Home

Chapter 1 - Worcester Polytechnic Institute

1. Seliktar s design yielded TEBVs that exhibited improved cellular alignment and high collagen expression but experienced large decreases in volume length and wall thickness Still despite the changes in size the realignment of cells and collagen in response to mechanical conditioning yielded great increases in 23 Page mechanical integrity Samples conditioned for only four days exhibited an 87 higher yield stress and eight day samples showed a 200 higher yield stress than static samples Seliktar et al 2000 2 Kelm improves production time using microtissues In 2009 Kelm sought to build a bioreactor system that would yield healthy scaffoldless small diameter TEBVs while reducing the production time of previous designs The team used myofibroblasts and endothelial cells to construct microtissues as an initial platform for the tissue culture Kelm suggests that microtissues produce ECM quicker and more efficiently This natural tendency to produce ECM combined with pulsatile mechanical conditioning of the tissue would reduce the required maturation time of the cells and therefore expedite production of mature tissues Kelm et al 2010 Mechanical Stimulation Flow Flow Outlet Inlet Culture Media TEBVs Figure 3 A cross section of the tissue mount the Falcon tube housing unit and the path of media flow adapted from Kelm et al 2010 Kelm s bioreactor design consists of three main components a
2. amp Rolle M W 2011 Engineered Vascular Tissue Fabricated from Aggregated Smooth Muscle Cells Cells Tissues Organs doi 10 1159 000322554 Hahn M S McHale M K Wang E Schmedlen R H amp West J L 2007 Physiological Pulsatile Flow Bioreactor Conditioning of Poly ethyleneglycol Based Tissue Engineered Vascular Grafts Annals of Biomedical Engineering 35 190 Harvard Pump 22 2010 Retrieved 2 15 2010 from http www instechlabs com Pumps syringe 22 ph Huynh T N amp Tranquillo R T 2010 Fusion of Concentrically Layered Tubular Tissue Constructs Increases Burst Strength Annals of Biomedical Engineering 38 6 2226 doi 10 1007 s10439 010 0045 z Isenberg B amp Wong J 2006 Building Structure Into Engineered Tissues Materials Today 9 12 54 60 doi 10 1016 S1369 7021 06 71743 6 Isenberg B C amp Tranquillo R T 2003 Long Term Cyclic Distention Enhances the Mechanical Properties of Collagen Based Media Equivalents Annals of Biomedical Engineering 31 8 937 949 doi 10 1114 1 1590662 Kanda K Matsuda T amp Oka T 1993 Mechanical Stress Induced Cellular Orientation and Phenotypic Modulation of 3 D Cultured Smooth Muscle Cells American Society for Artificial Internal Organs 39 3 686 Kelm J M Lorber V Snedeker J G Schmidt D Broggini Tenzer A Weisstanner M Hoerstrup S P 2010 A Novel Concept for Scaffold Free Vessel Tissue Engineering Self Asse
3. 1993 and release of cellular growth factors Cheng et al 1997 Sudhir et al 2001 Liu s 1998 studv found that under circumferential strain rat blood vessel SMCs arrange themselves in the circumferential direction Kim 1999 found that cyclic mechanical strain 7 distension at 1 Hz for 4 davs applied to smooth muscle tissue grafted onto polvmeric scaffolds increased SMC proliferation and collagen and elastin expression This resulted in enhanced mechanical properties and long term tissue organization Cheng 1997 found that mechanical strain influences fibroblast growth factor 2 FGF 2 release from human SMCs As the amplitude of deformation rose more FGF 2 was released in response from 4 amplitude 90 cycles at 1 Hz releasing 0 140 190 FGF 2 to 14 90 cycles at 1 Hz releasing 5 740 5 FGF 2 In order for engineered musculoskeletal vascular and cardiac tissues to function successfully without failure in vivo mechanical modulation must be considered Ideally a combination of mechanical and biochemical factors will lead to engineered constructs whose functionality and mechanical properties closely mimic those of native tissue Perfecting mechanical conditioning techniques is of the utmost importance for cardiovascular tissue engineering before in vivo applications can be seriously considered Rubbens et al 2009 Butler et al 2009 Mechanical forces are becoming as important as growth factors or cytokines in the overall growt
4. As before the only conclusion that could be made was that the strength statistically decreased from 3 to 7 days The p values for the other groupings were too low to determine statistical differences UTS Trial 2 T EL p u 3 Day 7 Day O Static BI conditioned Figure 36 UTS second trial N values are 3 3 2 and 3 respectively In addition to UTS we also measured the mean thickness of the unconditioned and conditioned samples Displayed in Figure 37 are the results from these experiments The average thickness of static samples after 3 days in culture was 0 491 0 069 mm and the 3 day conditioned samples average thickness was measured as 0 548 0 045 mm p 0 16 therefore not statistically different After 7 days in culture the static samples were found to have an average thickness of 1 045 0 19 mm whereas the conditioned rings had a mean thickness of 1 041 0 078 mm p 0 47 therefore not statistically different All day 3 samples were compared to all day 7 samples this statistical comparison yielded a p value of 3 43 E 6 therefore confirming a statistical difference between the thicknesses of the samples 62 Page Thickness Trial 1 E E z T 3 Day 7 Day O Static BH Conditioned Figure 37 Thickness first trial N values are 2 3 2 and 4 respectively Thicknesses were also measured for the second trial Figure 38 After three days of culture the average thicknesses for
5. Crimp push in wire connector pieces to the ends of these lengths of wire and fit into the same plastic alignment case ensuring wires of the same color are all secured on the same side see Figure below 9 Cutand strip a 60 cm length of wire still zip connected together and attach one end of the negative black wire and positive red wire to the power supplv and secure with a screw see orange box in Figure below 90 Page 10 Cut and strip a 5 cm long piece of negative black wire and twist together with all other free ends of negative black wires securing together with tape see Figure below 11 Attach the free end of the positive red wire to the contact block nearest the 22 mm button enclosure s exit hole see orange arrow in the Figure in step 3 12 Attach the 16 AWG power cable to the power supply see green box in Figure in step 9 and cover all wires with electrical tape 13 Strip free ends of 6 foot long wire sealed into 6ORPM 12V DC motors and crimp push in wire connectors onto lengths of wire and fit into plastic alignment case ensuring wires of the same color are all secured on the same side see Figure below 91 Page Final Product 92 Page Appendix D Building Our Device The following engineering drawings profile the geometry and dimensions of the components of the device that were machined These include the tissue chamber base motor mount and removable tray All of these components we
6. Problems arose when we attempted to secure these luers into the wall of the tissue chambers The luer pictured on the right in Figure 22 was nearly impossible to permanently mount into the tissue chamber wall 48 Page Figure 23 Threaded svringe silicone tubing connectors Due to the problems encountered with the 1 16 inner diameter barbed luers we considered alternative interlocking luers We chose to use a l 28 threaded luer to fix into the tissue chamber wall that would interface with a 1 16 inner diameter barbed luer lock tip Figure 23 These luers allowed for permanent fixture into the tissue chamber and easily interfaced with the 1 mL syringes we had been using throughout bench top testing The 1 16 inner diameter barbed luer lock tip was heat shrunk to the silicone tubing in the same manner as described in Ali 2009 The threaded luers were leakproof based on bench top testing over a period of 12 hours This was accomplished by threading the luers into the tissue chambers attaching a 1 mL syringe to the outside opening of the threaded luer and filling the chambers with water The fittings are manufactured from autoclavable plastics and come equipped with a barbed tip which aids in securing the silicone tubing onto the luer fitting and prevents fluid flow beyond the barb Leakproof testing involving manual fatigue testing of the luers under fluctuating silicone tube pressure for 20 minutes as well as 16 hours of motor cam
7. The goal of this project is to create a cartridge to house a tissue engineered blood vessel that interfaces with a pressurization system that imparts cyclic circumferential strain on the tissue during culture The cartridge should include an external medium flow loop to provide continuous nutrient supply to the tissue Ideally the cartridge should be inexpensive and easy to manufacture such that multiple cartridges can be used in a single experiment to culture batches of tissue engineered blood vessels Finally the cartridge should be interchangeable with the luminal flow cartridge under development by another MQP team and members of the Rolle Laboratory 3 2 Objectives Based upon our initial research of past MQPs and published literature we developed the following objectives 28 Page e Accurate Must produce a strain magnitude within 10 1 e High Throughput The ability to condition many rings or tubes simultaneously run multiple media conditions simultaneously and or vary culture duration Ali et al 2009 e Adjustable Able to condition TEBVs of different diameters and lengths at different frequency and strain levels e Easily Secured TEBVs should be easily secured prior to testing e Easily Removed TEBVs should be easily removed after testing e Inexpensive Both to build using cheap materials parts and to maintain using less media durable and reusable materials energy efficient less time expense e Easy
8. tube providing a sterile culture environment for tissue samples and offering a number of customizable features to the user Also as our device promotes advances in medicine and patient wellness it addresses several greater social and ethical concerns 6 1 Bioreactor design 6 1 1 Successfully achieved 10 1 distension The silicone tube was statically inflated and changes in outer diameter were measured in order to calculate the percent distension It was determined that the displacement of 75uL of water by the 1 mL syringe would yield an average distension of 9 8 0 2 6 1 2 Uniformity of distension leads to high throughput The preloaded 10 cm silicone tube was marked at every centimeter and subjected to inflation of 75uL While the tube exhibited inconsistent distension values at the proximal and distal ends 10 1 distension was consistently achieved at markers 2 8 This suggested that the tube achieves uniform distension across at least 6 cm thereby defining the length of usable tubing on which tissue rings may be loaded and conditioned In a client interview it was determined that six tissue samples could be loaded on each silicone tube Therefore the bioreactor may culture and condition a total of 30 tissue samples simultaneously 6 1 3 Customizabilitv The bioreactor is designed so the user can run up to five isolated svstems at one time Each unit has its own mechanical conditioning svstem the main components of this svstem
9. MI as needed Non autoclaved sterile materials Latex gloves as needed 10 amp 25 mL setological pipets as needed Sterile Monoject 1mL TB syringe x5 25 cm tissue culture flask x2 90mm or larger if preferred tissue culture dish x2 140mm tissue culture dish Culture media DMEM 1X with 4 5 g L glucose L glutamine amp sodium pyruvate 50 mL Complete culture media DMEM 1X with 4 5 g L glucose L glutamine amp sodium pyruvate modified with 10 FBS 1 Penicillin Streptomycin 300 mL Tools amp materials cleaned with 70 ethanol solution Pipetter Aspiration hose Base of device including motors cams syringe holders Tray of device 97 Page Incubator Set up Procedure 1 Sprav and wipe down the base component of the device and position in the incubator with the motors furthest from and parallel to the front of the incubator 2 Feed the wires through the hole at the back of the incubator 3 Arrange the switches and power supplv on top of or next to the incubator near an electrical outlet and away from any liquids 4 Connect the male and female wire connectors red wires to red wires black wires to black wires being very careful not to bend any of the pins in the process if bent motors may not turn on can be fixed by straightening or pulling on pins 5 Plug in the power supply 98 Page Ring Loading onto Silicone Tube Procedure Note All procedura
10. Polytechnic Institute Retrieved from http gordonlibrary wpi edu vwebv holdings Info bibld 678959 Buschmann M D Gluzband Y A Grodzinsky A J amp Hunziker E B 1995 Mechanical Compression Modulates Matrix Biosynthesis in Chondrycyte Agarose Culture Journal of Cellular Science 108 1497 Butler D L Goldstein S A Guldberg R E Guo X E Kamm R Laurencin C T Tranquillo R T 2009 The Impact of Biomechanics in Tissue Engineering and Regenerative Medicine Tissue Engineering Part B Reviews 15 4 477 484 doi 10 1089 ten TEB 2009 0340 Cheng G C Briggs W H Gerson D S Libby P Grodzinsky A J Gray M L amp Lee R T 1997 Mechanical Strain Tightly Controls Fibroblast Growth Factor 2 Release from Cultured Human Vascular Smooth Muscle Cells Circulation Research 80 1 28 Chiquet M Matthisson M Koch M Tannheimer M amp Chiquet Ehrismann R 1996 Regulation of Extracellular Matrix Synthesis by Mechanical Stress Biochemistry and Cell Biology 74 6 737 Dartsch P C amp Hammerle H 1986 Orientation Response of Arterial Smooth Muscle Cells to Mechanical Stimulation European Journal of Cell Biology 41 2 339 346 DeLoach S S amp Townsend R R 2008 Vascular Stiffness Its Measurement and Significance for Epidemiologic and Outcome Studies Clinical Journal of the American Society of Nephrology 3 1 184 192 doi 10 2215 CJN 03340807 77 Page Diam
11. SAUNAS GL WT Valu 211315 4 0S yua 00 X 89 0 SUINI SU0FI I5 Says JOU UBZJJ A77 dd dWEqa PULA JIWOUOHIJ BIMIBILIJA 3509 NOY mM paweygo sway uondsoq way 4 aJQEXULUS JEBU Ll4 SIM eydy uol up JSISWeIP B T aaas 1eay sjquedwosoig yO TT ui uat efl Z BS SLM SEDO tT JS23Welp Jaqno Sud UoIlIsssidwoy 55 1 05 JOA ZT YU OOO X M TTO Amy Zitt 31989 23640A M07 Il ASE3 y2e q Insejd WWF yU JOpaJ35 yd 00T dd dulqm gI 9T T qeq 01 an Jan AJEWA4 yd 00T dd dan ajew 01 JUNOLU Daul ggZ T Jan ajeway Use OQ 5T 0 2589 IC PESI fT DI 4834 Lana dd 51015 OT Jaiauipnp J3400 F T Gwe Ise BUUNI 40 OTX Uldus Tt JIIBWEIP 8 T uid JBMOP ee 9TE 2610 1 gf Jaye gi S O aloggt ET Sulqni 2UUUS ABaU Uljajo jjod JEas 23JN1SIOA 20SpId Uun ajnsojppua uoung UUUISZ 3J0H 5 Jugg Suega SUISEJUJ JO BAIS3UPV JOSAH DI gS aaisaupe Axod3 a21aa0 JEJIP3A dUTZT A JOSAH AXxod3 Apddns Jamog INdINQ alius MTZ AZT UEM SZ ixoqiear anbJoL USIH Wd 09 20 ATT 10101 Jas LEO Del Id 148451534 UOISEJQV J3BUS SUYM YIU ST X UIPIM ZT X Ysa ET JAMMHHM Dal 135 FIA LI JEJJO HEYS 3108 LULUQ 3315 S59JUIEJ CO uondussag way 108 Page
12. and the tissue chamber This validation is proof of concept suggesting that the bioreactor could be used to successfullv impart mechanical conditioning on tissue engineered ring samples 8 1 Design Features The design of a novel bioreactor which cvclicallv imparts mechanical conditioning was accomplished The preliminarv tests in conjunction with the bench top validations lead to the conclusion that a 10 cm long silicone tube sample could uniformly be distended by 10 and therefore can impose cyclic circumferential strain on tissue engineering ring constructs Compared to other devices currently available our bioreactor design is beneficial because it allows for a great deal of variability and customizable features Changes in culture conditions can be varied by altering the dimensions of the easily replaceable cam component of the mechanical stimulation system Additionally the individual tissue chambers allow several culture media conditions to be altered at once Motors run independently from one another so several static and mechanically conditioned samples can be cultured simultaneously and for different periods of time Another benefit to our device is that with the total number of tissue chambers a high throughput is achieved Our device is capable of housing up to six tissue rings per chamber and the entire system is capable of maintaining five chambers yielding in total 30 tissue rings being conditioned in vitro during a single exp
13. are the motor and cam The motors interface with a set of power switches so some motors may run at 60 RPM while others are shut off This also presents the possibilitv of conditioning individual samples in intervals e g one hour on one hour off The size of the cam mav also be changed in order to adjust the volume of medium displaced bv the svringe A smaller cam will displace a smaller flow volume and likewise a larger cam 67 Page will displace a larger volume The individual tissue chambers provide isolated culture environments for each set of tissue rings The user mav adjust the tvpe of culture media and additives in each svstem 6 1 4 Device and materials are biocompatible After 3 and 7 dav culturing periods cells remained viable and healthv without evidence of contamination This is partially due to the biocompatible and sterilizable material choices UHMWPE sterile syringes stainless steel dowels polypropylene pinch clamps Additionally the tissue chamber s loose fitting Petri dish style lids allow gas exchange and the removable tray allows the samples to be easily transported to a biosafety cabinet where cell culture media can be replenished Finally the bioreactor meets the defined size constraints and is able to fit in an incubator 6 1 5 Easy to secure and remove samples During the loading phase of cell testing 88 21 of 24 of tissue engineered blood vessel rings were successfully loaded onto the silicone tube T
