Fabrication of dental and orthopedic implants by prototyping based bio-ceramic and bone powder

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(1)FABRIC ATION OF DENTAL AND ORTHOPEDIC IMPLANTS BY PROTOTYPING BAS ED ON BIO-CERAMIC AND BONE POWDER. LUIS CARLOS PARRA CALVACHE. Tesis presentada a la Universidad de los Andes como requisito parcial de grado del programa de pregrado en Ingeniería Mecánica. Asesor: FABIO A. ROJAS M. Dr.Eng.Mec. UNIVERS IDAD DE LOS ANDES FACULTAD DE INGENIERÍA DEPARTAMENTO DE INGENIERÍA MEC ÁNICA BOGOTÁ D.C. 20 DE ENERO DE 2009.

(2) To my parents and sisters who made this possible. To my whole family, for the great support they are in my life. To Mari, for all you did to help me without thinking in the time or the distance. To Shandis, for bringing that little cute thing to this family. To La Mona, for sharing all your sweetness with me. To my grandfathers who are in heaven now, and are taking care of us. To Fer and all the support you have given me since we met. To my friends who have always been there for me especially to “el ñañerio”.. ii.

(3) “Teachings that do not speak of pain have no meaning… ...because humankind cannot gain anything without first giving something in return” Hiromu Arakawa. iii.

(4) INDEX. 1. INTRODUCTION.........................................................................................................1 2. PROJECT DESCRIPTION ...........................................................................................2 2.1 General Objective ....................................................................................................2 2.2 Specific Objectives ..................................................................................................2 3. CONCEPTS...................................................................................................................3 3.1 Bio-ceramic..............................................................................................................3 3.1.1 Calcium Sulfate ................................................................................................3 3.2 Bone Powder............................................................................................................4 3.3 Prototyping ..............................................................................................................4 4. PREVIOUS WORK.......................................................................................................6 5. MATERIALS AND PROCESS ....................................................................................9 5.1 BONE POWDER PRODUCTION..........................................................................9 5.1.1 BONE SELECTION.........................................................................................9 5.1.2 STERILIZATION.............................................................................................9 5.1.3 MACHINING.................................................................................................10 5.1.3.1 SAFETY ..................................................................................................11 5.1.4 BONE POWDER M ORFOLOGY.................................................................11 5.2 M IXTURE.............................................................................................................13 5.3 Prototyping using a manual method ......................................................................14 5.3.1 Characteristics of the specimens.....................................................................15 5.3.2 Analysis of the prototyping using a manual method ......................................17 5.4 PROTOTYPING....................................................................................................17 5.4.1 SCHP PROTOTYPING..................................................................................17 5.4.2 CONVENTIONAL PROTOTYPING............................................................18 5.4.2.1 ZP130 CHARACTERIZATION .............................................................18 5.4.2.2 SURFACE CHARACTERISTIC............................................................19 6. CHARACTERISATION OF THE SPECIM ENS PRODUCED BY PROTOTYPING .........................................................................................................................................19 6.1.1 APPARENT POROSITY...............................................................................19 6.1.2 APPARENT POROSITY RESULTS.............................................................20 6.2.1 ISO-IT QUALITY..........................................................................................20 6.2.2 M EASUREM ENTS........................................................................................21 6.2.3 ISO- QUALITIES...........................................................................................21 6.2.4 STATISTICAL ANALYSIS ..........................................................................22 6.3.1 THREE POINT FLEXURAL TEST ..............................................................23 iv.

(5) 6.3.2 THREE POINT FLEXURAL TEST RESULTS............................................25 6.4.1 COM PRESSION TEST..................................................................................26 6.4.2 COM PRESSION TEST RESULTS ...............................................................27 6.5.1 CHRONIC CYTOTOXICITY........................................................................27 6.5.2 CHRONIC CYTOTOXICITY RESULTS .....................................................29 7 CONCLUSIONS .........................................................................................................34 8. REFERENCES ............................................................................................................35 Annex 1 M easurement data .............................................................................................38 Annex 2M ATERIAL SAFETY DATA SHEET .............................................................40. v.

(6) LIS T OF FIGURES. Figure No. 1 Z CORP prototyping machine ......................................................................5 Figure No. 2 Prototyping Process. [6]. ..............................................................................5 Figure No. 3 Tool bit angles ..............................................................................................7 Figure No. 4Bone Powder produced by Rojas [4].............................................................7 Figure No. 5 Diameters to determine the Shape Factor.....................................................8 Figure No. 6 Bovine Bone .................................................................................................9 Figure No. 7 Bone being cleaned. .....................................................................................9 Figure No. 8 Bone mounted to be machine .....................................................................10 Figure No. 9 M echanism mounted in the lathe................................................................10 Figure No. 10 M echanism mounted in the lathe depicting how the powder is collected11 Figure No. 11 (40X), Bone Powder.................................................................................12 Figure No. 12 Calcium Sulfate .......................................................................................14 Figure No. 13 M old for manual prototyping...................................................................15 Figure No. 14 Specimens made by manual prototyping..................................................15 Figure No. 15 Specimen made by manual prototyping failed in the Instron machine ....16 Figure No. 16 Z 406 prototyping the specimens .............................................................17 Figure No. 17 SCPH Screws made by 3D printing.........................................................18 Figure No. 18 ZP130 Screws made by 3D printing.........................................................18 Figure No. 19 (40X), ZP 130..........................................................................................19 Figure No. 20 (25X) ZP 130 Screw Filet. .......................................................................19 Figure No. 21 Screw (not at scale) ..................................................................................21 Figure No. 22 Digital calliper..........................................................................................21 Figure No. 23Three point flexural specimen ...................................................................23 Figure No. 24 INSTRON machine 3367 .........................................................................24 Figure No. 25 Three point flexural specimen being failed ..............................................24 Figure No. 26 Compression specimen.............................................................................26 Figure No. 27 INSTRON machine 5586 .........................................................................26 Figure No. 28 Compression specimen during compression failure test ..........................27 Figure No. 29 BioRad micro plate reader........................................................................28 Figure No. 30 BioRad micro plate reader........................................................................29 Figure No. 31 Culture 2mg/mL .......................................................................................30 Figure No. 32 Culture 1mg/mL .......................................................................................30 Figure No. 33 Culture 0.8mg/mL ....................................................................................30 Figure No. 34 Culture 0.5mg/mL ....................................................................................30 Figure No. 35 Culture 0.2mg/mL ....................................................................................31 Figure No. 36 Culture 0.1mg/mL ....................................................................................31 Figure No. 37 Culture 0.0.5mg/mL .................................................................................31 Figure No. 38 Control Culture.........................................................................................32. vi.

