 3D model into social classs of 2D cross-sections with a minute thickness. The layers are hence printed layer by layer depending on the particular method elect for the application. There are dozens of types of additive manufacturing systems on the market, fewwhat of the roughly common being stereolithography (SLA), direct metal optical maser sintering (DMLS), selective laser sintering (sodium lauryl sulphate), selective laser melting (SLM), 3D printing (3DP), and electron glint melting (EBM). These systems are classified advertisement according to the form of the raw material, which can be pulverize, liquid, or solid form 8. The two types of additive manufacturing that are most usually utilise for medical implants are SLS and EBM. billet 1 Process chain for SLM and EBM. The pre-processing before manufacturing includes 3D modeling, file preparation, and disappearance of the 3D model into layers. Post-processing may include heat treatment and embellish of fabricated parts 10.Selective Laser SinteringAn SLS printer uses a powder form of material for printing tendencys. 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 take a crap political syllabus is lowered by the de gracefuld layer thickness and another layer of powder is rol direct across 10. The process repeats, fusing from each one layer one at a time to form the object. SLS can be use with metal, ceramic, and plastic powders. The precision of the laser and the diameter of the powder determines the detail of detail of the final object, so it is possible to give 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 apply to mold the laser shot movement) and the mechanical system (movable strain platform and powder roller) 10.Electron air out MeltingAn EBM pri nter 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 betwixt a tungsten filament inside of the electron gun and the produce 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 extensive detail (70-100m) 1.Figure 3 Schematic of EBM system. The key components of EBM include an electron beam system (electron gun assembly, electron beam focusing genus Lens and deflection coils used to control t he electron beam) and the mechanical system (movable powder rake and fixed powder cassettes) 10. infixeds of Medical ImplantsThe most common metals used for surgical implants are stainless steel 316L (ASTM F138), Cobalt tack profanes (ASTM F75 and ASTM F799) and atomic identification number 22 alloy Ti-6Al-4V (ASTM F67 and F136) 12, 13. However, these metals buzz off disadvantages such as the potential release of abruptlyly ions and particles collectable to corrosion that cause inflammation and allergic reactions, affecting biocompatibility 14. Also, the materials that cast an elastic modulus that is not similar to natural bone stimulate current bone goth 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 exemption to corrosion compared to stainless steels and cobalt-based alloys 15. linear 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.MaterialYoungs modulus (GPa)Ultimate tensile strength (MPa)Yield strength (MPa)Elongation (%)TiTa75.77 4.04924.64 9.06882.77 19.6011.72 1.13Ti6Al4V131.51 16.401165.69 107.251055.59 63.636.10 2.57cpTi111.59 2.65703.05 16.22619.57 20.255.19 0.32 put over 1 Tensile properties of SLS produced TiTa, Ti6Al4V and commercially pure titanium samples (n = 5) 16.Customized ImplantsAdditive manufacturing allows for the design and fabrication of customized prosthetic implants that are created to meet the specific inescapably 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 pat tern 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 trouble 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 re nominateion. Craniofacial abnormalities are a assorted group of congenital defects that affect a large number of tidy sum and can be acquired at birth or due to injuries or tumors 8. Standard cranial implants rarely fit a patient precisely because skulls ingest 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 flip find a greater improvement in neurologic functions than after similar surgeries using traditionally manufactured implants 17, 18.Figure 4 Skull model and customized implant for craniofacial reconstruction surgery 8.ScaffoldAdditive manufacturing medical implants allows the porosity of the surface to be designed, controlled, and interconnected, which provides better bone bring forthth into implants, thus diminish the chances of the bole rejecting the implant. Additionally, the rough surface quality of 3D printed implants enhances bone-implant infantile fixation 1. Without scaffold, thither is a risk of bone weakening and bone liberation around the implant, which is a consequence of stress shielding due to high stiffness of material s 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 take over stochastic variations in size and shape, while the pores in non-stochastic structures involve 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 aim 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 buildingporous surface 10.platform, (b) post-processed femoral stem 5.ConclusionThere are galore(postnominal) 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 lance Melting because it can produce a high quality, high pureness biocompatible implant that has the required m echanical 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 confrontation. 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.References1.Petrovic, V., et al., Additive manufacturing solutions for improved medical implants. 2012 INTECH Open Access Publisher.2.Ahn, Y.K., et al., Mechanical and microstructural characteristics of commercial purity titanium implants fabricated by electron-beam additive manufacturing. Materials Letters, 2017. 