14. be induced in TEBVs in appropriate culture environments The use of collagen gel based grafts has been studied as a potential means for the reduction of the dependency on SMCs The collagen gel is easier to control and improves the strength of the graft Similar to the elastin networks remodeling enzymes promote the synthesis of these networks Mauck et al 2009 11 Page In a 2009 studv which compared the effectiveness of TEBVs to that of autologous grafts of saphenous and mammary arteries cyclic strains were proven to increase the production of collagen networks which is indicative of higher tensile strength This study specifically examined burst pressure suture retention strength and compliance of mechanically conditioned TEBVs in comparison to native saphenous veins Konig et al 2009 The TEBVs were superior to the autologous grafts in burst pressure and suture retention but the compliance ratios of the TEBVs were much lower The long term study showed that the cells appeared to react to their environment and maintain mechanical homeostasis After six months of implantation the TEBV compliance ratio increased by over 100 whereas the native tissue samples remained constant This increase in compliance validates the notion that a tissue sample exposed to mechanical manipulation is capable of remodeling and improving its mechanical properties Konig et al 2009 In this instance the body functions as the ideal bioreactor condi
15. distension confirmed the ability of interlocking luer fittings to prevent fluid leakage from the system By using both male and female luer fittings we can change out overused or damaged silicone tubes for new ones without having to disassemble or replace any major components of the device The female 1 16 inner diameter barbed bayonet fittings can be permanently connected to silicone tubing autoclaved in single use packets used for experimentation and then disposed of The more easily replaceable standard parts we use in our design the better the durability and lifespan of our device before standard wear renders it non functional 6 Mechanism for attaching silicone tubing to syringe tip When preloaded with water and clamped at its free end the silicone tube slides off of the syringe tip due to pressurization from depressing the syringe plunger To attempt to correct this we considered three different approaches for sealing the silicone tubing to the syringe tip s to prevent leaking and 49 Page detachment of the tubing First a suture was tied around the silicone tube above the barb on the tip of the 1 16 female luer fitting Figure 24 This method failed Silicone glue was then applied to the tip shaft of the 1 16 female luer fitting below and above the barb The silicone tube was then forced onto the luer tip over the barb and set to dry for 48 hours A second sample using the 2mm blunt end metal svringe tips was also used Silic
16. efficacv of individual materials and interfaces between design components were then tested through a series of qualitative and quantitative bench top experiments 53 Page Chapter 5 Design Verification To validate specific features of our design we categorized the desired functions into two categories those functions to be conducted by the tissue chamber and those to be accomplished by the mechanical conditioning system The experiments used to determine the appropriate means for these features are explained in the following chapter 5 1 Syringe Selection We considered three different types of syringes that were readily available to use as a means of displacing specific volumes of water into the silicone tubing of our device to achieve 10 1 distension The three syringes we examined were sterile Monoject 3mL luer lock syringes sterile 1mL luer lock syringes and sterile Monoject 1mL push connect or luer slip syringes all pictured in Figure 27 The volume of water required to achieve 10 distension for a 10 cm long 1 9578 mm inner diameter autoclaved silicone tube was found to be approximately 0 075 mL through extensive bench top testing of multiple samples This volume was measured through observing the specific volume displaced by the 1 ml syringe that resulted in 10 distension of the outer diameter of the silicone tube The experiment was performed by filling a 1 ml syringe with water completely tapping all air bub
17. from the 0 2mL point to 0 125mL instead of from 0 3mL to 0 225mL decreasing the overall length of plunger left unsupported by the syringe barrel Due to these factors summarized in Table 4 we decided to use a 1mL syringe in our design The 4 5 mm 0 075mL on a 1mL syringe 4 5mm distance required by the 1mL syringe to inject 0 075mL of water into the silicone tubing to achieve 10 distension makes it far simpler to manufacture and operate the cam It is more difficult to move a syringe by 1 5mm 3mL syringe than by 4 5mm 1mL syringe The larger working area over which to mechanically distend the silicone tubing is preferred making it possible for us to use a larger cam if necessary The 1mL syringe also has far smaller measurable volume increments along the barrel of the syringe making it far simpler to measure small volume changes in the system Between the luer slip and luer lock 1mL syringes we decided to use a 1mL luer slip syringe as seen in Figure 28 The 1mL luer lock syringes we viewed for comparison in catalogs were bulkier at the tip than the luer slip The twist required to secure the luer lock syringe tips into place was trivial compared to a push connect tip Locking luers were an unnecessary precaution that would add an additional complication to loading and preparing silicone tube samples during procedural set up 1mL luer slip syringes are autoclavable reusable and inexpensive at roughly 17 cents per syringe Table 4 Su
18. greater the chance of leaks loose fittings and difficultv in monitoring air flow and air levels Ali et al 2009 Our second option was to use a solenoid coil actuator to displace a specific volume of water within a silicone tube to achieve 10 1 distension Solenoids work by creating a magnetic field around a solenoid coil which moves a metal armature positioned in the middle of the circular coil The armature is forced down by a spring pushing the stem and base onto a silicone tube filled with water positioned beneath the solenoid The entire system is pictured in Figure 17 The magnetic field can be controlled to push the armature at a rate of 1 Hz based on the number of loops in the solenoid coil There are several problems associated with this system namely that solenoids operate in a very abrupt manner J O Rourke personal communication October 2010 The movement of the Armature armature and flexion of the spring are not smooth in motion which could cause damage to the silicone tube and friction in the system Using a solenoid in a moist humid environment may disrupt solenoid function as well The Figure 17 Solenoid actuator Edited from affects of electromagnetic fields on TEBV growth is also tpub com unknown and could potentially be problematic Our final option is a motor pictured in Figure 18 Motors can effectively push syringe plungers when attached to a cam and or flatbar assembly One motor could
19. hopes of creating a mechanically sound replacement for human tissue These grafts however are limited to vessels with larger diameters In vessels smaller than 6 mm in inner diameter the synthetic grafts often failed due to thrombosis Atala et al 2008 Thrombosis is the formation of blood clots that occlude the vessel The synthetic materials used in the vascular replacement procedures often initiated an immune response This response would result in high levels of inflammation and failure of the graft O Donnell et al 1984 In an attempt to improve the biocompatibility of these materials scientists have researched biomaterials that when combined with autologous tissues could result in biointegration of the graft Mitchell et al 2003 The current standard for vessel replacement is the CABG or coronary artery bypass graft method The CABG procedure requires a type of graft called an autologous graft These grafts are sections of blood vessels less than 6 mm inner diameter such as the saphenous vein which are harvested from the patient The graft is then implanted into the coronary circulation system to redirect flow past the damaged or occluded vessels This approach is viable for ten to twelve years any longer could result in narrowing of the vessel resulting in a need for a second surgery L Heureux 2003 Goldman et al 2004 Tissue engineered blood vessels are a potential solution to the complications that arise from the use o
20. pulsatile pump a medium reservoir and a structural unit The pulsatile pump used is interfaced with control unit powered with a 1 bar inlet pressure it uses cell media as a flow medium The two stated objectives of the novel assembly device are to grow the microtissues into a three dimensional tubular shape and to enable circumferential mechanical stimulation This assembly device is housed in a 50 mL falcon tube with a custom made Stainless steel cap The assembly component is interfaced with the rest of the system through the steel 24 Page cap using silicon tubing Figure 3 Finallv a nutrient medium reservoir is used as a junction between the assembly device and the pump to allow recirculation of the flow medium Kelm s design demonstrates that self assembling microtissue constructs in conjunction with a pulsatile flow loop accelerates ECM production and tissue maturation as the tissues showed very high collagen expression after only 14 days This novel concept brings the industry one step closer to the quick and efficient production of autologous TEBV constructs Kelm et al 2010 3 Syedain enhances ECM composition Syedain s team has developed a bioreactor that imparts cyclic mechanical strain on tissue engineered heart valves in an attempt to optimize compositional properties Syedain s design consists of a latex tube which houses the tissue engineered heart valves The valves receive mechanical stimulation through a cell m
21. static and conditioned samples were calculated to be 0 7018 0 05 mm and 0 72 0 02 mm respectively p 0 20 therefore not statistically different After being cultured for seven days the thicknesses of the rings increased to 1 08 0 08 mm for the static samples and 1 154 0 09 mm for the conditioned samples p 0 10 therefore not statistically different All day 3 samples were compared to all day 7 samples this statistical comparison yielded a p value of 9 6 E 10 again confirming a statistical difference between the thicknesses of the samples 63 Page Thickness Trial 2 1 2 1 0 8 0 6 0 4 oi i Ou 0 2 n 3 Day 7 Day Q Static B Conditioned Figure 38 Thickness second trial N values are 5 6 6 and 5 respectively 5 6 Histological Analysis Rat aortic smooth muscle tissue rings WKY 3M 22 were harvested at 3 and 7 days Figure 39 for both sample sets Figure 39 Conditioned A and static B tissue rings on silicone tubes after 7 days of culture The tissues were sectioned mounted and stained with hematoxylin and eosin and imaged under 20x magnification Cell count and area of the tissue in square millimeters were calculated using ImageJ software Mechanically conditioned samples yielded a density of 12 28 cells mm after 3 days Figure 64 Page 40 A and 18 26 cells mm after 7 davs B Static samples vielded densities of 22 16 cells mm C and 15 87 cells mm D a
22. syringe 1 5mm of syringe plunger movement to achieve 10 distension in the attached silicone tubing The tip of the syringe is fitted with a luer lock which secures luer based syringe tips to the syringe by twisting the detachable tips onto the barrel of the syringe This ensures that the syringe tips remain attached during use and prevents the syringe tips from being pulled from the syringe when a force is applied Figure 28 1 mL push connect or luer slip syringe The 1mL luer lock syringe is identical to the 1mL push connect or luer slip syringe with the exception of the luer fitting at the tip of the syringe A side by side comparison of luer slip and luer lock syringe tips can be seen in Figure 28 One mL luer slip and luer lock syringes come pre packaged and sterilized The barrels of these syringes are marked at 0 01mL increments 0 01mL corresponding to 0 017 mm of space between graduations The overall length of the syringe from luer tip to plunger base is 91 5 mm when the syringe plunger is fully depressed and 109 5 mm when at a pre distension volume of 0 3mL Further fatigue testing indicated that the shorter the syringe plunger during mechanical conditioning with the motor cam and spring the less likely the plunger is to bend and permanently deform over a 55 Page period of 2 days constantly running within an incubator The safety factor was thus later changed to 0 125mL instead of 0 225mL so the plunger would travel
23. to Use With minimal manual input using fewer simpler steps With the completion of the literature review the team then developed a Pairwise Comparison Chart PCC which helped us determine which objectives needed to take priority when we reached the conceptual design stage Chapter 4 In total nine objectives were analyzed in order to establish a set of design priorities Additionally the PCCs were issued to our advisor and a graduate student who as actual clients of the device could further validate the design priorities Our PCC can be found in Appendix A Pairwise Comparison Chart PCC As seen in Table 2 three areas were determined to be a priority for both clients and the team accuracy media supply and waste removal Accuracy was defined as our device s ability to induce 10 1 cyclic strain As found in literature strains of this magnitude significantly increased the mechanical and structural integrity of TEBV samples Seliktar et al 2000 Both media supply and waste removal were related to our device s ability to sustain cell constructs It was considered a necessity to maintain an ample supply of nutrients for the cell samples during conditioning We all agreed that our design must provide constant nutrient supply and allow for repeated replenishment of nutrients Additionally all users felt that waste removal was equally important as to maintain cellular homeostasis These areas of interest are highlighted yellow in Tab
24. 305 doi 10 1089 ten 2006 12 3285 Fujiwara K 2003 Mechanical Stresses Keep Endothelial Cells Healthy Beneficial Effects of a Physiological Level of Cyclic Stretch on Endothelial Barrier Function American Journal of Physiology Lung Cellular and Molecular Physiology 285 4 L782 4 doi 10 1152 ajplung 00142 2003 Gassman A A Kuprys T Ucuzian A A Brey E Matsumura A Pang Y Greisler H P 2010 Three Dimensional 10 Cyclic Strain Reduces Bovine Aortic Endothelial Cell Angiogenic Sprout Length and Augments Tubulogenesis in Tubular Fibrin Hydrogels Journal of Tissue Engineering and Regenerative Medicine doi 10 1002 term 323 Gimbronejr M Andreson K amp Topper J 1999 The Critical Role of Mechanical Forces in Blood Vessel Development Physiology and Pathology Journal of Vascular Surgery 29 6 1104 1151 doi 10 1016 S0741 5214 99 70252 1 78 Page Goldman S Zadina K Ovitt T Copeland J Krasnicka B amp Anderson R 2004 Long Term Patency of Saphenous Vein and Left Internal Mammary Artery Grafts After Coronary Artery Bypass Surgery Results from a Department of Veterans Affairs Cooperative Study 44 11 2149 Guilak F Sah R L amp Setton L A 1997 Physical Regulation of Cartilage Metabolism In V C Mow amp W C Hayes Eds Basic orthopaedic biomechanics 2nd ed pp 179 Lippincott Raven Gwyther T A Hu J Z Christakis A G Skorinko J K Shaw S M Billiar K L
25. 8 Uniformi it VIrE SUL EE 59 Six tissue rings loaded onto silicone tubing sariei a a a a a 59 Tissue rings mounted on silicone tubing and loaded into the tissue chamberS 00008 60 Uitimate tensilestkenetin tirsttbial eet 61 RSC o qf hg E EE 62 MPV NCE 6 Ss TGS E led late aces a evans aati 63 Thickrmess Ssecona AN EE 64 Conditioned and static tissue rings on silicone tubes after 7 days of culture 20s ss sseeeennn 64 Tissue samples stained with hematoxylin and eosin at 20x magnification ss ss seeeennnnennnz 65 Tissue samples stained with Fast Green Picrosirius Red at 40x magnification esscr 66 Final GSSIS MCAD EE 71 SIE e DEE 72 Mechanical Stimtlatio EE 73 9 Page Chapter 1 Introduction Most current technologies used to develop small diameter tissue engineered blood vessels TEBVs do not meet the clinical needs for a mechanically sound graft without extensive time and conditioning in culture Ideally autologous tissue engineered vessels or grafts would be cultured in vitro then applied in vivo This concept has motivated researchers to study engineered vessels and is the basis for this project There are currently a variety of methods for the development of small diameter vasculature however many current technologies present a number of concerns and limitations Originally engineers developed synthetic grafts made from materials such as Dacron and polytetrafluoroethylene Zacharias et al 1987 in
26. Design of a Bioreactor to Cvclicallv Strain Tissue Engineered Blood Vessels A Major Qualifving Project Report Submitted to the Facultv of the WORCESTER POLVTECHNIC INSTITUTE In partial fulfillment of the requirements for the Degree of Bachelor of Science By Kenneth Adams Keith Bishop Elizabeth Casey Date April 28 2011 Approved 1 Mechanical Conditioning 2 Tissue Engineered Blood Vessels Prof Marsha W Rolle Major Advisor 3 Bioreactor Table of Contents MOVE CORE a A RA a 1 Oe ene a t 4 neknowiedoe et EE 5 e e EE 6 NOV OW Ee 7 Teor TEE 8 EhapterF aa tele elu eg D 10 Chopter 2 lt BEE 14 2 1 Mechanical ee elle de TE 14 2 D KT le ele e ia ai ko at E 14 2 1 2 Impact on TEBV Structure and Properties sse sse eennnnnzzunnnnzantenzzntenzzntenzznttnzattrzzantnzzanenzznnenzza 15 2 1 3 Types and Studies Demonstrating Mechanical Conditioning Techniques ss ss seeeessnsennzsnnnnni 18 2 LACON U o L A 21 42 et ege 22 2 2 1 Current bioreactor designs cccccesscccccssscccceseccceenececceesccecsusececeeeeceeseueccecsusecesseneceesaueeessuneceesenes 23 2 2 2 Areas for improvement and design opportunities ss ssesssnnnnanzsnnnenzznntnzzzatrenzssttenzzzntrezzznnnenzz 26 Chapter Project SU aC i nsdanaeestadnaguenteoanintesoere eect 28 SEN Date EUR TEE E GIN EE 28 SEENEN 28 EECH 30 3 4 Revised Client Statement 31 Bn ENNERT 32 4 1 F nctions Specifications ki on OESE E E E