(7) LIS T OF TABLES. Table 1 Work parameters...................................................................................................6 Table 2 Work parameters...................................................................................................6 Table 3 Reference to produce the bone powder................................................................7 Table 4 Bone powder produced.......................................................................................13 Table 5 Specimens Properties (M anual prototyping) ......................................................15 Table 6 Specimen hardness .............................................................................................16 Table 7 Apparent porosity ...............................................................................................20 Table 8 ISO IT Qualities [7]............................................................................................20 Table 9 ISO-IT Qualities obtained ..................................................................................22 Table 10 Cylinder M ax quality........................................................................................22 Table 11 Cylinder M in quality ........................................................................................23 Table 12 Filet M ax quality ..............................................................................................23 Table 13 Filet M in quality...............................................................................................23 Table 14 ...........................................................................................................................34. vii.

(8) 1. INTRODUCTION An orthopedic or dental implant is evaluated depending on the speed of recovery of the patient and the amount of material needed for its manufacture. When the implant is placed, the recovery of the persons will take days or weeks depending on how invasive the implant is. The durations of the healing process is related to the reaction of the body to the implant, taking into account as well that sometimes the body rejects the implant when this is not compatible with the immune system of the person. Some implants that need to be in the body for a long period require a considerable volume of manufacturing material. These implants are usually made from bone, but in some cases the volume necessary to produce the implant is bigger than the volume of the bone used.. In this project, the study is focused on continuing the work done by Rojas in the last few years. Peñaloza and Rojas [1] started to develop a material based on bone powder and a bio-ceramic, which could solve the problems mentioned in the previous paragraph. The material proved to be non toxic, with properties that are good enough to believe that it can be used in medical applications. Peñaloza [1] developed a material based on Calcium Sulfate and Bone Powder, the material was named SCPH which is its acronym in Spanish (Sulfato de Calcio que aglutina Polvo de Hueso). SCPH has 80 % in weight of Calcium Sulfate and 20 % of Bone Powder. Once the material is selected, the goal is to find a process capable of reproduce any kind of geometry with a good surface quality. The process has to ensure that the properties of the material are not affected, so that, it can be used successfully in the future.. The process to be considered is called Prototyping, a method that uses 3D printing to produce complex geometries. In previous studies the material was processed by injection [1], obtaining low IT qualities in the order of 11 to 13. IT refers to International tolerance grade; the quality varies from 01 to 18, being a tolerance of 01 the best quality and 18 the worst [10]. Considering that the surface topography influences the reaction of the body to an implant, this project intends to obtain a lower IT quality accompanied by a good surface shape, which could be related to the healing process in future works. A cytotoxic analysis will be held to see the influence of the IT quality on the material. 1.

(9) 2. PROJECT D ES CRIPTION 2.1 General Objective 1. The M ain objective of this study is to produce objects capable of being used as implants. These implants could temporally replace an osseous structure lost by a patient. The implant should induce new bone growing with structural properties that allow the patient to recover lost abilities.. 2.2 S pecific Objectives The specific objectives related to the main objective are: 1. To produce orthopedic and dental implants by prototyping, using bone and bio-ceramic powders. 2. To quantify the prototypes characteristics by evaluating its bio-mechanic and cytotoxic behavior with normalized tests. 3. To make groups of prototypes to copy standardized implants found in the industry. Some prototype ISO-IT quality will be quantified (Statistically). 4. To characterize the graft in its cytotoxic behavior in order to evaluate how it works in a in-vivo environment.. 2.

(10) 3. CONCEPTS The body always has a reaction against a foreign body inside of it. It is necessary then to clarify the following concepts: • Bioinert: refers to any material that once placed within the human body has minimal interaction with its surrounding tissue. Generally a fibrous capsule forms around a bioinert implant [2] • Bioactive: refers to a material, which upon being placed within the human body interacts with the surrounding bone and in some cases, even with the soft tissue. [2] • Bioresorbable: refers to a material that upon placement within the human body starts to dissolve and is slowly replaced by advancing tissue, in this project this tissue is to be newly formed bone. [2] • Osteogenic: refers to a material that is able to form bone due to its content on living cells (osteocytes or osteoblasts). [1] • Osteoconductive: refers to a material that provides an inert scaffold upon which osseous tissue can regenerate bone, but it has no capacity to form bone [2] • Osteoinductive: refers to a material that is able to stimulate cells to undergo phenotypic conversion to osteoprogenitor cell types capable of bone formation [2]. 3.1 Bio-ceramic Ceramics have a good osteoconductive capacity additional to its good compatibility with the human body. When a bone is damaged it can be fixed with a bio-ceramic implant. The bio-ceramics are mainly used in the following cases: • Fracture • Skeletal disease (osteoporosis ) • Removal of a tumor that leaves a cavity inside the bone. 3.1.1 Calcium S ulfate SCPH is 80 % in weight of Calcium Sulfate, which is a bio-compatible material that has good osteoconductive properties. The Calcium Sulfate dissolves in a fast way 3.