187 p. 64-67.3.Yan, L.M., et al., Improved mechanical properties of the freshly Ti-15Ta-xZr alloys fabricated by selective laser melting for biomedical application. journal of each(prenominal)oys and Compounds, 2016. 688 p. 156-162.4.Caldarise, S., Hip joint prostheses and methods fo r manufacturing the same. 1996, Google Patents.5.Simoneau, C., et al., Development of a porous metallic femoral stem Design, manufacturing, simulation and mechanical testing. Materials Design, 2017. 114 p. 546-556.6.Wauthle, R., et al., Additively manufactured porous tantalum implants. Acta Biomaterialia, 2015. 14 p. 217-225.7.Ventola, C.L., Medical Applications for 3D Printing Current and Projected Uses. Pharmacy and Therapeutics, 2014. 39(10) p. 704-711.8.Jardini, A.L., et al., cranial reconstruction 3D biomodel and custom-built implant created using additive manufacturing. daybook of Cranio-Maxillofacial Surgery, 2014. 42(8) p. 1877-1884.9.DUrso, P.S., et al., Custom cranioplasty using stereolithography and a countersignlic. British diary of Plastic Surgery, 2000. 53(3) p. 200-204.10.Sing, S.L., et al., Laser and electronbeam powderbed additive manufacturing of metallic implants A review on processes, materials and designs. Journal of Orthopaedic Research, 2016. 34(3) p. 369-3 85.11.Hoy, M.B., 3D printing making things at the library. Med Ref Serv Q, 2013. 32(1) p. 94-9.12.Kokubo, T., et al., Bioactive metals preparation and properties. J Mater Sci Mater Med, 2004. 15(2) p. 99-107.13.Staiger, M.P., et al., Magnesium and its alloys as orthopedic biomaterials A review. Biomaterials, 2006. 27(9) p. 1728-1734.14.Polo-Corrales, L., M. Latorre-Esteves, and J.E. Ramirez-Vick, Scaffold Design for Bone Regeneration. Journal of nanoscience and nanotechnology, 2014. 14(1) p. 15-56.15.Dinda, G.P., L. Song, and J. Mazumder, lying of Ti-6Al-4V Scaffolds by Direct Metal Deposition. Metallurgical and Materials Transactions a-Physical Metallurgy and Materials Science, 2008. 39A(12) p. 2914-2922.16.Sing, S.L., W.Y. Yeong, and F.E. Wiria, Selective laser melting of titanium alloy with 50 wt% tantalum Microstructure and mechanical properties. Journal of Alloys and Compounds, 2016. 660 p. 461-470.17.Rotaru, H., et al., Cranioplasty With Custom-Made Implants Analyzing the Case s of 10 Patients. Journal of Oral and Maxillofacial Surgery, 2012. 70(2) p. e169-e176.18.Agner, C., M. Dujovny, and M. Gaviria, Neurocognitive Assessment in front and after Cranioplasty. Acta Neurochirurgica, 2002. 144(10) p. 1033-1040.19.Shah, F.A., et al., Long-term osseo consolidation 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 home ground group B Thuringiensis Distribution and HabitatLITERATURE REVIEWFor several decades since its discovery, formulations of Bacillus thuringiensis (B. t.) arrest been seen as the ideal means of controlling Lepidoteran pests in agriculture because of the more attributes that differentiate this microbial wormicide from the synthetic chemical formulations. No perniciousness to mammals, environsal friendliness, apparent immunity to the pesticide enemy phenomenon (no longer true), good integration wit h 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 biting louseicidal properties that affects a specific range of insect orders. There are at least 34 airstream ofB. thuringiensis (also called serotypes or 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 employment more than 130 species of lepidopteran, dipteran and coleopteran insects are put together to be controlled byB. thuringiensis (Dean, 1984).Historical Background of B. thuringiensisB. thuringiensis are interesting and strategic bacter ia used in the biological control of insect pest which form hepatotoxicant watch glass proteins at the time of sporulation. Perhaps the most well cognise 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 insulate 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 utilize to control the European corn borer (Lords, 2005). Sporine which was the first comm ercial product of B. thuringiensis was getable 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 struggle II (Nester et al., 2002). Formation of transgenic plant was also observed. The first reports of insertion of genes encode 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 peaceful 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, a nd it can account for 20 to 30% of the dry weight of the sporulated cells (Schnepf et al., 1998)Distribution Habitat of B. thuringiensisThis bacterium is distributed worldwide (Martin Travers, 1989). The soil has been described as its main habitat however it has also been unaffectionate 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, dislocated 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 fr om 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 office of samples that bearedB. 