27. E ENOT 33 4 2 Gonceptual RT EE 34 4 2 1 Mechanical COnGItIONING SYSTEM iii ini si a a iad a N 34 l 22 KE e E ne EE 37 AD E EENEG 39 4 3 Comparison of Design COMPONENTS semea ee teen 41 Chapter S Design Verificatio Miesen rene TE E NS 54 5 Eeer dee GE 54 5 2 Quantification ot Displa cea Volume iii a 56 5 2 L EXperimenta Procedure ee ed tere eebe ee 57 52 2 Experimental ROSUILS sisa iet aditu oni a EeE 57 DUNOMO Tubine DISTENSION sisien ed di ees tauau aaah eats 58 DS l Experimental Procedure EE 58 Die EXE MIME Mal E 59 S4 WISSUS RIG UOC IVS EE 59 Dil EXPE hime lita elei 59 SE A Experimental EE 60 5 5 Preliminary Impacts of Mechanical Conditioning sess sseennnnnnnnnzznenenzznnnnnnzzattenzzn ata nnnna ann nz nanna taa 60 5 5 l Experimental ee TEE i 60 5 5 2 EXperimental EE 61 Boistologj al A nalVSiS iii ii tatiana ta i be kt a e kan nt ut to tn an 64 Chapter Eeer ee 67 6 1 Bioreactor GESIEN EE 67 6 ECOnomic Lon le EE 68 69 Environmental impa huaa a a a f aa 69 See EECH elt 69 SAEN Ann EENEG deed ee See ed ee E ee 69 SE e Elte BC ISSUCS ii ua ke a au ta 69 637 Manuracturabiliti EE 69 SE Ee ie side a a ft ka e th b ee tti tati N 70 Chapter 7 Final Desigh tt VGA GION isib teens a A aka U FA Chapter 8 Conclusions and Recommendations 6 ena rna ntEn rna nnEna nn ant nz nanna 75 Sal DESEN Cal Ul CS ei ne i E EE 75 8 2 Future RECOMMENGALIONS EE 75 RETE rONCO ee 77 Appendix A Pairwise Comp
28. Loading Procedure Note All procedural steps take place within a sterile biosafety cabinet unless otherwise specified Whenever leaving the biosafety hood be sure to apply 70 ethanol before re entering to lessen contamination risk 1 Set up biosafety cabinet as seen in Figure below adding more tissue chambers if necessary 101 Page 2 Fill tissue culture flask with 725 mL of culture medium using pipetter 3 Remove svringe plunger place compression spring around plunger and reinsert plunger into the svringe barrel 4 Depress svringe plunger until spring is fullv compressed insert svringe into tissue culture flask and fill svringe to the 0 9 mL mark on the svringe barrel 102 Page 5 Invert the syringe and tap out any extra air bubbles that may exist within the syringe barrel 6 Depress the plunger to remove the excess air that floats to the syringe tip during tapping and place the syringe in a sterile petri dish 7 Fill the tissue chamber with 25 mL of complete culture medium with FBS and P S added 8 Using foreceps remove a tissue ring loaded silicone tube from the 140 mm culture dish and carefully place it into the tissue chamber 103 Page 9 Using foreceps grip the luer attached to the silicone tube and fit in tightly over the luer threaded into the chamber wall 10 Insert the 1 mL push connect syringe into the outer opening of the threaded luer in the tiss
29. Native 0 029 78 45 gt 97 273 31f 2 87 0 14 PP pulsed MP nonpulsed number represents weeks Data insufficient Measured from bovine muscular arteries stripped of adventitia Measured from bovine arteries by fluorometric assay 2 Other Types of Mechanical Conditioning Magnetic stress dynamic compressive loading flow mediated shear stress continuous mechanical tension and stretching on membranes are all effective methods commonly used in the tissue engineering community to alter the physical and mechanical properties of tissue engineered constructs Jon Dobson 2006 developed and prototyped a unique method to mechanically condition stem cells in vitro using biocompatible magnetic nanoparticles MNPs and ion channels present within the stem cells themselves Dobson s device applies stress to the cell membrane using forces applied directly to magnetic micro and nanoparticles attached to the cell membrane via surface receptors Quantitative results of this bioreactor system s effects on tissue mechanical properties do not yet exist but are currently being modeled in vitro Dobson et al 2006 Dynamic compressive loading is another effective means of conditioning cell constructs Ballyns 2010 investigated the effects of dynamic compression loading on engineered bovine meniscal fibrochondrocytes seeded and crosslinked in alginate hydrogels and shaped as menisci A custom bioreactor applied sinusoidal displacement in alternat
30. als of Biomedical Engineering 30 10 1221 1233 Wilson E Mai Q Sudhir K Weiss R H amp Ives H E 1993 Mechanical Strain Induces Growth of Vascular Smooth Muscle Cells via Autocrine Action of PDGF The Journal of Cell Biology 123 3 741 747 Zacharias R K Kirkman T R amp Clowes A W 1987 Mechanisms of Healing in Synthetic Grafts Journal of Vascular Surgery 6 5 429 436 82 Page Appendix A Pairwise Comparison Chart PCC Adjusta ble size Medi Accura of High i i a Waste he te 10 ring tub Through Programma SC remov E Tota Bioreactors 1 p ble Is Gef 17 j 1 OEE 2 94 Accurate 10 1 1 Adjustable size of ring tube High Throughput E easily secured 1 0 5 eea a aa Wi TEBVs 1 0 5 0 5 8 82 easily 20 S 1 1 CN removed 16 1 0 5 Media supplv Waste removal PRESSE a da o 0 00 Inexpensive 83 Page Appendix B A Guide to Heat Shrink Tubing Goals e Effectively seal silicone tubing to 1 16 inner diameter male barbed bayonet luer fittings Materials e Scissors scalpel e Heat gun preferably with low and high heat settings e Tweezers forceps e Gloves e Biocompatible heat shrinkable tube 1 8 diameter e Silicone tubing e Male barbed bayonet luer fitting for 1 16 inner diameter tubing Procedure 1 Using gloves to handle the silicone tubing cut the tubing to desired length 10cm Note For more accurate clean cuts use a scalpe
31. antouros S Flanagan T C Finocchiaro T Deichmann T Wilhelmi M Schmitz Rode T amp Jockenhoevel S 2009 Design of a Bioreactor System for the Mechanical Conditioning of Small Diameter Tissue Engineered Vascular Grafts International Journal of Artificial Organs 32 7 458 Diamantouros S E Flanagan T C Finocchiaro T Deichmann T Wilhelmi M Schmitz Rode T amp Jockenhoevel S 2009 Development of a System for the Performance and Evaluation of Mechanical Conditioning on Tissue Engineered Vascular Grafts 10 146 149 doi 10 1007 978 3 642 03900 3 43 Dobson J Cartmell S H Keramane A amp El Haj A J 2006 Principles and Design of a Novel Magnetic Force Mechanical Conditioning Bioreactor for Tissue Engineering Stem Cell Conditioning and Dynamic in Vitro Screening IEEE Tranactions on Nanobioscience 5 3 173 177 Electric Solenoid Actuators Retrieved 12 13 2010 2010 from http www tpub com content doe h1013v2 css h1013v2 166 htm Ferrechio L Lopez A Skelly J amp Thompson J 2009 Bioreactor Design for Vascular Tissue Engineering MQP Worcester MA Worcester Polytechnic Institute Retrieved from http gordonlibrary wpi edu vwebv holdings Info bibld 298751 Freed L E Guilak F Guo X E Gray M L Tranquillo R Holmes J W Vunjak Novakovic G 2006 Advanced Tools for Tissue Engineering Scaffolds Bioreactors and Signaling Tissue Engineering 12 12 3285 3
32. arison Chart PC 83 Appendix B A Guide to Heat Shrink TUBING asenna A a A 84 Appendix C A Guide to Wiring and Sealing Motors ccccccsssecccessececcesecceeeeseceseeseceeeeeceeseeecessunecesseees 86 Potne ana Sealinie ee 86 Wirine Motors to Power ele TEE 88 Appendix D Building our DeViCE EE 93 Appendix E User EE EE 97 Pr pare autoclaved ele EE 97 Non alito laved sterile Ma e CN 97 Tools amp materials cleaned with 70 ethanol solution nn nnnnnnnnnnnnnnnnnannnnn nn nanna n rama nn nn nnnznnnn na 97 incubator Set up PrOCe GUNG ee 98 Ring Loading onto Silicone Tube Procedure ise kisi taa ka ki ita bad G kg be 99 Tissue Chamber Loading toen ise a e A 101 Appendix Billion Materials iii ita ta G dk 108 3 Page Authorship The final version of this paper was written in equal parts by all group members All group members were involved in editing all sections of the paper 4 Page Acknowledgements We thank our advisor Marsha Rolle for her guidance and support throughout our project We would like to thank Lisa Wall for her continuous support in the acquisition of lab materials and equipment We gratefully acknowledge Tracy Gwyther for her guidance and aid in data collection and user feedback during advisor meetings We d like to thank Craig Jones and Jason Hu for their assistance with lab equipment and DVT respectfully Many thanks to Michael Fagan for his help machining our device and sealing the motors
33. ate 3mm inner diameter tissue engineered vascular grafts These grafts were subjected to 7 weeks of mechanical conditioning under pulsatile flow at 2 mL sec Results showed significantly higher collagen levels resulting in higher tensile strength and improved elastic moduli compared to static samples Overall TEBVs show much promise as alternatives to autografting for vascular replacements based on their burst strength suturability and mechanical similarities to native vessels 2 Drawbacks of Mechanical Conditioning Mechanical conditioning in culture is a difficult challenge Systems have been developed which apply mechanical forces via piston compression systems substrate bending hydrodynamic compression and fluid shear Dobson et al 2006 Problems exist with these systems namely long term sterility and cells receiving adequate nutrients through the thickness of the vessel In systems that use scaffolded tissue constructs the scaffold takes on a portion of the mechanical load Therefore the tissue in culture 17 Page does not receive the full impact of the mechanical conditioning and as a result the production of ECM is hindered Dobson et al 2006 Studies have shown that excessive cvclic stretch is detrimental to cells Fujiwara et al 2003 Gassman et al 2010 Fujiwara 2003 modeled this phenomenon using endothelial lung tissue Lung tissue naturally stretches by an estimated 5 during normal breathing in vivo
34. atever specifications are desired Flat arms can be as long as necessarv to accommodate for individual motor spacing although the longer the flat arm the larger the overall device The cam and flat arm design can be seen in Figure 20 Motor Syringe Flatarm Figure 20 Top view of cam and flat arm design left and side view of cam design right for motor syringe attachment Our second conceptual design was based on using just the cam without a flat arm The cam dimensions would be calculated in the same way as for the cam and flat arm design The cam would rotate depressing the plunger of the syringe with its longer side A compression spring controls the plunger s 46 Page extension as the cam rotates to its shorter side Springs will be necessarv to ensure that the svringe plunger maintains contact with the cam and that the plunger fullv extends back to its original position within the one second time frame required for running at a frequencv of 1 Hz The spring must be big enough to fit around the plunger vet not exceed the size of the plunger head in order to remain positioned correctly between the syringe barrel and plunger during conditioning By switching out the cam for a different sized one the volume of water displaced by the syringe can be catered to the user s needs The elimination of the flat arm in this design allowed for us to shorten our design considerably In theory the cam design is more compact it allows for t
35. atic inflation tests 75 ul of water resulted in 9 80 0 23 distension of a 10 cm length of 1 96 outer diameter silicone tubing Figure 30Error Reference source not found Therefore a 75 uL displacement volume was used for subsequent studies 57 Page 4 U1 LA O c 2 A CG w si 2 Q oN LP so Injected Volume uL Figure 30 Required volume to achieve 10 distension of silicone tubing n 5 9 8 0 23 Red lines represent desired max and min distension values Green line represents target value 10 distension 5 3 Uniformity of Tubing Distension The goal of this experiment was to determine how uniform the distension was along the length of the silicone tube This allowed us to visualize the amount of working space for the placement of tissue rings 5 3 1 Experimental Procedure We marked each sample with nine markings numbered 0 8 with 1 cm spacing between them Figure 31 Then similarly to the Quantification experiment we statically distended the silicone tube by injecting 75 uL and measured the induced diameter by taking an image through the Leica Software This was repeated for every reference point along the length of the tube 1 cm 1 mi luer slip syringe Luer 10 cm Pinch clamp fitting silicone tube Figure 31 A 1 mL luer slip syringe connected to a 1 16 barbed luer lock tip fixed to 10 cm silicone tube sample by heat shrink tubing The 10 cm sample is marked with a black marker seal
36. ave exhibited the potential of enhancing ECM production and structural integrity of TEBVs through mechanical conditioning It is imperative to address the shortcomings of each design in order to optimize the biological performance and economical and industrial efficiencies of these devices These issues were partially addressed by Kelm 2010 and they were able to reduce the total production time for a matured vessel the design still retained a very low throughput for the amount of work that was required to yield a desirable tissue product The only profiled bioreactor that exhibits the capability to yield a high throughput is the air pressurization design that is currently being used in the Rolle lab at WPI Even so the current device has sacrificed sterility threads warp in the autoclave It is not designed for efficient and constant media exchange and does not have the customizable features that are 26 Page needed for future studies interchangeable with a growth chamber abilitv to use different tube diameters etc The MQP team seeks to alleviate these complications bv developing and prototvping a design that allows for high throughput of isolated tissue cultures while maintaining the chemical biological and mechanical conditions that optimize cell growth and alignment and ECM production comparable to native tissue 27 Page Chapter 3 Project Strategv Our initial client statement challenged us to design a device that
37. be used to move multiple syringes at once or multiple motors can be used to move syringe plungers individually The movement of these plungers at 60 RPM results in distension of the silicone tubes at a frequency of 1 Hz When preloaded with water or media the silicone tubes can be distended by 10419 depending on the length of the flatbar or dimensions of the cam 43 Page Figure 18 12V DC 60 RPM high torque gear box electric motor We chose to use the motor as our method of mechanicallv distending the silicone tube due to its low cost see Appendix F for pricing adjustability of frequency smooth and quiet operation size 37 mm in diameter 70 mm in height and simplicitv over the other two design alternatives Using multiple motors further improves the design bv allowing for variation of separate tissue chamber conditions during experimentation By using multiple motors individual chambers can be shut off or set to run at a slower speed with the flip of a switch or the turn of a knob We found during bench top testing that our motor Error Reference source not found ran very quietly and without complications for 7 days on its own otors can be positioned in a variety of different ways to achieve the same function allowing for more design flexibility 3 Tissue chamber We narrowed our design options down immediately when deciding between using tissue chambers where the cells would be seeded on a vertical silicone tube such as in
38. bles out of the syringe barrel and attaching two interlocking male and female luers The female luer was heat shrunk to 10cm of silicone tubing see Appendix for manufacturing instructions The system was flushed with water until the plunger read 0 3mL and clamped at its distal end with a pinch clamp We used approximately 0 3 mL of water to preload the silicone tube and connectors prior to sealing the distal end of the tube and pressurizing it Due to using such a small volume of water 0 3mL 0 075mL a safety factor of 0 225mL 0 6mL we limited our syringe options to between 1 and 3 mL syringes The safety factor was used in the event that the user accidentally pushed the syringe plunger too hard and ejected too much water It also allows for a larger cam to be used in our design further on leaving room on the syringe barrel for larger volumes of water to be displaced by larger cam sizes in future studies 54 Page Figure 27 3mL luer lock syringe left Luer slip vs luer lock syringe tips right The 3mL Monoject syringes came pre packaged and sterilized The barrel of the syringe was marked at 0 1mL increments 0 1mL corresponding to 0 06 mm of space between graduations The overall length of the syringe from luer tip to plunger base is 80 mm when the syringe plunger is fully depressed and 84 5 mm when at a pre distension volume of 0 3mL the safe minimum volume used for distension studies It would require 1 5mm 0 075mL on a 3mL
39. but anything higher than 17 22 may damage the endothelial cells of the lungs The key is to condition cells at an ordinary tolerable percentage that won t over stimulate or harm them Fujiwara et al 2003 Despite these limitations mechanical conditioning remains a widely used method to prepare tissue engineered constructs for the rigorous environment of the human body as it improves ECM synthesis cellular alignment and mechanical properties 2 1 3 Types and Studies Demonstrating Mechanical Conditioning Techniques Several different mechanical conditioning techniques have been shown to alter the mechanical and physiological characteristics of tissues These techniques include magnetic stress dynamic compressive loading flow mediated shear stress continuous mechanical tension stretching on membranes and cyclic mechanical distension Different tissue types require different mechanical stimuli to produce tissues with specific characteristics For instance mechanical conditioning that focuses on applied compressive forces would enhance a tissue s compressive strength while mechanical tension or stretching would improve elasticity and compliance Cyclic mechanical distension is the type of conditioning the team chose to use for the purposes of our project 1 Cyclic Mechanical Distension A study conducted in 2000 by Seliktar cyclically strained collagen gel blood vessel constructs composed of adult rat aortic SMCs mounted on etc