(11) due to its bioresorbable characteristics. This property may lead to an unsuccessful new bone growing since the new bone does not have time to achieve a solid structure [3]. M oreover, the dissolution the graft has as consequence a loss of mechanical resistance. This fact can lead to future bone fractures.. 3.2 Bone Powder SCPH is 20 % in weight of Bone Powder. This bone powder is mainly composed of Hidroxyapatite (HA) which is a calcium carbonate. It also contains collagen and integrator proteins, which along with Hidroxyapatite are bio-compatible with good osteoinductive properties so that it creates a good fixation of the implant [3].. 3.3 Prototyping The first prototyping machine was developed by the M assachusetts Institute of Technology. The machine spread a thin layer and then applied a binder to the powder in order to produce a defined geometry. The process is explained in detail to understand the magnitude of the process, in figure 1 two different chambers are depicted, the chamber on the left has the powder that feeds the process and the chamber on the right is where the printing process takes place. Every chamber has a piston so that the chamber can move up or down depending on the movement required. It is important to note that the process can not start until the powder in both chambers is flat and uniform. To have the machine ready for printing, 500 g of powder are necessary as a minimum quantity to start the process. Once the feeding chamber has enough powder and all is set, the process can start and go through the next steps. For a better understanding see figure 2. The steps (depicted in figure 2) are: •. Step 1: The piston in the feeding chamber moves up, and the powder that is raised above the chamber is spread in to the next chamber. •. Step 2: The binder is applied into the powder in the next chamber.. •. Step 3: The chamber on the right moves down the same distance that the other was raised.. This process is repeated several times until the prototype is completed layer by layer.. 4.

(12) Figure No. 1 Z CORP prototyping machine. Figure No. 2 Prototyping Process. [6].. 5.

(13) 4. PREVIOUS WORK Rojas [4] in his previous work made a series of experiments. Every experiment has its own parameters of work. Each experiment produced a different type of bone dust. In this project the work is focused on the bone powder 91. Table 1 and 2 show the parameters needed to produce such powder.. Tool ID. Tool. Tool. description. material. Rectified. High. High. Speed. Speed. Steel. Tool bit. 12% Co. Support. γn. Side rake. [°]. κn. [°]. R. [mm]. κ in. α i n [°]. [°]. angle [°]. FP3. No need. 10. 5. 65. 0. 10. 5. Table 1 Work parameters. The angles can be seen in figure 3, where: •. γ n Side rake angle. •. α n Side clearance angle. •. κ n Side cutting edge angle. •. R Nose radius. •. κ i n End cutting edge angle. •. α i n Front clearance angle. Advance. Cut Speed. Cut Depth. (mm/rev). (m/min). (mm). 0,115. 30. 0,1 Table 2 Work parameters. 6. Cut fluid. Application of fluid. Air. Static.

(14) Figure No. 3 Tool bit angles. a) Original Photography 30X. b) Original Photography 125X. Figure No. 4Bone Powder produced by Rojas [4]. Figure 4 shows the structure of the bone powder. As it can be seen it has a geometry of high volume, elongated shapes with sharp endings. Another characterization was made and it is shown in table 3. The geometry and parameters are the goal to be obtained in this work Biggest mean. 243,84. diameter (µm) Standard deviation. 79,91. MD Shape Factor. 0,5. Standard Deviation. 0,09. SF Table 3 Reference to produce the bone powder. 7.

(15) Figure No. 5 Diameters to determine the Shape Factor. The Shape Factor is determined by tracing two circles in a dust particle, as seen in figure 5. The outer circle is the smallest circle that contains the whole particle. And the inner circle is the biggest circle that can be made inside the particle. The particle is observed in a microscope, and then a picture is taken. The image is then processed with the Software SOLID EDGE V15, to finally measure the diameters of a particle. The process has to be done several times so a mean value and a standard deviation can be made.. 8.

(16) 5. MATERIALS AND PROCESS 5.1 BONE POWDER PRODUCTION 5.1.1 BONE S ELECTION Bovine bone was selected to produce the powder; the bone has to be fresh. A fresh bone is the one obtained from a cow recently sacrificed; figure 6 shows an example of a bovine bone. The bone is cut through the black lines, and then the interior medulla is removed. The bone has to be clean with no smooth tissue at all.. Figure No. 6 Bovine Bone. 5.1.2 S TERILIZATION The cleaned bone is then submerged in Hydrogen Peroxide during two days, figure 7 shows how the bone must be during this process. Then the bone is let in salt for two weeks. Finally the bone is ready to be machined.. Figure No. 7 Bone being cleaned.. 9.

(17) 5.1.3 MACHINING At the beginning, the bone powder was obtained by the method described by Peñaloza [1]. As the bone has an irregular shape it was introduced in mint so it can have cylindrical shape to be machined, in order to allow the tool beat to do a constant cutting process, then the product of mint and bone powder was dissolved in water to separate them, and finally the bone powder was dried in a pot. But after two weeks of work it was concluded that the method was inefficient, at times the powder produced was nothing but mint, besides the process required an additional time to separate the powder from the mint, not to mention the change in color of the bone powder. The method used was changed and no mint was involved in the process. Figure 8 shows the mechanism to produce the powder; this is mounted as seen in figure 9 and 10. The capsule collects the powder as the process is taking place. Finally the bone powder is ready to be mixed with Calcium Sulfate (Jade Stone). Figure No. 8 Bone mounted to be machine. Figure No. 9 Mechanism mounted in the lathe. 10.

(18) Figure No. 10 Mechanism mounted in the lathe depicting how the powder is collected. 5.1.3.1 SAFETY The particles that compound the bone could be harmful to human health. In order to prevent diseases the following safety elements are mandatory in the manufacturing area: • Safety goggles: In order to prevent the contact of particles with the eyes, that could cause irritation • M ask: In order to prevent the inhalation of the bone particles. The personal experience of Rojas tells that the inhalation of the bone causes respiratory problems, the problems usually are manifested as the symptoms presented by a person with influenza. 5.1.4 BONE POWDER MORFOLOGY Figure 11 shows the powder observed in the microscope; this image is process in the software SOLID EDGE V15 to measure the dust particle diameters, 30 measurements were made to obtain the dust morphology. Table 4 presents the properties of the bone powder.. 11.