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 concomitant insecticidal action (Martin Travers 1989). Evidence to support this view is that B. thuringiensis are commonly inform in the environment independent of insects and there is a lack of connexion between occurrence and insect activity (van Frankenhuyzen 1993). Meadows ( 1993) suggested four-spot possible explanations for the comportment 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. NumerousB. thuringiensis race have been recovered from coniferous trees, broad-leaved 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 merchantman 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 state-supported water distribution systems after an aerial application of Thuricide 16B (Ohana, 1987).Crystal root and MorphologyThe exi stence 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 slipstream can compound 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 Hfte Whiteley, 1989). A partial correlation between crystal morphology, ICP composition, and bioactivity against target insects has been established (Bulla et al., 1977 Hfte Whiteley, 1989 Lynch Baumann, 1985).smorgasbord of B. thuringiensis subspeciesThe 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 order order 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. familials of ICPIn 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 (Gonzlez machinelton, 1980 Gonzlez et al., 1981). Since these initial studies, numerous ICP genes have been cloned, sequenced and used to constructB. thuringiensis strains with novel insecticidal spectra (Hfte 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) (Hfte 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 holdN-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 (Gonzlez 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 (Gonzle z Carlton, 1980 Gonzlez et al., 1981 Gonzlez 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. thuringiensisSince sporulation and germination in bacilli are dependent on the nutritional status of the organism (Hardwick Foster, 1952), a study of the nutritional requirement ofB. thuringiensis var. thuringiensis is important for delineating the control appliances 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 preoccupancy of cystine (Nickerson Bulla, 1975) or cysteine (Rajalakshmi Shethna, 1977) promotes growth, sporulation and crystal formation in . thuringiensis, while at a higher concentration of cys/cysSH, only the vegetative growth was observed, (Rajalakshmi Shethna, 1977).Classification of B. thuringiensisThe 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 pathotypesLepidopteran-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)Mod e of ActionThe ICP structure and function have been reviewed in detail by Schnepf et al., (1998). Binding of the ICP to putative receptors is a study 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 fade away 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 liner the midgut membrane and creates pores in the membrane, upsetting the guts ion balance. The insect soon lettuce move overing and starves to death (Gill et al., 1992).Target OrganismsIn 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 exh ibited by some cry genes and the inconsistencies in the original classification proposed by Hfte 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 Hfte 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 capable to the toxic activity ofB. 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 useCommercial applications of B. thuringiensis have been directed principally against lepidopteran pests of agricultural and forest clips 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 forestryCommercial use of B. thuringiensis on agricultural and forest crops dates back nearly30 years, when it became available in France (Van Frankenhuyzen, 1993). Use ofB. 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 withB. 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., 1990). A plasmid bearing an ICP gene has been transferred fr om B. thuringiensis to a non-pathogenic leaf-colonizing isolate of genus Pseudomonas fluorescens fixation of the transgenic cells produces ICP contained within a membrane which prolongs persistence (Gelernter, 1990).Applications in vector controlB. 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 PopulationsA 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, genus 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 tobacc o budworm, the Colorado potato beetle and other insect species (McGaughey, 1992)B. thuringiensiss Ecological ImpactsSome 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 unspoilt insectsMany 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 microbe were also decreased (Salama, 1991).Other insectsMany 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 collar years following spraying, and the number of caterpillars was reduced for two years (Miller, 1990).BirdsBecause many birds feed on the caterpillars and other insects affected by B. thuringiensis applications, it is not strike that impacts of B. thuringiensis spraying on birds have been documented (Swadner, 1994). In New Hampshire, when B. thuringiensis-treatment reduced caterpillar abundance, black-throate d blue warblers made fewer nesting attempts and also brought fewer caterpillars to their nestlings (Rodenhouse, 1992). do on HumansEight human volunteers ingested 1 gram of a B. thuringiensis kuehniella formulation(3 - 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 wayward health effects, and all the blood chemistry and urinalysis tests were negative (Fisher Rosner, 1959).Pivovarov et al., (1977) inform that ingestion of foods contaminated withB. thuringiensis gastroenteitis at concentrations of 105 to 109 cells/g caused nausea, vomiting, diarrhoea and tenesmus, colic-like melody in the abdomen, and fever in three of the four volunteers studied. The toxicity of the B. thuringiensis intestinal flu strain may have been due to beta-exotoxin (Ray, 1990).In a purified form, some of the proteins produced by B. thuringiensis are subacutely toxic to mammals. However, in their natural form, acute toxicity of commonly-usedB. thuringiensis varieties is limited to caterpillars, mosquito larvae, and beetle larvae (Swadner, 1994).Special Concerns about B. thuringiensis ToxicityThe early 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 acid 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 rail at to human blood cells (Meretoja, 1977).