40. ct The bioreactor is composed primarilv of durable plastics that are abrasion resistant and mav be sterilized in an autoclave with the only exceptions being bulk products such as pinch clamps and luer fittings The use of sterilizable reusable materials minimizes the waste production associated with the device 6 4 Societal influence According to the World Health Organization WHO 2010 heart disease is the second leading cause of death in the world killing about 2 5 million people annuallv The development of patient derived TEBV grafts could therefore have a positive impact on a tremendous number of patients worldwide 6 5 Ethical concerns The use of laboratorv animals as models for experimental purposes is a common topic of debate Tissue engineered constructs grown in vitro offer an alternative to animal models during research and development Furthermore this device and all relevant research mav lead to saving lives and improving the conditions of millions of patients 6 6 Health and safetv issues Our bioreactor facilitates research that may develop usable patient derived blood vessel grafts Self derived grafts are generally safer than synthetic grafts or donor transplants where mechanical failure infection compliance mismatches and rejection of the graft are prominent issues The production of this bioreactor and the resultant TEBVs will ultimately lead to advances in the medical industry and improved patient care 6 7 Ma
41. d Tendon Tissue Engineering Biomedical Materials 2 3 169 173 doi 10 1088 1748 6041 2 3 001 Schutte S C 2010 Cyclic Strain Improves Strength and Function of a Collagen Based Tissue Engineered Vascular Media Tissue Engineering Part A 16 10 3149 Seliktar D Black R A Vito R P amp Nerem R M 2000 Dynamic Mechanical Conditioning of Collagen Gel Blood Vessel Constructs Induces Remodeling In Vitro Annals of Biomedical Engineering 28 4 351 362 doi 10 1114 1 275 Smith D H Wolf J A amp Meaney D F 2001 A New Strategy to Produce Sustained Growth of Central Nervous System Axons Continuous Mechanical Tension Tissue Engineering 7 2 131 139 doi 10 1089 107632701300062714 Sudhir K Hashimura K Bobik A Dilley R J Jennings G L amp Little P J 2001 Mechanical Strain Stimulates a Mitogenic Response in Coronary Vascular Smooth Muscle Cells via Release of Basic Fibroblast Growth Factor American Journal of Hypertension 14 11 1128 81 Page Svedain Z H amp Tranquillo R T 2009 Controlled Cyclic Stretch Bioreactor for Tissue Engineered Heart Valves Biomaterials 30 25 4078 4084 doi 10 1016 j biomaterials 2009 04 027 The Top 10 Causes of Death 2008 Retrieved 3 1 2010 from http www who int mediacentre factsheets fs310 en index html Tower T T Neidert M R amp Tranquillo R T 2002 Fiber Alignment Imaging During Mechanical Testing of Soft Tissues Ann
42. de of the motor mount and pinch the heat shrink tubing and hysol adhesive encasing closed around the wire 86 Page 13 Let drv for 24 hours Before After 87 Page Wiring Motors to Power Supplv Materials Motors sealed into motor mount and base 5 hole 22mm button enclosure plastic Selector switch 22mm plastic x5 contact blocks Easv ID low voltage cable 24 2 AWG 0 11 W x 0 06 thick 12 VDC 50 L Wire cutters Wire connectors male and female pins x10 16 AWG power cable Procedure 1 Assemble the button enclosure as per instructions given with purchase 2 Measure cut and strip all wires should look like this when stripped the ends of four 5 cm long pieces of positive red 24 2 AWG 12 VDC wire see Figure below 3 Attach and screw the pieces of wire between the 5 selector switches see blue arrows in Figure below 88 Page 4 Measure cut and strip the ends of five pieces of positive red 24 2 AWG 12 VDC wire so all wires feed out of the same side of the 5 hole 22mm button enclosure and end at approximately the same point see Figure below lt Screw in Here 6 Strip the other end of the same lengths of wire and crimp push in wire connector pieces to the ends of each wire and fit into plastic alignment case ensuring wires of the same color are all secured on the same side 7 Cutand strip 5 lengths of negative black 24 2 AWG 12 VDC wire to equal lengths 89 Page 8
43. dure Using the experimental procedure outlined in Appendix E four tissue chambers were prepared In two of the four chambers rotating cams were turned on to induce mechanical conditioning while the other two were left off and represented static culture control samples The four chambers where then placed into the bioreactor system and run for two different time periods One set consisting of a static and conditioned chamber was allowed to culture for 3 days while the other ran for a total of 7 days To ensure that the cell constructs received proper nutrients the media DMEM 1X with 4 5 g L glucose L glutamine amp sodium pyruvate modified with 10 FBS 1 Penicillin Streptomycin 60 mL per chamber was changed every two days Following the end of the culture periods the tissue rings were harvested for uniaxial tensile testing and histology to measure ultimate tensile stress UTS thickness and cell density Thickness was measured using DVT Imaging Software while UTS was 60 Page determined through the use of an Instron machine The static samples were compared to the conditioned samples to determine the effectiveness of our device 5 5 2 Experimental Results Two individual trials were conducted each consisting of two time points 3 and 7 day in which both static control and mechanically conditioned experimental samples were examined for UTS thickness and cell density For the first trial Figure 35 the average UTS o
44. e importantly retain their functionality post autoclaving The pinch to open mechanism for this clamp is far more ergonomic than the previous clamp eliminating the need to hold the clamp steady with a second hand during opening The seal on the silicone tube is visibly much tighter than the previous clamp as well The hole located in the base of these clamps also makes it possible for us to manufacture a peg or dowel and hole to mount the clamps within the tissue chamber This would make the clamps removable and eliminate the need to permanently adhere the clamps to the chamber surface which is helpful in the event of clamp malfunction or during sterilization of individual parts The smaller ergonomic pinch clamp was chosen for our device due to size restrictions within the tissue chamber 8 Tissue chamber materials Our options for tissue chamber materials were limited by the fact that the material had to be biocompatible autoclavable and or sterilized by 70 ethanol easily machinable and relatively inexpensive We needed to choose a type of plastic for our tissue chambers base component of our design removable tray and tissue chamber lids Figure 26 Ultra high molecular weight polyethylene left and polycarbonate right We decided to use ultra high molecular weight polyethylene UHMWPE Figure 26 left for our tissue chambers due to its high impact strength and durability weather resistance humid incubator environment ope
45. e fitting has been installed into the cap The final piece of the cap system is a sterile air filter that allows for gas exchange as the tissues are being cultured and conditioned The bottom of the silicone tubing contains a 25 Page needle end cap onto which a small weight is secured This grounds the tubing and consequently the TEBVs and helps control the mechanical distension of the svstem Ali et al 2009 One unique feature of the air pressurization design in comparison with the other profiled current A 6 Distributer _ 4 VII PUSH TO CONNECT PIECE ADAPTER VIII AIR FILTER 5 Needle Valve 1 Regulator i CAP IX BARBED CONNECTOR Ill HEAT SLEEVE X MEDIA CHAMBER IV SILICONE TUBE XI VASCULAR CELL V CELL MEDIA RINGS ll NEEDLE ENDCAP VI END WEIGHT Figure 4 The Ali design uses tube as a tissue chamber and allows up to eight isolated bioreactor systems to operate at one time Ali et al 2009 technologies is the high throughput that it allows Figure 4 B The air pressurization design uses a manifold to distribute the bursts of compressed air among up to eight bioreactor cartridges This design allows for several isolated tissue cultures to be active and conditioned simultaneously Ali et al 2009 2 2 2 Areas for improvement and design opportunities Several different bioreactor designs both in the general industry and in the microcosm of the Rolle lab h
46. eas for improvement and potential design opportunities 22 Page 2 2 1 Current bioreactor designs 1 Seliktar s cyclic strain bioreactor Solenoid Flow Loop Outlet Silicone Cell Seeded Tube Scaffold Figure 2 The Seliktar team s final bioreactor design houses the tissue construct on a silicon tube as an external pneumatic control device imparts cyclic strain Two filters installed into the device allow for gas exchange adapted from Seliktar et al 2000 Seliktar recognized the importance of mechanically conditioning TEBV constructs in order to improve their ECM production and mechanical properties His team cultured adult rat aortic SMCs embedded in a collagen gel scaffold once they were mechanically stable the tissue constructs were transferred into a sterile bioreactor that interfaced with a controlled pneumatic flow loop to impart cyclic strain on the tissue samples Seliktar s design Figure 2 consisted of a bioreactor chamber that contained an inlet and outlet port to allow for the mechanical flow The team s TEBVs were installed on a flexible silicone sleeve and fastened to the ports using sterile sutures Gas exchange was made possible by two 0 2 um filters that were installed into the top wall of the chamber An external compressed air system connected to the bioreactor s inlet and outlet ports using solenoid valves and provided 5 and 10 cyclic strain to the inner silicon tubing Seliktar et al 2000
47. ed and by a pinch clamp 58 Page 5 3 2 Experimental Results Static inflation of autoclaved tubing samples n 20 suggested that 10 1 distension could achieved Five reference points points 2 6 were found to always be within 104196 distension After an injection of 75 ul of fluid all points were within 10 1 6 Error Reference source not found Figure 32 Distension 3 4 5 Length Along Tube cm Figure 32 Uniform distension along silicone tubing n 20 Red lines represent desired max and min distension values Green line represents target distension 10 5 4 Tissue Ring Loading 5 4 1 Experimental Procedure To verify the desired objectives of a high throughput and easy sample loading four tubing samples were each loaded with 4 6 TEBV rings Figure 33 and secured within our tissue chamber Figure 33 Six tissue rings loaded onto silicone tubing 59 Page The chambers were filled with culture media and placed in the tissue chamber housing unit Figure 34 For a complete procedure see Appendix E Figure 34 Tissue rings A mounted on silicone tubing B and loaded into the tissue chambers C 5 4 2 Experimental Results Tissue ring samples n 45 were successfully mounted 45 53 85 success rate onto 4 silicone tubes n 4 6 per tube After 3 and 7 day conditioning periods samples remained viable and uncontaminated 5 9 Preliminary Impacts of Mechanical Conditioning 5 5 1 Experimental Proce
48. edia flow via a syringe pump that has been custom made for this system A needle valve assists in redirecting the fluid back through the syringe Media is also supplied to the latex chamber through a perfusion loop that is driven by a MasterFlex peristaltic pump Using a second loop for media exchange allowed the mechanical frequencies and strains to be adjusted without completely altering the nutrition and oxygen supply to the tissue Syedain et al 2009 Syedain s bioreactor yielded tissues that exhibited homogeneous cellularity which proves that all of the cells were well nourished and that the dual flow loop system was successful Furthermore the conditioned heart valves expressed 86 more collagen than static control samples but still fell short by 37 in comparison to native heart valves Syedain et al 2009 4 Current device used in the Rolle lab The current technology used in the Rolle lab at WPI consists of a polystyrene BD Falcon conical tube Figure 4 A as a tissue chamber and housing unit for a flexible silicone tube on which the vascular constructs are installed The cap of the tube has been modified in order to contain a threaded barb which acts as a platform for the junction between the compressed air chamber which provides the mechanical loading and the bioreactor cartridge The cap system is also fixed with a heat sleeve and anti leak o rings in order to ensure the security of the cartridge Furthermore an air pressur
49. eplaced with liquid before attaching the syringe While these designs would have worked well in a vertical tissue chamber setting they are far more difficult to accomplish in a horizontal setting These may all seal the silicone tube effectively but there is nothing present to hold the silicone tube taut and steady relative to the base of the tissue chamber Clamping or sealing the endcap inside the tissue chamber would keep the tube taut Figure 25 Pinch clamp from World Precision Instruments left and Z Man Corp middle amp right For this reason our silicone tubing sealing mechanisms were narrowed down to various types of clamps We chose to clamp the tubing inside of the tissue chamber in order to prevent complications that could arise from running the tube outside such as media leakage due to the presence of extra outlets in the walls of the chamber Clamps effectively seal tubing without leaks and simultaneously can hold the tubing taut and off the bottom of the tissue chamber securing its position in 3D space The first type of SijPage clamp we tested was a pinch clamp from World Precision Instruments Figure 25 left This clamp was 100 leakproof based on pressurization testing involving distending silicone tubing with a syringe past 20 yet lost much of its flexibility post autoclaving This led us to a patent pending model for a pinch clamp from Z Man Corp Figure 25 middle and right These clamps are autoclavable and mor
50. er than the current design s lids which simplv rest on top of the tissue chambers We feel that this will aid in the prevention of spilling during transport from biosafetv cabinet to incubator and will further reduce the chance of contamination During the manufacturing stage we learned that the UHMWPE sheets used for the tissue chambers was a relativelv soft plastic compared to other materials available in the market For our purposes the material was sufficient however it was not the easiest to machine due its softness We recommend that alternative plastics be considered to avoid issues with milling tapping and cutting 76 Page References Ali H Breindel J Bullock S 8 Herrera J 2009 Bioreactor to Dvnamicallv Condition Tissue Engineered Vasculature MQP Worcester MA Worcester Polvtechnic Institute Retrieved from http gordonlibrary wpi edu vwebv holdings Info bibld 298689 Atala A Lanza R Nerem R amp Thomson J 2008 Principles of Regenerative Medicine Burlington Mass Elsevier Academic Press Ballyns J J amp Bonassar L J 2011 Dynamic Compressive Loading of Image Guided Tissue Engineered Meniscal Constructs Journal of Biomechanics 44 3 509 Bourne P Favreau J Mawhiney K amp Ortiz A 2010 Design and Testing of a Novel Cell Seeding and Bioreactor System for the In Vitro Growth of Scaffold Free Tissue Tubes for Tissue Engineered Blood Vessels MQP Worcester MA Worcester
51. eriment 8 2 Future Recommendations Having conducted one successful full system experiment the team has developed the following recommendations The current hose clamps being used as syringe holders are very stiff and have been shown to crack over time with repeated use We suggest that an alternative be designed or purchased Even though the hose clamps successfully support the syringe during the conditioning cycles it is very difficult to insert and 75 Page remove the svringe barrels Metal svringe holders would be an appropriate alternative to the current holders Additionallv the svringe plunger was found to be prone to deformation due to the high levels of shear stress created at the plunger and cam interface We suggest that either a metal svringe or a stiffer svringe be used Small amounts of leaking were also observed in two of the four chambers during testing It was determined that an adjustment of the threaded luer located in the wall of the tissue chamber resolved this issue However in order to avoid this from occurring during future experiments we suggest that a rubber o ring be placed around the thread of the luer Another alternative could be to permanently fix the threaded luer into the wall of the tissue chamber preventing the possibilitv of user error during assembiv Even though the team experienced no contamination during our week long experiment we suggest that tissue chamber lids be machined with overhang rath