(19) Figure No. 11 (40X), Bone Powder. Histogram 1.Biggest Diameter Distribution. 12.

(20) Histogram 2.Smallest Diameter Distribution. Grater mean diameter (µm). 255.83. Standard deviation M D. 41.02. Shape Factor. 0,47. Standard Deviation SF. 0,09. Table 4 Bone powder produced. The histograms present the diameters distribution, none of them is close to have a normal distribution, the distributions have peaks that are not presented in normal or lognormal distributions. The histograms are closer to be a Weibull distribution.. 5.2 MIXTURE The mixture is 20% in weight bone powder, and 80 % in weight bio-ceramic. It was made just by taking the two elements and shaking them, until there was no evidence of bone particles, the process tried to make the mixture as homogenous as possible, it took approximately 5 minutes to produce a mixture.. Calcium Sulfate was observed in the microscope too, the particles are around 25 µm. The particles tend to stay together and are easily attached to any surface. Calcium Sulfate in figure 12 A in shown in particles with no other element around, meanwhile in. 13.

(21) 12 B the circle shows particles surrounding a flake of bone, showing how the bioceramic adheres perfectly to the bone powder.. A). B). Figure No. 12 Calcium Sulfate. 5.3 Prototyping using a manual method In order to understand the process that could take place during the impression, two specimens were made. The process has the next steps: 1. M anufacturing a mold 2. Spread some water in to the mold 3. Spread SCPH powder in to the mold 4. Shake the mold 5. Repeat step 3 and 4 several times 6. Wait until the specimen dries 7. Remove the specimen. Figure 13 shows the specimen inside the mold used. The mold was fabricated with a vinyl acetate sheet to reproduce a cylindrical shape. Parts of the sheet were cut and then a piece of tape was put to conserve the cylinder form. After this, some water was spread inside the cylinder, to proceed later with the addition of powder. Powder was poured into the water until there was no more water left, so the powder could not get wet. The specimen was shaken in order to have uniform layers.. 14.

(22) Figure No. 13 Mold for manual prototyping. Figure 14 has two specimens after being removed from the mold. There can be seen some layers that are different from the rest of the cylinder, but it is mostly uniform. The surface quality is better than the one obtained by Peñaloza in previous work. With the vinyl acetate mold the specimens have a shiny appearance. The mold material could be use in the future in order to obtain a better surface finish.. Figure No. 14 Specimens made by manual prototyping. 5.3.1 Characteristics of the specimens Density The density of the cylinders was obtained from the weight and measures of the specimens. Table 5 has the measurements that were made, and the resultant density.. Cylinder 1. Cylinder 2. Diameter (cm). 1.82. 1.02. Height (cm). 3.2. 3.2. Weight (g). 13.6. 4.6. Density (kg/m3). 1633. 1759. Table 5 Specimens Properties (Manual prototyping). 15.

(23) Compression resistance. Figure No. 15 Specimen made by manual prototyping failed in the Instron machine. A specimen was compressed in the laboratory of the Universidad de los Andes with the INSTRON machine, following the norm ASTM D695 with a velocity of 1.3 mm/min. The specimen was tested as shown in figure 15; the resultant hardness is 7.25 MPa, value that is close to the one obtained by Peñaloza that is around 7.5 M Pa. Hardness The hardness was taken under the norm ASTM 2240. The hardness was measured at some points of the second cylinder, the result are show on table 6.. Hardness Shores D 80 74 69 80 66 74 82 Table 6 Specimen hardness. The mean value of the Shore D Hardness is 75, that is close to the one obtained by Peñaloza that is around 70.. 16.

(24) 5.3.2 Analysis of the prototyping using a manual method • The results are promising in order to find a 3D impression method that could reproduce in some way the manufacturing process. •. The process did not affect the characteristics studied of the SCPH. •. The manual method helps to understand better the 3D impression method. •. It is important to have a powder wet enough, in order to have a binder between particles. •. The Vinyl Acetate helped to improve the surface quality of the specimens fabricated.. 5.4 PROTOTYPING. 5.4.1 SCHP PROTOTYPING Once the SCPH mixture was obtained the powder was processed with the impression machine Z 406 from Z CORP, owned by IMOCOMOM . Figure 16 shows the printing process, the areas with different color are the prototypes being formed. Figure No. 16 Z 406 prototyping the specimens. In figure 17 the screws manufactured are shown. Screws show that the process is not convenient for producing defined geometries. The specimens made by prototyping had a weak structure. The weakness of the prototypes can be observed on the constant powder that falls of the prototype surface.. 17.

(25) Figure No. 17 SCPH Screws made by 3D printing. 5.4.2 CONVENTIONAL PROTOTYPING In order to explore the prototyping technology a lot of 30 screws were manufactured by this method. Z Corp Corporation provides the powder and binder, the powder reference is ZP 130, and the binder is ZB58. Specimens are shown in figure 18 better and defined screws were made. Even when the screws were better, the geometry shown by it was not as good as it was thought.. Figure No. 18 ZP130 Screws made by 3D printing. 5.4.2.1 ZP130 CHARACTERIZATION In figure 19 it could be seen the shape of the powder used, that has a size around the 200 µm. Grain s ize that is similar to the bone powder size, and 10 times bigger than the grain of Calcium Sulfate. 18.

(26) (a). (b). (c). Figure No. 19 (40X), ZP 130. 5.4.2.2 SURFACE CHARACTERISTIC Figure 20 shows the topography of the screws made. The results show that the shape is nothing alike the file made in CAD. Although the filet is differentiated its definition is low.. Figure No. 20 (25X) ZP 130 Screw Filet.. 6. CHARACTERIS ATION OF THE S PECIMENS PRODUCED BY PROTOTYPING. 6.1.1 APPARENT POROS ITY The apparent porosity is determined with the following equation: [8] Weight of the wet specimen Weight of the dry specimen Weight of the specimen suspended in water. 19.

(27) To determine the apparent porosity, the specimen is weighted when is dried, and then is submerged in to water for an hour. The wet specimen is weighted, and then is suspended in to water and with a dynamometer to determine its weight. The number of pores inside the material the material is important. The pores allow the blood to penetrate the implant. The blood then permits the osteocytes and osteoblasts to begin the new bone structure to grow.. 6.1.2 APPARENT POROS ITY RES ULTS Table 7 shows the results obtained. The apparent porosity is around 45.36 %, this value is higher than the one obtained by Peñaloza [1] that was 12.77 %. Wd 4,8 5,7 2,6. Ww 3,6 4,4 2. Ws 6,1 7,5 3,3. AP 48 41,93 46,15. Table 7 Apparent porosity. As the AP is higher it could be thought that the production of new bone in the prototypes is better than in the specimens produced by Peñaloza [1].. 6.2.1 IS O-IT QUALITY To determine the ISO-IT quality table 8 is used. Table 8 ISO IT Q ualities [7]. 20.

(28) 6.2.2 MEAS UREMENTS The measurements were made only on the screws obtained by conventional prototyping. The screws made with SCPH did not have a defined geometry that could be analyzed. The screws had circular shape in the CAD archive, but the geometry obtained was an oval instead of a circle. Because of this fact two IT qualities are going to be obtained. With the largest measurement that can be made in the oval, and with the smallest.. Figure No. 21 Screw (not at scale). The biggest diameter of the screw on the filet zone (Cad archive) is 3.96 mm, and in the cylindrical shape is 3.4 mm; these are the zones that are going to be used to determine the ISO-IT quality.. The measurements were made with a digital calliper shown in figure 22. Figure No. 22 Digital calliper. 6.2.3 IS O- QUALITIES All the results of the measurements are presented in table 9. It can be seen that in some cases there is almost no variation on the quality. It results helpful in order to fabricate prototypes with this process.. 21.

(29) Screw 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30. Cylinder M in 14 14 14 14 14 14 14 13 14 14 13 14 14 14 14 13 14 14 13 13 14 14 14 14 14 14 14 13 14 14. Cylinder M ax 14 15 15 15 15 15 15 15 15 15 15 15 15 15 14 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15. Filet M in. Filet M ax. 7 10 11 9 10 7 7 11 9 9 9 11 9 12 12 9 11 11 9 11 9 9 13 11 11 9 9 7 O1 7. 15 15 16 15 15 15 16 15 16 15 15 15 16 15 15 16 16 15 15 15 16 15 16 15 16 16 15 15 15 15. Table 9 ISO-IT Qualities obtained. 6.2.4 S TATIS TICAL AN ALYS IS. Quality 13 14. Frequency 6 24. Table 10 Cylinder Max quality. 22.

(30) Quality 14 15. Frequency 2 28. Table 11 Cylinder Min quality. From the cylinder data, it can be noted that the best quality that can be obtained is 13. In some cases the quality is worst that the obtained by Peñaloza [1], that was between 11 and 13. Quality O1 7 9 10 11 12 13. Frequency 1 5 11 2 8 2 1. Table 12 Filet Max quality. From the smallest measurement of the cylinder there is a range that shows better qualities than the ones obtained before [1], but this is not the quality of the totally of the oval. In the other hand we have table 13, shows qualities lower than 13. Quality 15 16. Frequency 20 10. Table 13 Filet Min quality. 6.3.1 THREE POINT FLEXURAL TES T The test was held following the norm ASTM D790 “Standard Test M ethod for Flexural Properties of Reinforced Plastics and Insulating M aterials”. This norm requires rectangular specimens as the one shown in figure 23.. Figure No. 23Three point flex ural specimen. The machine use for this test is the INSTRON 3367, that belong to the Department of M echanical Engineer of Universidad de los Andes. The machine could be seen in figure 23.

(31) 24. The velocity used for this test was 0.4mm/min. The specimen was mounted as shown in figure 25 where the distance between points is 40 mm. Figure No. 24 INSTRON machine 3367. Figure No. 25 Three point flexural specimen being failed. 24.

(32) The stress, the strain and modulus of elasticity are calculated as follows:. [8] [8] [8]. Where: = Stress in outer fibers at midpoint (MPa) = Strain in the outer surface (%) = Flexural M odulus of elasticity (MPa) P = Load at a given point on the load deflection curve (N) L = Distance between point (mm) b = Width of the specimen (mm) d = Depth of the specimen (mm) D = M aximum deflection of the center of the beam (mm) m = Slope of the tangent to the initial straight-line portion of the load deflection curve (N/mm) 6.3.2 THREE POINT FLEXURAL TES T RES ULTS The results of the test are show in the next graphic; the Stress is around 1.5 MPa, and. 802 M Pa. Graphic 1 25.

(33) 6.4.1 COMPRES S ION TES T The test was held under the norm ASTM D695 “Standard Test M ethod for Compressive Properties of Rigid Plastics”. This norm requires cylindrical specimens as the ones shown in figure 25.. Figure No. 26 Compression specimen. The machine use for this test is the INSTRON 5586, which belongs to the Department of M echanical Engineer of Universidad de los Andes. The machine can be seen in figure 27. The velocity used for this test was 1.3mm/min. The specimen was mounted as shown in figure 28. Figure No. 27 INSTRON machine 5586. 26.

(34) Figure No. 28 Compression specimen during compression failure test. 6.4.2 COMPRES S ION TES T RES ULTS The results of the test are show in the next graphic; the compression resistance is close to the 3 M Pa. Graphic 2. 6.5.1 CHRONIC CYTOTOXICITY Cytotoxicity was measured. by. a colorimetric method. using 3-[4,5-. dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (M TT) according to M osmann (1983). It was performed in Chinese hamster ovary K1 cells (CHO-K1) exposed to the different concentrations of the SCPH. The CHO-K1 cells were grown as monolayer in 27.