52. erimental testing and general knowledge obtained during the course of our project 41 Page 1 Distensible tubing We were given fiftv feet of clean not sterile 1 4732 mm inner diameter x 1 9558 mm outer diameter silicone tubing from Specialtv Manufacturing Inc SMI at the start of our project We decided to build our design around this source of distensible tubing due to the Rolle Lab s experience handling this particular size and brand Silicone tubing has been shown to be an effective means of distending tissue engineered constructs Niklason et al 1999 The inner diameter of the tissue rings being used in our project 2 mm match almost preciselv with the outer diameter of the silicone tubing 1 9558 mm leaving an extra 0 04 mm to aid during tissue ring loading Loading involves delicatelv placing a tissue ring or tube around the outer circumference of a sterile distensible tube The tube is then inflated or pressurized continuously at 10 1 of its outer diameter at a frequency of 1 Hz over a 3 7 day period in our studies In the event that the tissue rings adhere to the surface of the silicone tubing during distension they can be detached simply by stretching on either end of the silicone tube M Rolle personal communication September 2010 Silicone tubing is autoclavable inexpensive at 2 cents per 10 cm length disposable can interface with syringe tips and will not leach when soaked in cellular media for long p
53. eriods of time 2 Mechanical conditioning device We considered three different device designs before reaching a final decision for a means of distending the silicone tubing First an air pressurization system designed by the Ali MQP in 2009 was considered as a means of distending silicone tubing via air flow This device used a pressure control system which provided a constant high pressure of 26 psi which obtained the 10 strain on the silicone tubing A three way solenoid valve controlled by an electrical timer switched back and forth between the system s high 26 psi and low 4 psi pressure inputs at a frequency of 1 Hz The use of air pressurization proved to have its drawbacks with the Ali MQP s system The entire system was large and complicated involving numerous connections between the air compressor pressure control devices distribution manifold and tissue chambers The pressurization system shown in Figure 5 consists of every part of the system except for the tissue chambers outlined in yellow The overall size of this system makes it incredibly difficult to run within an incubator as well as incredibly noisy While there is an option for running tubing outside of the incubator the size of the distributor labeled as 1 in Figure 5 and bulk of 42 Page the remainder of the svstem would make it difficult to arrange within and around an incubator The greater the number of connections between the svstem and the tissue chambers the
54. erstanding of our objectives and constraints we then began defining potential functions and means which could be applied to design alternatives We decided to separate the design functions into two categories functions means for the tissue chamber and functions means for the mechanical conditioning svstem Below is an itemized list of our proposed design functions Tissue Chamber Functions e Provide nutrients to cells e Remove cell waste e Allow access to and removal of tissue rings tubes e Allow gas exchange e Compatible with a pump Mechanical Stimulation System Functions e Cyclically distend tissue samples by 10 e Run during the duration of mechanical conditioning of samples e Consistent must strain at 10 1 per cycle e Adjustable pressure controls per tissue chamber e Compatible with tissue chamber In order to visualize these functions after making a list we then created a Morphological Chart Table 3 and continued to expand upon the potential means for each function 33 Page Table 3 Morphological Chart Functions Mean 1 Mean 2 Mean 3 Mean 4 Tissuechamber i Submerge in Media Provision Media Rotisserie Media Pool Porous Mandrel Cell Waste Removal Drainage Flow Loop Filtered Reservoir a Individual Tissue Tissue Ce Access Removable Lid Chambers Ee Container Gas E HEPA Filter Petri Petrilid o Exposure to Humiditv Regulation Incubator Mechanical CAM Motor Air Pressurization Pump Compatibil
55. esent in their tissue constructs as many other studies have claimed to have found Isenberg et al 2003 Niklason 1999 used pulsatile radial flow to cyclically distend silicone tubing loaded with PGA scaffolds seeded with neonatal SMCs at 2 75 Hz and 5 strain for 8 weeks Niklason argued that pulsatile flow conditions 165 beats per minute and 5 radial distension would more accurately represent conditions seen in vivo and thus produce vessels better suited for eventual in vivo use This study resulted in SMCs with an increase in modulus collagen and elastin deposition suture retention and calponin and myosin 19 Page heavv chain contractile components Vessel wall thickness was also increased with longer culture time and pulsatile culture conditions After treating the conditioned SMCs with prostaglandin Fa the structure physically contracted as a natural blood vessel should Table 1 summarizes the different characteristics measured in Niklason s study Niklason et al 1999 Table 1 Characteristics of engineered vessels in Niklason s 1999 study Vessel Wall thickness Collagen Suture SMC density type cm dry weight retention g 10 cells ml 5 P 0 019 0 006 29 6 AQ 16 0 40 0 16 n 4 n 4 n 4 n 4 P 0 001 5 NIP 0 009 t a7 1 2 t a 1 n 3 BP 0 038 0 004 SO 5 91 26 0 93 0 37 DES n 6 in 6 DEE P 0 005 P 0 005 8 NP 0 023 0 004 3 d EB 1 19 0 14 DEE n 4 n 6 0 3
56. f the previously mentioned vascular repair approaches TEBVs are constructed by assembling cells taken from the patient into tubular constructs These constructs can then be implanted into the patient and in theory should have mechanical properties similar to healthy tissue Two dimensional models such as skin grafts have been successfully created three dimensional models are currently limited due to the Samples poor structural integrity and low mechanical strength In order to be deemed successful TEBVs 10 Page should be able to withstand physiological loads from blood pressure and fluid dynamics within the body Additionally the samples must be able to resist tearing especially during suturing and handling Mitchell et al 2003 In 2003 a tissue engineering technique was patented that used a cell sheet based method that cultured fibroblasts and smooth muscle cells SMCs around a cylindrical mandrel The result was a fully autologous graft that was capable of withstanding mechanical loads greater than that of blood pressure over 2200 mm Hg and was tear resistant Since the graft was composed of living cells it was self renewing resulting in an increased healing potential Additionally the graft was completely biological and could be remodeled by the body during the healing process Remodeling is a response of the cells to the mechanical manipulations enacted by the surrounding environment L Heureux 2003 Even though the
57. f unconditioned samples after a three day culture period was 555 92 129 78 kPa whereas conditioned samples after 3 days were found to have a mean UTS of 429 60 107 33 kPa p 0 19 therefore not statistically different For the 7 day samples unconditioned rings were found to have a UTS of 82 79 13 29 kPa while the conditioned rings were measured to have a UTS of 122 42 32 96 kPa p 0 052 therefore not statistically significant The UTS for both static and conditioned ring samples were significantly stronger at 3 days compared to 7 days p lt 0 001 UTS Trial 1 T EL p R u 3 Day 7 Day O Static BH Conditioned Figure 35 Ultimate tensile strength first trial N values are 2 3 2 and 4 respectively These calculations were repeated for the second trial Figure 35 After three days of culture the static samples exhibited an average UTS of 128 23 36 8 kPa whereas the conditioned samples were found be able to withstand 160 27 31 kPa before failing p 0 18 therefore not statistically different After seven days of culture the static samples experienced a UTS of 84 19 19 4 kPa compared to the 61 Page conditioned samples which had a mean UTS of 84 9 8 9 kPa p 0 48 therefore not statistically different All day 3 samples were compared to all day 7 samples this statistical comparison yielded a p value of 0 004 therefore confirming a statistical difference between the strengths of the samples
58. for incubator use Thanks are also due to Patrick Brodeur for his assistance wiring our electrical components We would like to thank Jack Ferraro and Douglas White for use of the Goddard machine shop We would also like to thank Sakthikumar Ambady for sharing his knowledge of cell culture techniques and proper use of lab equipment Finally we would like to thank James O Rourke and David Comeau President Albright Technologies for their aid in the design process 5 Page Abstract Cvclic mechanical loading improves strength and extracellular matrix ECM svnthesis in tissue engineered blood vessels TEBV The goal of this project was to design a device to impart cvclic circumferential stretch on cell derived TEBV rings cultured on flexible silicone tubing Our device cvclicallv displaces water 75 ul volume to inflate tubing at a frequencv of 1 Hz During static inflation tests the tubing diameter increased by 10 1 6 TEBV rings were loaded onto silicone tubing and subjected to mechanical conditioning for 3 7 days Our device conditioned samples in an incubator at 10 distension at a frequency of 1 Hz Static and conditioned tissues remained viable and uncontaminated and exhibited high cell densities and increased thicknesses from 3 to 7 days 6 Page Table of Tables Table 1 Characteristics of engineered vessels in Niklason s 1999 study L eessnnnenenzennenzannnenzzsnenenzzna 20 Tabie 2 Clientana Team PEC COomparisot seines dii
59. fter 3 and 7 davs respectivelv Data is based on sample sizes of 2 for each time period and culture condition with the exception of conditioned 7 davs which had 1 sample This preliminary analysis suggests that cell density increased by 49 from three to seven days for conditioned samples while static samples exhibited a decrease of 29 cell density according to ImageJ calculations df En Gare ol di Ki yoy oe sl kee G 4 Conditioned SC d d GE y Zi Ne D i An U CH EL Static Figure 40 3 and 7 day tissue ring samples stained with hematoxylin and eosin at 20x magnification Collagen staining was also conducted using Fast Green Picrosirius Red for all tissue ring samples Figure 41 Upon review under 20x and 40x magnification little to no collagen appeared to be present In order to definitively make a conclusion the samples should be recut deeper into the tissue rings or use a biochemical assay to quantify the amount of collagen in each ring sample 65 Page Conditioned Static Figure 41 Tissue samples stained with Fast Green Picrosirius Red at 40x magnification Overall all tissue rings maintained a high cell densitv remained viable and were uncontaminated for all sample sets 66 Page Chapter 6 Discussion Each component of the final assembly was designed to meet or help meet a project objective or constraint including being easy to use and assemble achieving the desired distension along the silicone
60. h and development of TEBVs Freed et al 2006 2 1 2 Impact on TEBV Structure and Properties 1 Benefits of Mechanical Conditioning Naturally occurring vascular SMCs are not exposed directly to shear stress by normal blood flow they are primarily affected by pulsatile distension during the cardiac cycle Isenberg et al 2003 Due to this tissue engineered blood vessels are primarily conditioned based on the dominant stress encountered in vivo cyclic mechanical distension Studies have shown that cyclic mechanical distension strengthens engineered vessels Niklason et al 1999 Freed et al 2006 Isenberg et al 2006 Syedain et al 2008 Seliktar et al 2000 Huynh et al 2010 and promotes collagen production and cell alignment Niklason et al 1999 Freed et al 2006 Isenberg et al 2003 Schutte et al 2010 Seliktar et al 2000 It also has enormous beneficial effects on SMC phenotype ECM deposition growth factor release and vascular tone Isenberg et al 2003 Biomechanical factors influence tissue growth Isenberg et al 2003 Niklason et al 1999 Syedain et al 2008 development Guilak et al 1997 Niklason et al 1999 maintenance Butler et al 2009 Syedain et al 2008 degeneration Butler et al 2009 Fujiwara et al 2003 and repair Cheng et al 1997 Fujiwara et al 2003 15 Page Cells receiving mechanical stimuli will align themselves in the direction of the stretch or f
61. he motor to be inverted and anchored into the base of the device to further save space The use of less material for the base of the device saves material costs and the odds of mechanical failure are reduced by limiting the number of mechanical connections in the overall system 5 Syringe tips We considered three different syringe tips as a means of connecting our syringe to the 1 9558 mm inner diameter silicone tubing Blunt end metal syringe tips 2 mm diameter Figure 21 right were first considered due to their similarities in diameter to the silicone tubing we were using These tips effectively interface with a large variety of syringes including the 1mL luer slip and luer lock Figure 21 Pictured left to right 1 16 inner diameter female barbed luer lock tip 1 16 inner diameter male barbed bayonet tip and 2mm blunt end metal syringe tip Problems arose with this type of syringe tip when trying to build it into the wall of the tissue chamber Any tip would have to be sealed into the wall of the tissue chamber to allow for silicone tube submersion and easy connection from the sterile tissue chamber to the syringe and mechanical 47 Page conditioning svstem Mounting tubing connectors also helps keep the entire svstem in line so fluid flows in one direction out of the svringe and the tubing is held taut off the bottom of the tissue chamber to prevent tissue ring damage In the event of svringe tip or silicone tube failure
62. hed silicone sleeves coated in type collagen and chitosan The sleeves and constructs were sutured into in a sterile air tight bioreactor where media was added and 0 2 um filters served as vents for gas exchange The tissue constructs were cycled within an incubator at a frequency of 1 Hz and 10 cyclic strain for 4 and 8 days The silicone sleeve was distended using a regulated compressed air supply and a solenoid valve located outside the incubator The vessels showed increased contraction and mechanical strength when compared to statically grown control constructs The vessels conditioned for 8 days showed significant increases in ultimate tensile stress the maximum load divided by the initial cross sectional area of a sample and material modulus 18 Page stress divided bv strain Morphologicallv the mechanicallv conditioned vessels showed higher circumferential orientation of the vessel cells Seliktar et al 2000 Another studv focused on the effects of applied stretching on rabbit pulmonarv arteries Thev found that after four davs of stretching the amount of pro collagen tvpe l positive cells grew along with higher levels of protein svnthesis within the smooth muscle cells SMCs and cell replication within the construct Kolpakov et al 1995 A similar studv bv Liu in 1998 used rat blood vessels Thev discovered that continuous tensile strain on the vessels tested in 10 dav increments over a period of 30 davs resulted
63. his confirms that the 10 cm silicone tube OD 1 968 ID 1 9558 successfully houses the tissue rings and provides a simple user friendly assembly platform Furthermore the luer fittings allow the user to access the tubing and rings which facilitates the loading and unloading of culture samples into and out of the tissue chambers 6 1 6 Time and budget met The entire bioreactor was designed and manufactured within the 21 week time constraint and the team produced a working prototype for less than the 368 budget 6 2 Economic impact This bioreactor design has the potential for economic impacts in the research community and in clinical use The bioreactor was manufactured for less than 400 For comparison the Harvard 22 Syringe Pump which uses two syringes compared to five and may only contribute as a single component of other bioreactor designs is advertised as the industry standard and costs over 3000 Harvard Pump 22 2010 The production of our high throughput bioreactor would supplement the production rate of cell culture and research This may lead to earlier FDA approved TEBV grafts that could be used to treat cardiovascular disease As these types of grafts would consist of tissues that are derived from the 68 Page patient fewer instances of rejection can be expected This will ultimately result in the user enduring fewer costs associated with pharmaceuticals follow up procedures and hospital stavs 6 3 Environmental impa
64. hut using a polypropylene pinch clamp which is secured to the bottom of the chamber using a 1 8 stainless steel dowel see Section 4 2 2 3 Figure 42 Final design CAD 71 Page Figure 43 Final design The base also contains five hose clamps 5 and a milled section in which five 12V 60 RPM hobbv motors are mounted 6 Each motor contains a cam and interfaces with a reverse engineered 1 mL syringe pump 7 as described in 4 1 1 4 The design s mechanical conditioning component consists of five 12 volt 60 RPM gear box hobby motor Figure 42 amp Figure 43Error Reference source not found 8 which s fixed with a circular cam 9 As the cam rotates it depresses the plunger of the fixed 1 mL syringe 10 It has been determined that the distance the plunger needs to be depressed in order to displace enough water to provide a 10 distension in the silicone tubing is 4 5 mm Therefore the axis of rotation for the cam is offset from the center by 4 5 mm 72 Page Figure 44 Mechanical stimulation Between the plunger head and the svringe bodv is a spring 11 which allows the plunger to retract as it is in contact with the short leg of the cam This essentiallv creates a svringe pump When interfaced with the silicone tubing through the luer fittings in the new tissue chamber design the displacement of the svringe plunger will cause the silicone tubing in the tissue chamber to distend the amount of distension depends
65. in better alignment of the SMCs Liu 1998 Brett Isenberg and Robert Tranquillo 2003 studied the effects of long term cvclic distension on collagen based media equivalents denatured cross linked type collagen consisting of adult rat aortic SMCs The SMCs were grown and incubated on Teflon rod mandrels in standard cell culture medium changed once per week The tissue constructs themselves were 1 3 mm in length 0 2 0 3 mm thick and 8 mm in inner diameter The tissue constructs were conditioned on distensible latex mandrels inflated by pressurized air within an incubator The collagen based media equivalents MEs were strained incrementally to the target percentage over 3 days and remained at that frequency 0 5 Hz and strain level 12 5 for 5 weeks The tissues were tested for various mechanical properties and collagen content They found that mechanical conditioning for 2 weeks yielded little change in elastic modulus E or ultimate tensile strength UTS but conditioning for 5 weeks resulted in significant increases in both Isenberg and Tranquillo found that cell age has no influence on response to stimulation and that 2 5 and 5 strain values are best for high UTS They also found that a 10 strain value during mechanical conditioning is best for obtaining a high elastic modulus and higher elastin content within samples This study failed to confirm whether or not an increase in cell number collagen content or fibril alignment was pr
66. ination in non sterile settings Both the single unit and individual chambers would facilitate media change quite easilv with these design considerations The single unit acts as a trav 45 Page in itself while the individual compartments are housed on a removable trav allowing for all chambers to be moved at once for both designs We chose to use the individual removable chambers on a removable trav to do its greater flexibilitv for a varietv of situations that could occur in the laboratorv during testing 4 Mechanical attachment to motor We considered two different options for a means of mechanicallv pushing the svringe plunger using a motor The first conceptual design used a cam and flat arm design We would manufacture a cam of appropriate dimensions and attach and clamp it to the shaft of the motor via a D shaped hole at the center of the cam In order to achieve 10 distension the syringe plunger would have to be moved by a certain distance depending on the size of the svringe and the volume of fluid needed to distend the silicone tubing The distance that the plunger must be moved is equal to the distance between the center of the D shaped hole and the center of the hole drilled to connect the flat arm to the cam The other end of the flat arm connects to a svringe plunger the movement of which controls the distension of a silicone tube and the tissue rings encircling it Cams and flat arms can be manufactured relativelv easilv to wh
67. ing one hour increments 3 times a week for 6 weeks total After 2 weeks collagen content in conditioned samples increased 1 8 fold compressive 20 Page modulus bv 1 8 2 3 fold and ECM content bv 2 3 2 fold compared to static samples Ballvns et al 2010 Flow mediated shear stress is another method In vitro models are limited in nutrient deliverv due to a lack of blood supply All nutrient media delivery must occur through diffusion Dynamic media flow within or around tissue engineered constructs offer the best solution to enhance media delivery and waste exchange They also simultaneously deliver flow mediated shear stresses to cells seeded within the constructs improving cell strength and structure Butler et al 2009 Fluid shear stress at physiological rest conditions is estimated to be 1 50 dyn cm in human arterial endothelial cells Fujiwara et al 2003 A recent study has demonstrated that placing axons under continuous mechanical tension increases axon growth rates up to 1cm day This was accomplished by using a microstepper motor system to incrementally separate two neural membranes allowing for soontaneous growth of axons from one membrane to another This research is useful in the repair of nerve injuries using transplantable neurons with 10cm long axons for central nervous system repair Butler et al 2009 Pfister et al 2006 Smith et al 2001 Research was conducted concerning the effects of mechanical
68. ited from pub com 43 Figure 18 12V DC 60 RPM high torque gear box electric motor sess ssnnennzznnenzzznenenzanenenzznnnenzzznnnnnznna 44 Figure 19 Individual removable chambers on a removable fra 45 Figure 20 Cam and flat arm design for motor svringe attachment ss ssessenenenzasnennzzsnnenzasntenzzsntnznzzni 46 Heure E KIEREN 47 Figure 22 Svringe silicone tubing conpnectors AAA EE HAAEEn Ana AnEnA za nnnnnznatnnn Anna 48 Figure 23 Threaded svringe silicone tubing CONNECTOSS ss ss seeeennnnnnenzanenenzznnnnnzzzannenzna tenna ann nnnnan nn nz nn 49 Figure 24 Using sutures and silicone glue to seal syringe tips to silicone tubing 0 ss sesssenenenzzenenenzzni 50 Figure 25 Figure 26 Figure 27 Figure 28 Figure 29 Figure 30 Figure 31 Figure 32 Figure 33 Figure 34 Figure 35 Figure 36 Figure 37 Figure 38 Figure 39 Figure 40 Figure 41 Figure 42 Figure 43 Figure 44 Pinch clamp from World Precision Instruments and Z Man Comp 51 Ultra high molecular weight polyethylene and polvcarbonate ss eessneeenzznnnnnennnannenzznna 52 3mL luer lock syringe Luer slip vs luer lock syringe Ups 55 BUEDEN 55 Svfjnee ana UDINE ASSCINIDIV i errn N EN 57 Required volume to achieve 10 distension sse sse eeennnnnnnnzentnnnzznnrnnnzzntnenzznntenzznnnnnnzznnnenz nn 58 Experimental set up for uniformity trials 00 ss ss eessennnnnnzonnnnnzennnnnzznntnznzzntnenzzntnnznzzntnnnzznenenza 5
69. itv Svringe Pump Peristaltic Pump Pump Svstem Mechanical Conditioning Svstem Cvclic Circumferential Mechanical CAM Motor Air Pressurization Loading Svringe Pump Peristaltic GE GEESS 87 Consistent Work Output Programmable Leak TE A ie SNE Ee ege penn rrogammable Jm Jee JI Adjustable Pressure Programmable TRIM Pot Resistors Switches Kee E DE Compatibilitv Valve Couplings 4 2 Conceptual Designs In order to meet our outlined objectives we broke up our bioreactor svstem into two major components a mechanical conditioning svstem and a tissue culture device Each had their own set of objectives and functional requirements 4 2 1 Mechanical conditioning svstem The purpose of the mechanical conditioning svstem is to use a flow medium to distend an elastic tube on which engineered tissue samples are mounted Distensible tubing has been used in past research as a means of imparting mechanical strain on tissue engineered constructs Niklason et al 1999 The elastic tube is generally seeded with tissue constructs and inflated by air or liquid to distend the tube and thus the tissues mounted on the tube 34 Page 1 Peristaltic pump Using the Svedain heart valve bioreactor design Svedain et al 2009 as a model the team considered the use of a peristaltic pump as a means to provide circumferential mechanical stimulation to the TEBVs The pump operates bv using a rotating cam Figure 6 1 to flex plastic tubing 2
70. l on a flat surface alongside of a measuring device 2 Cut heat shrink tubing to desired length 3 5mm or long enough to cover the barb and silicone tubing on the luer fitting see Figure below for approximate scaled sizing 3 Using gloves to handle the silicone tubing attach the silicone tubing to the barbed end of the luer fitting ensuring that the tubing is secured above the barb see Figure below 4 Using gloves cover the silicone tubing and barbed tip overlap with heat shrink tubing see Figure below 84 Page 5 Remove vour gloves in case of heat exposure and use tweezers forceps to pick up the luer fitting bv its hollow end see Figure below 6 Position the heat gun pointing awav from vourself and turn it on depending on the tvpe of heat gun used this mav require an additional person to hold the heat gun while the original person completes steps 7 9 see Figure below for model heat gun we used 7 Hoist the silicone tubing heat shrink tubing luer fitting combination approximatelv 1 5 inches from the mouth of the heat gun using the tweezers forceps and spin continuously for 10 30 seconds to adhere the heat shrink tubing to all sides of the luer fitting BEWARE of melting the luer fitting due to positioning the parts too closelv to the heat gun see Figure below 8 Repeat for multiple tubing assemblies 9 Switch the heat gun to the cool setting for a minimum of one minute before shutting the heat gu
71. l steps take place within a sterile biosafety cabinet unless otherwise specified Whenever leaving the biosafety hood be sure to apply 70 ethanol before re entering to control contamination risk 1 Fill base of 140mm tissue culture dish with 25mL culture media 2 Aspirate media out of tissue ring culture molds 3 Carefully remove tissue rings from culture molds using narrow 90 forceps and submerge in tissue culture dish media 4 Repeat for all tissue rings 5 Open autoclaved silicone tubing and place within tissue culture dish with tissue rings 6 Using narrow forceps place tissue ring arrow around one open grip of the blunt curved forceps and angle blunt curved forceps upwards in media to prevent ring sliding 99 Page 7 Use narrow forceps to place the open end of the silicone tubing tightly over the open grip containing the tissue ring 8 Use narrow forceps to slide the tissue ring arrow onto the silicone tube and position it approximately 2 cm from the luer 9 Let silicone tube and tissue rings remain submerged in culture media while loading other rings and samples 100 Page 10 Repeat steps 6 9 until 5 6 tissue rings have been successfully loaded 71 cm apart on the silicone tube 11 Repeat steps 5 10 until 5 silicone tubes have been loaded with 5 6 tissue rings each Note if using less tissue chambers use fewer silicone tubes lids and syringes Tissue Chamber
72. l system and the environment and therefore significantly reduces the risk of contamination Also the use of the luer fittings allows for easv removal and attachment of silicone tubes and TEBV samples Our device offers several unique features that overcome the limitations of current devices Our device offers high throughput options by allowing for the conditioning of up to 30 tissue rings simultaneously The device has also been designed so the cam syringe and silicone tubing can be removed and replaced with different sized components in order to adjust the flow volume and frequency and to accommodate different sized tissue rings Tissue chambers are isolated from one another and the motors run independently This allows several different culture conditions and experimental variables to be altered during a single experiment Standard replaceable parts have been incorporated into the design in order to allow the user to easily replace damaged or contaminated components Internalizing the motors in the polycarbonate base helps align and secure several design components Internalized motors keep mechanical systems separate from tissue chambers so they may remain within the incubator while the tissue samples are transported for feeding using the removable tray 74 Page Chapter 8 Conclusions and Recommendations Through component verification and bench top tests both aspects of the bioreactor were validated the mechanical conditioning svstem
73. le 2 Highlighted red in Table 2 is an additional area of interest and refers to the amount of effort required to secure tissue samples to our device As a team we did not feel this was as important as some of the other objectives however after receiving the client completed PCC we realized that it may be more important than initially thought Newly cultured tissue samples are very fragile and during the process of transferring the samples from their culture molds to our device they 29 Page can easily break Both clients decided that our device should facilitate this process This list of priorities and weighted objectives functioned as the backbone for the development of design alternatives Table 2 Client and Team PCC Comparison Media Supply 8 7 Waste Removal 7 5 Accurate 4 8 Easily Secured 6 5 High Throughput 25 4 4 3 5 Easily Removed 5 2 3 3333333 Programmable 0 5 1 5 2 166667 Adjustable 0 5 4 1 1 833333 Inexpensive 2 5 A O 0 833333 3 3 Constraints In addition to the development of objectives we also needed to determine our design constraints Our two most limiting constraints were time and budget The final deadline for project completion was April 21 2011 on Project Presentation Dav Our budget consisted of 368 00 which would have to cover all of our purchased materials for prototvpe and final design manufacturing Safetv was another prioritv for all of our alternative designs The design could not harm the user o
74. low to combat vessel weakness and increase cell length Weakness could occur when cells are aligned in a random pattern within the wall of a vessel versus aligning along the direction of blood flow Collagen and elastin fibrils will similarly align in a more compact manner in the direction of tension increasing vessel strength and stiffness Isenberg et al 2003 Tower et al 2002 TEBVs also respond to cyclic distension by producing more collagen and elastin changing the composition of the extracellular matrix and further strengthening and stiffening vessel walls Mechanical stretching has been shown to promote the expression of type and III collagens fibronectin and tenascin C in cultured ligament fibroblasts Sahoo et al 2007 Mechanical properties can be engineered to match those of natural blood vessels The burst pressure of a saphenous vein typically used in coronary artery bypass grafts in patients with heart disease is 1680 307 mmHg Konig et al 2009 A tissue engineered blood vessel cultured for 7 weeks with the same diameter and length as the tested saphenous vein had a burst pressure of 2594 501 mmHg Konig et al 2009 Vascular compliance AV change in volume AP change in pressure stiffness F force applied 6 displacement produced by force tensile strength calculated based on uniaxial tensile 2t S testing ultimate tensile stress UTS at failure and burst pressure P t wall thickness i
75. mbly of Microtissue Building Blocks Journal of Biotechnology 148 1 46 55 Kim B S Nikolovski J Bonadio J amp Mooney D J 1999 Cyclic Mechanical Strain Regulates the Development of Engineered Smooth Muscle Tissue Nature Biotechnology 17 10 979 79 Page Kolpakov V Rekhter M D Gordon D Wang W H amp Kulik T J 1995 Effect of Mechanical Forces on Growth and Matrix Protein Svnthesis in the In Vitro Pulmonarv Arterv Analvsis of the Role of Individual Cell Tvpes Circulation Research 77 4 823 831 Konig G McAllister T N Dusserre N Garrido S A lyican C Marini A L Heureux N 2009 Mechanical Properties of Completely Autologous Human Tissue Engineered Blood Vessels Compared to Human Saphenous Vein and Mammary Artery Biomaterials 30 8 1542 1550 doi 10 1016 j biomaterials 2008 11 011 Ku C H Johnson P H Batten P Sarathchandra P Chambers R C Taylor P M Chester A H 2006 Collagen Synthesis by Mesenchymal Stem Cells and Aortic Valve Interstitial Cells in Response to Mechanical Stretch Cardiovascular Research 71 3 548 556 doi 10 1016 j cardiores 2006 03 022 Kulik T J amp Alvarado S P 1993 Effect of Stretch on Growth and Collagen Synthesis in Cultured Rat and Lamb Pulmonary Arterial Smooth Muscle Cells Journal of Cellular Physiology 157 3 615 L Heureux N 2003 Tissue Engineered Blood Vessels and Methods and Apparatus for Their Manufactu
76. mmary of syringe characteristics Syringe Type Length of Syringe Distance Traveled for Syringe Luer Attachment Plunger Depressed 0 075mL Injection Accuracy 5 2 Quantification of Displaced Volume We tested our design alternatives to determine which approach was best suited at achieving 10 distension Having decided upon a water based pressurization system rather than compressed air we 56 Page needed to determine the volume of water required to distend a silicone tube Specificallv our design called for a silicone tube 10 cm in length for optimal tissue sample loading 5 2 1 Experimental Procedure To measure the volume of water displaced by the 1 mL syringe that achieved the target magnitude of 10 distension a water filled syringe was used to statically inflate a tube with defined volumes in increments of 25 uL Figure 29Error Reference source not found Tubing diameters were obtained from still images of the inflated tubes using a Leica EZ4 D microscope and Leica Application Suite For this experiment a total of 5 autoclaved samples were tested 1 mi luer slip syringe 10 cm silicone tube Luers Pinch clamp Figure 29 1 mL luer slip syringe connected to 1 16 barbed bayonet tip mated with a 1 16 barbed luer lock tip that is attached by heat shrink tubing to a 10 cm silicone tube sample A pinch clamp at the distal end of the tube was used as a means of fixation and sealing 5 2 2 Experimental Results In st
77. mplications specific instructions have been outlined in Appendix B A Guide to Heat Shrink Tubing for manufacturing these parts Despite these limitations heat shrink tubing is still the best option we tested for permanently sealing the silicone tubing to the syringe tip 50 Page 7 Silicone tubing sealing mechanism We had a varietv of options for sealing the free open end of the silicone tubing to ensure pressurization These included endcaps and a varietv of different clamps Endcaps are small rubber plugs fitted to the distal end of the silicone tube and held permanentiv in place with glue We considered pinch clamps for small diameter tubing due to their ease of use low cost and autoclavabilitv The Ali MQP 2009 had a large varietv of endcap designs including a needle endcap blunt endcap suture and a combination of suture and a blunt endcap The blunt endcap and needle endcap designs were multi functional Both aided in tissue ring loading by providing a point at the end of the silicone tube to place in the center of the tissue ring from where you can push the ring up the silicone tube with forceps The two endcaps also effectively sealed the silicone tubes from leaking due to air pressurization Sealing the distal end of the tube may also pose a challenge in preloading the silicone tube with liquid for distension by syringe The tube would have to be submerged completely in water or media to allow the air within the tube to be r