(35) Roswell Park M emorial Institute (RPM I) 1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (Gibco) and 2% glutamine (Gibco). First the SCPH was left in U.V. for 10 min in order to sterilize it. Later, in a 96 5 well flat bottomed plates, 3x10 cells per well were grown with eight replicate wells for. each treatment (except in column 9). After 24 h. of incubation cells were treated. The ninth column was the blank consisting of 100 µl of culture medium only. The first column was the negative control which consisted of 100 µl of culture medium. The remaining wells contained 100 µl of medium with a known concentration of the SCPH (0.05, 0.1, 0.2, 0.5, 0.8, 1 and 2 mg/ml). The plate was then incubated at 37ºC in a humidified 5% CO2 atmosphere for 48 h. After this time, M TT (5 mg/ml) was added to each well. Cells were incubated for further 4 h. Then, dimethylsulfoxide was added to dissolve Formazan crystals. After 5 min each well was analyzed in a BioRad micro plate reader at 595 nm, and a reference wavelength of 655 nm (Figure 29 and 30). Figure No. 29 BioRad micro plate reader.. 28.

(36) Figure No. 30 BioRad micro plate reader.. The results were expressed as the percentage of living cells as calculated from absorbance detected, assuming the absorbance of negative control as 100%. The same protocol was performed with the ZP 130 to evaluate cytotoxic effect at concentrations of 0.05, 0.1, 0.2, 0.5, 1 and 2 mg/ml. Statistical analysis Chronic cytotoxicity experiment was repeated two times. Two sample T test was performed to test differences between experiments, and a Pearson’s correlation test was performed to look for correlation among cell viability and the two powders concentrations with the statistical program Statistix 8.. 6.5.2 CHRONIC CYTOTOXICITY RES ULTS The results obtained for the two experiments with the SCHP and ZP 130, were very similar for each concentration used, with P values greater than 0.05 after performing a two sample T test. After 72 h exposure to the different concentrations of the SCPH (0.05, 0.1, 0.2, 0.5, 0.8, 1 and 2 mg/ml), CHO-K1 cells exhibit a dose-dependant viability decrease (Graphic 3). The Pearson`s Correlation (r = -0.4051; P < 0.01) suggest that the increase in the concentration is significantly correlated with a decrease in cell viability. All the concentrations evaluated exhibited viability above 60%,. 29.

(37) meaning that the SCPH do not has a deathly effect over this cell line after a long period of exposure at concentrations lower than 2 mg/ml.. Figure No. 31 Culture 2mg/mL. Figure No. 32 Culture 1mg/mL. Figure No. 33 Culture 0.8mg/mL. Figure No. 34 Culture 0.5mg/mL. 30.

(38) Figure No. 35 Culture 0.2mg/mL. Figure No. 36 Culture 0.1mg/mL. Figure No. 37 Culture 0.0.5mg/mL. 31.

(39) Figure No. 38 Control Culture. From figure 31 to 38 it can be seen the results of the experiments observed in the microscope.. Graphic 3 On the other hand, the CHO-K1 cell viability was negatively correlated (Pearson: r = 0.7999 and P < 0.01) with the increase in the ZP 130 concentration. In this case the lethal dose, at which the cell population is reduced in a half (LD50), could be around 6 mg/ml (Graphic 4). At concentrations higher than 0.7 mg/ml, the cell viability is drastically reduced; this is an indicator that the ZP 58 is more toxic in CHO-K1 cells than the SCPH. 32.

(40) Graphic 4. 33.

(41) 7 CONCLUS IONS Property. Compression Resistance MPa Flexion Resistance MPa. Compound Cortical 1 [25] Bone Lyophilized and irradiated [4] 125 a 175. Compound Compound 2 [5] 2 [26]. 2.7-7. 9.25-29.14. 220-340. 9 a 85. Cortical Bone [13]. BioCompound [19]. 33-193. Sponge Bone [24]. Current work. 6. 2.9-3.1. 252.2-270. Table 14. Table 14 presents some values of different studies, this helps to compare the characteristics obtained in this study. The machine that processed the material was optimized for a different powder; this machine could be manipulated in order to obtain a better quality in the tests and specimens. Or the powder could be manipulated, trying to simulate the characteristics of the powder used in 3D printing. In the same order of ideas the binder used could be replaced to obtain a better and compact structure of the specimens. The main conclusions that can be made out of this study are: •. The material has an apparent porosity that could be good for the blood to flow through the implant, and induce the growth of new bone.. •. Once again the cytotoxic behavior of the material is promising. Another method to process SCPH has to be found and evaluated in order to produce a material that could be used successfully as an implant.. 34. 1.3-1.7.

(42) 8. REFERENCES [1] J. Peñaloza. “Diseño de un material compuesto para implantes óseos”, Tesis de grado, Departamento de Ingeniería M ecánica. Universidad de los Andes, Bogotá, Colombia 2007 [2] Teoh Swee Hin. “Engineering materials for biomedical applications” Hackensack, NJ ; Singapore : World Scientific Pub., c2004 [3] Celeste Abjornson. PhD & Joseph M . Lane, MD. ´´Demineralized Bone M atrix and Synthetic Bone Graft and Bone Graft Substitutes´´. American Academy of Orthopedic Surgeons. [4] F. Rojas “Fabricaçao de implantes ortepedicos a partir da usinagem de osso humano”, Tesis doctoral, Departamento de Ingeniería M ecánica. Universidad Federal de Santa Catarina, Florianópolis, Brasil 2000 [5] J. Rodríguez. “Producción y caracterización de elementos a partir de polvo de hueso por prototipeo rápido”, Tesis de grado, Departamento de Ingeniería. M ecánica.. Universidad de los Andes, Bogotá, Colombia 2004 [6] http://web.mit.edu/tdp/www/whatis3dp.html. [7] Universidad de Oviedo, Área de Expresión Grafica en la ingeniería, material de estudio “Tolerancias Normalizadas ISO” Recursos para estudiantes, 2005. http://aegi.euitig.uniovi.es/ficheros/21_m/teo/tolerancias_dimensionales_2.pdf [8] Donald R. Askeland “Ciencia e ingeniería de los materiales “traducción, Virgilio González y Pozo y Gabriel Sánchez García. [9] M osmann, T. (1983) Rapid colorimetric assay for cellular growth and survival – application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65, 55.63. [10] Joseph E. Shigley “M echanical engineering design”. M cGraw-Hill series in mechanical engineering. 35.

(43) [11] Celeste Abjornson. PhD & Joseph M . Lane, MD. ´´Demineralized Bone M atrix and Synthetic Bone Graft and Bone Graft Substitutes´´. American Academy of Orthopedic Surgeons. [12] Buddy D. Ratner. Biomaterial Science: An introduction to materials in medicine. Amsterdam: Boston 2004: Elsevier: Academic Press 2nd Edition. [13] White, Timothy D. The human bone manual.. Amsterdam: Boston: Elsevier:. Academic 2005 [14] Seok Bong Kima, Young Jick Kima, Taek Lim Yoonb, Su A. Parka, In Hee Choa, Eun Jung Kima, In Ae Kima, Jung-Woog Shina. “The characteristics of a hydroxyapatite–chitosan–PMMA bone cement.”,2004 [15] Oguzhan Gunduz, Eray m. Erkan, Sibel Daglilar, Serdar Salman, Simeon Agathopoulos, Faik Nuzhet Oktar. “Composites of Bovine Hydroxyapatite (BHA) and ZnO”, 2007. [16] Alaadien Khalyfa, Sebastian Vogt, Jurgen Weisser, Gabriele Grimm, Annett Rechtenbach, Wolfgang M eyer, M atthias Schnalbelrauch. “Development of a new Calcium Phosphate Powder-binder System for 3D Printing of Patient Specific Implants”. 2007 [17] Sandra Quevedo, Fabio Rojas, Argemiro M ichael Sanabria “Desarrollo de una metodologia para la fabricacion de injertos compuestos de polvo de hueso y un biopolimero”. Ingenieria y Desarrollo 2006 [18] ASTM D790 “Standard Test M ethod for Flexural Properties of Reinforced Plastics and Insulating M aterials” [19] ASTM D695 “Standard Test M ethod for Compressive Properties of Rigid Plastics” [20] Celeste Abjornson, PhD. & Joseph M . Lane, M D. “Demineralized Bone M atrix and Synthetic Bone Graft Substitutes, Bone Grafts and Bone Graft Substitutes”. American Academy of Orthopaedic Surgeons.. 36.

(44) [21] M angonon, Pat L. “The principles of materials selection for engineering design” Upper Saddle River, NJ : Prentice Hall, c1999. [22] M .M. Blaschke, F. Rojas, “Semi-destructive tests for determining properties of human bone”, en Memorias 2001 I Congreso Internacional de Materiales y II Encuentro Nacional de Ciencia y Tecnologia de Materiales, Bucaramanga, Colombia [23] J. J. Rodriguez, F. Rojas “M echanical and Physical Properties of ThreeDimensional Printed Elements from Bone Powder”, en Memorias 2004 III Conferencia cientifica internacinal de Ingenieria Mecanica, COMEC, Universidad Central “Marta Abreu” de las villas Cuba [24] Y. Shikinami, M , Okuno, “Bioresorbable devices made of forged composites of hydroxiapatite (HA) particles and poly-L-lactide (PLLA): Part I. Basic characteristics” ,Biomaterials, vol 20, pp 859-877, 1999 [25] D. Reina. “Implementación de un sistema de manufactura de injerto de polvo de hueso por inyección.”, Tesis de grado, Departamento de Ingeniería. M ecánica.. Universidad de los Andes, Bogotá, Colombia 2005 [26] Sandra Quevedo, Fabio Rojas, Argemiro M ichael Sanabria “Desarrollo de una metodologia para la fabricacion de injertos compuestos de polvo de hueso y un biopolimero”. Ingenieria y Desarrollo 2006. 37.

(45) Annex 1 Measurement data. Biggest S mallest Diameter Diameter 330 105 255 105 280 180 230 130 205 80 255 130 280 130 220 55 305 180 255 105 205 105 310 155 205 95 230 105 255 105 330 155 280 105 230 105 270 130 305 130 305 140 205 105 205 130 255 140 280 80 255 105 305 155 205 105 220 120 205 115. SP 0,3182 0,4118 0,6429 0,5652 0,3902 0,5098 0,4643 0,2500 0,5902 0,4118 0,5122 0,5000 0,4634 0,4565 0,4118 0,4697 0,3750 0,4565 0,4815 0,4262 0,4590 0,5122 0,6341 0,5490 0,2857 0,4118 0,5082 0,5122 0,5455 0,5610. Table 1 Measurements to determine the dust characteristics. 38.

(46) Screw 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30. Cylinder M in 3,62 3,64 3,65 3,61 3,62 3,68 3,62 3,56 3,64 3,62 3,55 3,62 3,61 3,62 3,62 3,55 3,62 3,65 3,58 3,57 3,62 3,61 3,6 3,6 3,6 3,68 3,69 3,58 3,63 3,64. Cylinder M ax 3,87 3,97 3,94 3,88 3,97 4,02 3,94 4,03 3,99 3,92 4,05 3,96 4,03 3,92 3,87 4,04 4,01 3,96 3,96 3,99 4,11 3,85 3,98 3,9 3,98 4,02 3,98 3,91 3,92 3,98. Filet M in. Filet M ax. 3,95 4 4,01 3,99 4,02 3,97 3,95 4,01 3,94 3,94 3,93 4,03 3,99 4,04 4,07 3,98 4,02 4,03 3,93 4,01 3,98 3,94 4,09 4,01 4,03 3,99 3,98 3,95 3,96 3,95. 4,3 4,47 4,84 4,52 4,68 4,62 4,77 4,69 4,74 4,51 4,58 4,64 4,71 4,6 4,61 4,78 4,78 4,62 4,66 4,59 4,77 4,66 4,72 4,62 4,72 4,95 4,79 4,61 4,59 4,7. Table 2, Measurements to determine the ISO IT quality. 39.