78. n S tensile strength of blood vessel PSI O outer diameter in can be altered and potentially controlled for based on the frequency and force used during conditioning Compliance and stiffness are important properties contributing to overall TEBV health Compliance refers to the ability of a blood vessel to recoil to its original shape after deformation by force Stiffness is the blood vessels resistance to this deformation Tensile strength is the maximum stress that a blood vessel can handle in a 2 D plane before failure Burst pressure denotes the maximum stress that a blood vessel can handle in a 3 D plane before bursting during pressurization Vascular stiffness correlates with increased cardiovascular risk DeLoach et al 2008 By controlling these parameters tissue engineered blood vessel properties can be catered to the needs of individual patients by altering the specific vascular type and size and decreasing susceptibility to cardiovascular disease Increased ECM synthesis is a positive effect of biomechanical conditioning Mauck et al 2000 Freed et al 2006 Buschmann et al 1995 Ku et al 2006 Niklason et al 1999 Seliktar et al 2000 Syedain et al 2009 Mauck 2000 studied dynamically loaded tissue engineered articular cartilage seeded on 16 Page agarose gels Tissue disks were dynamically compressed at 10 strain at a frequency of 1 Hz at a duty cycle of 1 hour on 1 hour off per day for 5 days a
79. n et al 2009 Cvclic mechanical distension of tissue engineered constructs has been shown to increase contraction and mechanical strength Seliktar et al 2000 Isenberg et al 2003 increase circumferential cellular alignment along the vessel length Seliktar et al 2000 Liu 1998 and increase ECM svnthesis and cell growth Kolpakov et al 1995 Mechanical conditioning has become a major goal of recent tissue engineering efforts and has led to a shift in some of the primarv bioreactor design goals Bioreactors have evolved from acting solelv as culturing devices to culturing while simultaneously conditioning tissue constructs 2 2 Bioreactors Bioreactors are biomimetic laboratorv devices that regulate a number of environmental factors in order to create an optimal platform for biological activitv In tissue culturing and engineering a regulated environment is crucial in order to promote cell growth and tissue structure and mechanics bioreactors can be designed to control variables including pH temperature hvdration nutrition and waste regulation and removal Manv of these conditions mav be provided bv an incubator In the past decade the focus of these bioreactors has expanded to include devices that induce mechanical stimulation which improves compositional and mechanical properties The following sections will profile several published bioreactor designs and experiments which will help in identifying design intent ar
80. n off and storing it 85 Page Appendix C A Guide to Wiring and Sealing Motors Potting and Sealing Motors Materials 12 V 60 RPM DC motor x5 mounted into motor mount Epoxy hysol M 121hp medical device epoxy adhesive 50 mL Hysol adhesive for encasing urethane 50 mL Potting gun Easy ID low voltage cable 24 2 AWG 0 11 W x 0 06 thick 12 VDC 50 L Wire cutters Heat gun Soldering kit Vice Moisture seal polyolefin heat shrink tubing 1 5 ID before 0 75 ID after cut into 2 5 lengths x5 Small diameter heat shrink tubing for wiring Procedure Note do everything listed for each motor individually POR p B 10 11 12 Cut wire to 6 foot lengths Strip wire and fix to motor leads Solder wire to motor leads Heat shrink positive and negative wires near soldering point to prevent further separation of zip wire Secure motor mount into vice facing upside down with wires coming out the top Pot motor holes with epoxy hysol M 121 medical device epoxy and potting gun Once all motors have been potted flip motor mount in vice so epoxy drips down wires and not into motor Let dry for 6 hours Attach moisture seal heat shrink tubing to outside of motors encasing both the gearbox and motor Use heat gun to permanently attach tubing around motor Fill remaining area within heat shrink tubing with hysol adhesive for encasing Position wires in the same direction perpendicular to the long si
81. nch clamp 5 The motors are arranged in a staggered pvramid formation in order to minimize the amount of occupied space while maintaining the linearitv of the svstem and allowing the user to easilv access the tissue chambers To accomplish this three different sized flat arms are required 6 In order to improve manufacturabilitv the team dismissed the flat arm component of the design en route to developing the following design alternative 2 Motor cam series The motor cam series builds on the concept of the pvramid assembiv but simplifies it bv reducing the number of parts and fittings improving manufacturabilitv and internalizing the mechanism in order to make a compact customizable user friendly device The assembly consists of a plastic base Figure 16 1 which houses six 60 RPM gear box Figure 16 Motor cam series motors 2 Each motor is fixed with a cam 3 which rotates and cyclically depresses the plunger of a 1 mL syringe 4 Again the design employs tissue chambers and pinch clamps as seen in Figure 13 Six of these tissue chambers 5 are housed in a removable UHMWPE tray 6 that fits into the base 4 3 Comparison of Design Components In addition to comparing alternative designs as a whole we compared individual components used within these designs to aid in our selection of pieces for a final working assembly We compared these components using research interviews with experts and clients exp
82. ng or knocking against the sides of the conical tube The only means of holding the silicone tube taut was via an end weight attached to the needle endcap which was glued into the free end of the silicone tube This endcap was still not effective at preventing the free swinging of the silicone tube during transport The vertical orientation of the tissue chamber makes the tissue rings susceptible to gravitational forces that can merge multiple rings together or possibly even slide them off of the silicone tube entirely A horizontal orientation would eliminate this problem Other drawbacks of the pressurization system include its size and user friendliness The pressurization system makes use of compressed air which is stored in large tanks in the lab This takes up lots of space in the lab and is loud and disruptive in the user s workspace Furthermore a heavy aluminum base 32 Page makes the device awkward to transport Finallv the device was designed so large that it cannot fitin a laboratorv incubator and therefore cannot be used for culture experiments Given these limitations of the initial design we chose to pursue a completelv novel approach to our project During design conception we made an effort to use common parts standard sized screws svringes tubes motors etc to reduce costs of manufacturing and materials expedite the design process and to minimize production times 4 1 Functions Specifications With a firm und
83. nufacturability The bioreactor primarily consists of components that can be machined using high speed milling techniques and a series of machining files Most other components in the bioreactor are standard parts 69 Page that can be purchased from catalogs such as luer fittings silicone tubing and 12V hobbv motors Minimal assembly is required by the user and all assembly only requires simple common tools such as a screwdriver 6 8 Sustainability The plastics that are used in the body of the bioreactor UHMWPE and polycarbonate are both recyclable materials Machining scraps and even the device itself can be reprocessed and used for other applications thereby minimizing the amount of waste produced in the production and use of the bioreactor 70 Page Chapter 7 Final Design 8 Validation A computer generated drawing of the final design assembly is shown in Figure 42 A photograph of the final design assembly is show in Figure 43 The primary architecture of the assembly consists of a polycarbonate base Figure 42 amp Figure 43 1 The surface of the base contains four corner barriers 2 to hold a polycarbonate tray 3 The tray has been machined to contain five wells in which milled ultra high molecular weight polyethylene UHMWPE tissue chambers sit 4 Each chamber houses a series of threaded luer fittings that are fitted with a 10 cm 1 968 mm outer diameter silicone tube The distal end of the tube is clamped s
84. od flow vessel wall expansion and contraction due to pressure as well as longitudinal tension along the vessel Isenberg et al 2006 A diagram of these forces is pictured in Figure 1 The goal of mechanical conditioning of tissue engineered blood vessels is to mimic these natural in vivo forces while TEBVs are growing in vitro Fluid Shear Stress Wall Tension Laplace O pim r d Figure 1 Forces experienced within natural blood vessels Mechanical conditioning is a widely used method for treating engineered tissue constructs to enhance their physical and mechanical properties Techniques for creating artificial tissues have used various cell types such as smooth muscle and endothelial as well as differing culture conditions to create grafts that emulate native tissue Isenberg et al 2006 Isenberg et al 2003 Cells are affected by an immense variety of chemical and mechanical stimuli Bone and muscle cells in particular are known to respond to these mechanical stresses in a positive way Neidlinger Wilke et al 1995 constantly remodeling based on the stresses they are exposed to Mechanical stretching of smooth muscle cells SMCs cultured in collagen based tissue equivalents has been shown to have immense effects on SMC orientation Dartsch et al 1986 Kanda et al 1993 Liu 1998 ECM deposition Chiquet et al 1996 Kolpakov et al 1995 Kim et al 1999 Niklason et al 1999 Kulik et 14 Page al
85. oeiidotabii re te 30 Table 3 Morphological ei E 34 Table 4 Summary of Syringe characteristics ccccccccccssscccseccceeececeuscseeeceeeuececeueceeeeceeenceseueceesuesseeneseeensss 56 7 Page Table of Figures Figure 1 Forces experienced within natural blood Vessels ss ssssssnenenzennnnnzennnenzzontrrnzzatrenzzstrenzzonenznzzna 14 Figure 2 Bioreactor system used by Seliktar adapted from 2000 sess sssneeennzennnenzzsnnanenznzzznannnznzzznaa 23 Figure 3 Kelm bioreactor adapted from 2010 24 Figure 4 Ali bioreactor system 2000 26 Figure 5 Air pressurization system Ali 2009 ccccccccccssseccceceeseccccceeesececeseeeseceeeseeeseceseseunseeeessuenseeess 32 Figure C F OPIS Cale DUMP si iii ii SA a 35 SI 7a e den D E 35 SS 8 Wheel and arm WOU ON EE 36 Figure 9 Solenoid E Me ie Te 36 Figure 10 Motor and Cam SYSTEM sesccicainencsosusicssandecadd ceatdeadsiavnssttasansuneparnedenbatdeusioedsvedeeincestvacdseddoentdeecenideess 37 Figure 11 Double threaded CAN GSO E 38 Figure 12 Removable compartment assembly sosssssssessseesesrrrssrreresrrrsssreresrrerssreresererssreresererssrereserersseerene 38 Fig re 13 Milledtiss echamDe EE 39 Figure 14 Wheel and arm aSSeMbly cccccccssseccceesececcesecceceesececeusecceseececseescceseesecesseecessenceessusesetseneces 40 Figure 15 Motorized pyramid assembly and pinch clamp LZhManCorp com 40 Figure Ecart Eege 41 Figure 17 Solenoid actuator Ed
86. oir for the 39 Page flow medium When the tissue chamber is put back into the svstem the flow can be directed again back into the chamber When the user wants to remove a single compartment he or she will simplv redirect the flow to the reservoir stopping the flow into that cartridge without interrupting the others This still leaves the problem of water leaking from the svstem into the incubator following removal of the compartment The remaining end of the three wav valve will Figure 14 Wheel and arm assembly be fitted with a quick coupling fixture that only allows one way flow 2 Pyramid assembly The team conceptualized modifications to the wheel and arm design In order to increase user control over each TEBV sample the team sought to create isolated systems for each tissue chamber In order to achieve this the team concluded to use small gear box Figure 15 Motorized pyramid assembly and pinch clamp ZManCorp com hobby motors Figure 15 1 40 Page These motors can be purchased off the shelf and run at 60 RPM Again a machine wheel 2 and flat arm 3 would be fixed to each motor The opposite end of the flat arm would attach to the plunger of a 1 mL svringe 4 As the motors rotate the wheel the flat arms would undergo a horizontal translation causing the plungers to depress and retract These svringes interface with milled tissue chambers see Figure 13 which contain an internalized pi
87. on the size of the cam Both the tray and the base can be manufactured using high speed milling techniques This assembly was chosen as the final design because its materials and architecture helps maintain the stability of the assembly Also the base internalizes most of the mechanical components in the design Furthermore the removable tray provides a definable housing unit for the tissue chambers and allows the user to easily remove and transport all tissue chambers at once These design aspects are appealing to both the designer and user because the small individualized motors minimize the size of the entire design while allowing for customizability and individual shutoffs Also by having isolated a single mechanical failure is not likely to compromise the whole system The removals of the wheel and flat arm are also expected to decrease failure rates as there are fewer mechanical interfaces in the system Finally this component of the design allows for increased customizability while the cam has been designed to displace only enough water to distend the silicone tubing and therefore the TEBV samples by 10 this cam may simply be changed out with another of a larger diameter to provide greater distension of the tubing 73 Page The final design component is the tissue chamber compartment This design is preferred to the other design alternatives because it completelv separates the silicone tubing and pinch clamping from the mechanica
88. one glue failed to prevent leakage at the svringe tip silicone tube interface Figure 24 Figure 24 Using sutures left and silicone glue right to attempt to seal svringe tips to silicone tubing Ali 2009 found an alternative to these methods Thev used biocompatible 1 8 diameter transparent heat shrinkable tubing with a 2 1 shrink ratio to seal the silicone tubing to the rest of their pressurization svstem After extensive testing involving preloading different svringes luers and silicone tubing with water clamping the distal end of the silicone tubing and pressurizing the tube past 20 distension Section 5 1 we determined that heat shrink tubing was our best option and proved to prevent any leaking or detachment of the silicone tubing from the 1 16 female luer fitting However one limitation to this method exists When manufacturing these syringe tip silicone tubing parts one must use a heat gun to adhere the heat shrink tubing to the syringe tip silicone tubing junction by heating the heat shrink tubing above 175 C see Appendix A for specific instructions At such high temperatures the plastic of the 1 16 female luer fitting begins to melt potentially sealing the flow opening of the luer fitting Excess heat directly applied to silicone tubing was observed to cause point defects along the affected section of tubing when the tubing was pressurized causing plastic deformation and failure of the tube To avoid these co
89. out of it and the second involved having separate removable chambers housed on a removable tray Figure 19 Both designs are relatively easy to manufacture through milling and sawing and both have removable components The tissue chamber with separate removable chambers was deemed to be a better option for our design By having separate chambers the user could have the option of removing a single chamber from the system in the event of sample contamination or a problem with the mechanical components In the event that a chamber is damaged in some way it can simply be replaced eliminating unused or contaminated space in the system Both tissue chamber designs would interface with the motor cam mechanical conditioning device through a hole in one of its walls An air and water tight seal would allow for the silicone tubing to pass into the tissue chamber for submersion Wi l l MA Figure 19 Individual removable chambers while maintaining a sterile environment within the tissue on a removable trav chamber itself One function of the tissue chambers is to allow for the changing and replacing of media Both the single unit and individual chambers function similar to culture dishes in which media must be manuallv aspirated and added to feed the cells contained within them The chambers would be complete with loose fitting lids which act as culture dish lids in permitting gas exchange and offering protection from potential airborne contam
90. overcomes limitations associated with current bioreactors that mechanicallv condition tissue engineered vascular constructs It was then our responsibilitv to conduct an in depth literature review in order to gain a full understanding of the current technologv As we developed an understanding of the problem we began compiling a list of objectives and constraints Utilizing a collection of client interview data we then applied these objectives and constraints and created a list of functions and means for our device outlined in Chapter 4 These means were then analvzed and several design alternatives were created The designs were further scrutinized and with continued research we decided on and refined a final design which we evaluated through a series of quantitative and qualitative bench top tests 3 1 Initial Client Statement The initial statement provided bv our project advisor Marsha Rolle was Bioreactors have been shown to improve the structure and function of engineered tissues bv providing conditions that simulate the in vivo environment in which the tissue normallv exists In addition bioreactors can facilitate seeding organization and culture of cells to support tissue growth and maturation For tissue engineered blood vessels bioreactors that provide cyclic circumferential mechanical loading have been shown to increase cellular alignment and increase extracellular matrix ECM synthesis leading to improved tissue mechanical strength