(47) Annex 2MATERIAL S AFETY DATA S HEET (Revision: 08/04/2008) 1. Identification of the Substance/Preparation and of the Company/Undertaking.  Product Type: Model Stones, Plasters and Die Materials  Trade Names: Bitestone Buffstone Die Stone, Iv ory FlowStone Jade Stone Hard Rock Laboratory Plaster Handi Mix Lean Rock Iv ory Microstone Mounting Plaster Mounting Stone Prima-Rock Quickstone ResinRock Silky-Rock Snap Stone SpinBase SpinStone Super Die CAD Stone Economy Stone Flow Stone, Black Orthodontic Stone* Orthodontic Plaster*  Company: Whip Mix Corporation 361 Farmington Av enue Louisv ille, Kentucky, USA 40209 Emergency Telephone Number: (502) 634-1451 Fax Number: (502) 634-4512 Transportation Emergencies: CHEMTREC 1(800) 424-9300 (U.S. and Canada) International Calls: 1- 703-527-3887 (Collect calls accepted) * All sections apply to this product, in addition, the items identified by an * are related specifically to Orthodontic Stone and Orthodontic Plaster only. 2. Hazard Identification. These products used in dental labs should pose no potential adverse health effects.  Industrial Hygiene Air Monitoring over the past 5 years indicates no detectable respirable silica during the manufacturing process of stones, plasters or rocks.  Acute health effects involve transitory upper respiratory or eye irritation and existing upper respiratory and lung disease such as, but not limited to Bronchitis, Emphysema and Asthma. Lungs and eyes are target organs.  Chronic health effects from inhalation of crystalline silica has been classified by IARC as carcinogenic for humans (group 1). Inhalation of crystalline silica is also a known cause of Silicosis, a non cancerous lung disease caused by excessive exposure to crystalline silica 3. Composition/Information on Ingredients. Substance CAS No. EINECS Symbols Concentration, % Plaster of Paris 26499-65-0 None None 95 – 100 Crystalline Silica 148-60-7 None None <1 Titanium dioxide 13463-67-7 236-675-5 None < 3 4. First-Aid Measures.  For inhalation: Remove exposed person to fresh air, drink water to clear throat and blow nose to evacuate dust.  For eyes: Flush with large quantities of water. If irritation persist s consult a physician. 5. Fire-Fighting Measures.  Nonflammable. Use whatever measure of extinction is appropriate for surrounding fire. Water may cause product to solidify.  Will decompose above 1450 ºC to SO2 6. Accidental Release Measures.. 40.

(48)  Vacuum spilled material. Avoid creating dust. Wipe surfaces with wet cloth  Avoid washing down drains as material can plug drains 7. Handling and Storage.  Minimize dusts generation and accumulation. Avoid breathing dust. Avoid contact with eyes. Seal broken bags immediately. Continue to follow all MSDS Label warnings when handling empty containers.  Insure proper respiratory protection 8. Exposure Controls/Personal  Exposure Limits (as respirable dust). All values are mg/m 3 OSHA-PEL ACGIH-TLV, 2008 Nuisance Dust (Respirable) 5 Withdrawn Crystalline Silica (Respirable) 0.1 0.025  Personal protective equipment: None required during normal laboratory use.  Engineering controls: Use local ventilation to keep employee exposure to respirable dust below 0.025 mg/m3.  Respirator: Use respirator approved to NIOSH/MSHA half face with HEPA cartridges for exposures up to 10 times exposure limits. 9. Physical and Chemical Properties.  Solid, odorless powder, with variety of colors Vapor pressure (mmHg) Not Applicable Vapor density (air = 1) Not Applicable Melting Point ºC 145º Boiling Point ºC Not Applicable pH Not Applicable Specific gravity/density 2.5 – 3.5 Solubility in water 0.2% Flash point ºC Not Applicable  No dangerous reactions are known to occur with proper handling and storage. 10. Stability and Reactivity.  Basically stable, may solidify and generate heat if in contact with water. Will decompose above 1450 ºC 11. Toxicological Information.  Route of entry: Inhalation. Inhalation of excessive dust over a prolonged period may result in lung damage.  Effects of acute exposure: None known.  Carcinogenicity: The International Agency for Research on Cancer (IARC) reports inhaled crystalline silica is a Group 1 carcinogen to humans. NTP has listed crystalline silica as carcinogen. 12. Ecological Data.  No ecotoxicological studies are available. Generally considered chemically inert in the environment. Not dangerous to water life. 13. Disposal Considerations.  Waste is not hazardous as defined by RCRA (40CFR 261). Avoid washing down drains as material can plug drain. 14. Transport Information.  No special transport requirements, non-dangerous goods 15. Regulatory Information.  SARA III information: For purposes of SARA III reporting, these products contain no ingredients on the extremely hazardous CERCLA, or section 313 lists.  SARA Extremely Hazardous Substances 40 CFR 370: Acute  CERCLA: This product is not listed with CERCLA (40 CFR 117,302)  OSHA Hazardous Communication Standard (29 CFR 1910.1200: Contains material considered hazardous. 16. Other Information.  HMIS Rating: Health 1 Flammability 0 Reactivity 0 Other 0 Hazard: 4-Severe; 3-Serious; 2-Moderate; 1-Slight; 0-Minimum Prepared By: Donna Ringo, CIH Translated By: Date: 8/4/2008. 41.

(49) Date:CPL90533 I:\msds\modelsto.nes(rev. 0/04/2008). 42.

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