91. r cells in anv wav The bioreactor must be biocompatible non toxic to the cells within it and contain no possible outlets for injurv The materials we use in our design must be sterilizable in order to avoid contaminating tissue samples In addition to sterilitv cell viabilitv must be maintained bv means of constant media supplv to the TEBVs during mechanical conditioning Waste regulation must also be accounted for in order to guarantee optimal cellular conditions After discussion with our client we determined that the bioreactor must be transparent to allow for imaging and observation during TEBV testing Transparencv aids in the detection of experimental failure such as ring failure culture contamination or component failure 30 Page Since our device doesn t have a means to self regulate physiological conditions such as temperature and humiditv the device must fit within an incubator Each shelf in the incubator in our laboratorv is 40 cm wide x 15 cm high x 40 cm deep This constraint impacted our designs by limiting the available space that our device can occupv 3 4 Revised Client Statement After an improved understanding of the assigned problem and an extensive knowledge of the current technologv we were able to applv our metrics and overall project strategv and improve upon our initial client statement The resulting client statement reads as follows To design a bioreactor used in conjunction with a pressuriza
92. r responsibility to conduct an in depth literature review in order to identify the limitations of bioreactors used to mechanically condition tissue constructs As we developed an understanding of the problem we began compiling a list of objectives and constraints Using a collection of client interview data we applied these objectives and constraints and created a list 12 Page of functions and means for our device These means were then analvzed based on client input material biocompatibilitv and manufacturabilitv and projected expenses and several design alternatives were created The designs were further scrutinized based on the plausibilitv of component interfaces and the availability of materials and manufacturing tools We were able to determine our final design which we prototvped and evaluated through bench top testing and culture experiments Additionallv sections of this paper examine the effects our device mav have on the environment and the economv Finallv future recommendations were made so that future teams could learn from our results and conclusions and further develop the device 13 Page Chapter 2 Literature Review 2 1 Mechanical Conditioning 2 1 1 Definition In order for tissue engineered blood vessels TEBVs to function successfully they must have high strength and endurance Butler et al 2009 capable of withstanding the natural forces of the human body These forces include shear stresses due to blo
93. rain Journal of Biomechanics 28 12 1411 Niklason L E Gao J Abbott W M Hirschi K K Houser S Marini R amp Langer R 1999 Functional Arteries Grown In Vitro Science 284 5413 489 493 doi 10 1126 science 284 5413 489 Niklason L E Yeh A T Calle E A Bai Y Valentin A amp Humphrey J D 2009 Enabling Tools for Engineering Collagenous Tissues Integrating Bioreactors Intravital Imaging and Biomechanical Modeling Proceedings of the National Academy of Sciences doi 10 1073 pnas 0907813106 O Donnell T F Mackey W McCullough J Maxwell S Farber S Deterling R amp Callow A 1984 Correlation of Operative Findings with Angiographic and Noninvasive Hemodynamic Factors Associated with Failure of Polytetrafluoroethylene Grafts 1 1 12 10 2010 136 148 Pfister B J Iwata A Taylor A G Wolf J A Meaney D F amp Smith D H 2006 Development of Transplantable Nervous Tissue Constructs Comprised of Stretch Grown Axons Journal of Neuroscience Methods 153 1 95 103 doi 10 1016 j jneumeth 2005 10 012 Rubbens M P Mol A van Marion M H Hanemaaijer R Bank R A Baaijens F P amp Bouten C V 2009 Straining Mode Dependent Collagen Remodeling in Engineered Cardiovascular Tissue Tissue Engineering Part A 15 4 841 Sahoo S Cho Hong J G amp Siew Lok T 2007 Development of Hybrid Polymer Scaffolds for Potential Applications in Ligament an
94. ranslation in the svringe heads causing them to depress and retract The flat arm can be fixed to the wheel at anv of several points 5 resulting in different radii and therefore different flow volumes Figure 8 Wheel and arm motor This design concept is appealing as it combines several simple existing technologies for a novel application It also serves as an effective high throughput alternative to the svringe pump which was deemed out of the team s price range Some predicted complications include size and isolating single svringes or assembiv svstems 4 Solenoid flexion device The next preliminarv design was a solenoid flexion device The design consists of a batterv operated solenoid Figure 9 1 suspended above the plastic tubing 2 which contains the tissue samples Fixed to the solenoid is a rubber press 3 The size of press would be chosen depending on the volume of water in the tubing that needed to be displaced in order to inflate the tubing and achieve the desired 10 distension of the tubing and TEBVs While this design assembly eliminates several parts and Figure 9 Solenoid flexion device fixture points and in turn eliminates opportunities for the device to fail it was deemed unfit for this project It was discovered that it is very difficult to program a solenoid to function at speeds as low as 60 RPM which is required for the mechanical conditioning to 36 Page take place at 1 Hz Fur
95. rating temperature range of 40 F to 275 F 250 F needed for autoclaving and that it meets ASTM D4020 D4976 and D6712 FDA and USDA specifications Although this material does not 52 Page fulfill the need for transparencv of the material to allow visualization of the sample within the chamber transparent tissue chamber lids will satisfv this requirement We decided to use transparent abrasion resistant polvcarbonate PC Figure 26 right for the base and removable travs of our device Based on client interviews we discovered that the base and trav of our device need not be autoclaved between uses because thev will not be in direct contact with tissue samples It would merely need to be wiped down with 70 ethanol before entering the incubator For this reason we chose abrasion resistant polvcarbonate to withstand harsh cleaners and solvents PC also has high impact strength and durabilitv is weather resistant and has an operating temperature range of 40 F to 200 F Due to its low operating temperature it cannot be sterilized within an autoclave 250 F and was thus not considered in designing tissue chambers Materials were selected based on several characteristics such as biocompatibilitv sterilizabilitv size ease of use and how well thev interface with other components in the device Using these material choices we designed and prototvped a device to mechanicallv condition tissue engineered blood vessels in vitro The
96. re Patent 6503273 Retrieved 10 4 2010 2010 from http www freepatentsonline com 6503273 html L Heureux N Paquet S Labbe R Germain L amp Auger F A 1998 A Completely Biological Tissue Engineered Human Blood Vessel The FASEB Journal Official Publication of the Federation of American Societies for Experimental Biology 12 1 47 56 Liu S Q 1998 Influence of Tensile Strain on Smooth Muscle Cell Orientation in Rat Blood Vessels Journal of Biomechanical Engineering 120 3 313 320 Mauck R L Baker B M Nerurkar N L Burdick J A Li W Tuan R S amp Elliott D M 2009 Engineering on the Straight and Narrow The Mechanics of Nanofibrous Assemblies for Fiber Reinforced Tissue Regneration Tissue Engineering Part B 15 2 171 Mauck R L Soltz M A amp Wang C C B 2000 Functional Tissue Engineering of Articular Cartilage Through Dynamic Loading of Chondrocyte Seeded Agarose Gels Journal of Biomechanical Engineering 122 3 10 5 10 252 261 doi 10 1115 1 429656 Mitchell S amp Niklason L 2003 Requirements for Growing Tissue Engineered Vascular Grafts Cardiovascular Pathology 12 2 59 64 doi 10 1016 S1054 8807 02 00183 7 80 Page Neidlinger Wilke C Stalla l Claes L Brand R Hoellen I Rubenacker S Kinzl L 1995 Human Osteoblasts from Vounger Normal and Osteoporotic Donors Show Differences in Proliferation and TGF Beta Release in Response to Cvclic St
97. re manufactured from raw plastic sheets using high speed milling techniques in a HAAS automated mill The following models and dimensions were programmed using SolidWorks and FeatureCAM softwares and were uploaded into the mill s computer system HAAS Milling Machine Partially Milled Tissue Chamber 93 Page e F635 mm Tissue Chamber 127 41 mm 133 31 mm 26 44 mim 24 48 mm 20 0 mm A 3 18 mm 49 42 mm W 3 16 mm 94 Page A Ae mm 12 70 mm 181 1 18 285 43 rom 843mm THRU ALL 1427 26 mim Mtn 406 40 mm 65 0 5 d mAr i 1 S Base amp Tray Holders aL eee 40 43 mm 12 0 mm 3 06 mm THRU ALL K THRU ALL co 7 0 mm r I l 4 i I I I l l h BS 290 0 mm Motor Mount 95 Page A JU mm TA P6 35 mm 189 07 mm 40 0 mm 19 64 mm 3 03 mm 8 96 Page Appendix E User Manual Prepare autoclaved tools materials Forceps narrow tip 457 and or 90 Forceps blunt curved tip Flat spatula for grease Aspirator tips 10 cm silicone tube heat shrunk to 1 16 inner diameter female barbed polypropylene luer x5 Tissue chamber x5 with stainless steel dowel pin fitted into pinch clamp hole and polypropylene female 14 28 threaded luer mounted into chamber wall Tissue chamber lids x5 Compression spring number dependent on number of mechanically conditioned tissue chambers in use Silicone based high vacuum grease Dow Corning Midland
98. s cap Because the tubing is fixed to the threaded caps before thev are both screwed onto the Falcon tube the tubing is subjected to torsion and possible tearing or deformation Therefore the biggest challenge associated with this design would be to not compromise the safetv of the silicone tube and the tissue sample when fastening the threads Figure 12 Removable compartment assembly 2 Removable compartments This design uses an original assembly of isolated removable plastic compartments Figure 12 1 that fit into a containment unit 2 The frame separates the compartments acts as a junction between the pump system and cartridges and provides hinged covers for each compartment 3 The hinged covers 38 Page looselv sit atop the compartments which allows for gas exchange and provides easv access to the interior for aspirating and changing out cell media Finallv the fronts of the compartments will contain a small opening 4 above the media pool to which the silicon tubing will be fixed bv a clamp located outside the tissue chamber 3 Milled tissue chambers This conceptual design consists of milled plastic tissue chambers Figure 13 1 which interface with a mechanical conditioning svstem s syringe 2 through a series of luer fittings 3 The flexible plastic tubing is attached to the luer fittings using heat shrink wrap tubing 4 The distal end of the silicone tubing is clamped using a pinch clamp 5 that i
99. s fixed to the bottom of the chamber using a stainless steel dowel 4 2 3 Design assembly 1 Wheel and arm assembly Figure 13 Milled tissue chamber The design is driven by the wheel and arm motor Figure 14 1 as described in Section 4 2 1 3 The flat arm is interfaced with a fixture that holds the heads of several BD 10cc syringe plunger heads 2 Displaced flow medium is transferred from the syringes into the tissue mounted silicone tubing located in the compartments of the removable compartment assembly 3 which will be constructed as described in Section 4 2 2 2 The combination of these design alternatives allows for high throughput isolated systems for the simultaneous operation of independent experiments and high customizability One problem that arises with this design is that a single compartment cannot be removed without shutting off the pump for the other compartments Also even if a single compartment can be removed there is nothing stopping the water in the silicone tubing from spilling out The team conceptualized a system that may overcome these design barriers Each syringe would pass the flow through a three way cross flow valve 4 The top end of the valve will lead into a BD Falcon polypropylene tube which would be fixed with a loaded silicone tube 5 When a tissue chamber needs to be removed the flow can be redirected using the cross flow valve into the Falcon tube which serves as a temporary reserv
100. stretching on collagen synthesis by mesenchymal stem cells and aortic valve interstitial cells Ku 2006 found that at 14 stretch there was a significant increase in collagen synthesis in both types of cells directly affecting the rate of ECM production and valve strength Collagen synthesis was dependent on the degree and duration of stretch in aortic valve tissue Ku et al 2006 In another study rat vascular smooth muscle cells were subjected to cyclic strain induced by a vacuum below the culture plates the cells were grown on Cyclic strain caused an increase in total cell count by 40 compared to control tests Wilson et al 1993 2 1 4 Conclusions The ultimate goal of blood vessel engineering is to create a nonthrombogenic nonimmunogenic suturable tissue that is able to withstand arterial pressures is compliant and can be remodeled in response to injury Schutte et al 2010 Mechanical stimulation improves the overall structure and function of native and tissue engineered vasculature alike this is imperative for achieving these goals Biomechanical forces have been shown to enhance the physical characteristics of TEBVs Mechanical cues cause Change in cell morphology physiology biochemistry and gene expression all of which 21 Page contribute to overall tissue function in vivo Butler et al 2009 Diamantouros et al 2009 Freed et al 2006 Isenberg et al 2003 Niklason et al 1999 Seliktar et al 2000 Svedai
101. that is fixed to a frame 3 The design would incorporate inlet and outlet channels 4 5 in order to allow for a continuous circuit of Figure 6 Peristaltic pump flow medium Using a peristaltic pump as a design component is very appealing as it is an existing technology and is programmable However one major drawback is that existing models do not offer high throughput capabilities 2 OctoPump The next design alternative was conceived by reverse engineering a syringe pump The design consists of eight syringes Figure 7 1 housed in a circular frame 2 The pump uses an offset rotating cam 3 located in the center of the frame to depress the plungers of the syringes The displacement of a Figure 7 OctoPump medium would inflate a plastic tube on which the tissue rings are housed thereby providing the desired distension This novel design allows for high throughput of isolated samples and the cam device could be manipulated to adjust flow frequencies and volumes However in order to remove a single sample the user would have to deactivate the entire system 35 Page 3 Wheeland arm motor Another design that mimics a svringe pump is the wheel and arm motor assembiv A small mechanical motor Figure 8 1 spins a machine wheel 2 attached to a flat arm 3 The flat arm s far end is attached to a housing device that is fixed to several svringe heads 4 As the wheel spins there is a horizontal t
102. the Ali 2009 MQP versus being seeded horizontally We eliminated a vertical tissue chamber design based on our assessment of the air pressurization system used in the Ali 2009 MQP introduced in the beginning of this chapter Our final concern with the vertical tissue chamber design of the Ali MQP is the amount of wasted cell media used in the 50 mL conical tubes 35 mL of media used if filled as shown in Figure 4 A horizontal design in which enough media would be used to submerge the tissue rings would reduce the costs related to cell media usage 44 Page The only stated concern of the Ali MQP team regarding horizontal tissue chamber arrangements is the chance that flexible tubes will not remain completelv horizontal when inserted into the media Ali et al 2009 We saw a way to overcome this problem and so we chose to pursue horizontal tissue chamber arrangements Our oniv foreseeable challenge with horizontal chambers was in finding a wav to submerge distensible tubing loaded with tissue rings within the media in the chambers without leaking All chamber designs must allow for tissue ring submersion and interface with the motor cam mechanical conditioning device We considered two different design variations for a horizontal tissue chamber based on our decisions to eliminate vertical designs and use motors as a mechanical conditioning device The first tissue chamber design involved using a solid block with separate chambers milled
103. there would be no wav to remove the 2mm blunt end metal svringe tip from the tissue chamber wall without replacing the entire tissue chamber Similarlv if the silicone tube were to become worn out due to repeated use damaged during autoclaving or otherwise harmed the 2mm metal syringe tip would have to be removed and replaced Figure 22 Syringe silicone tubing connectors Our plan to combat these problems was to use a two tip male female luer fittings The female tip would be secured into the wall of the tissue chamber and fixed there permanently while the male tip would be permanently attached to the silicone tubing and lock onto the female tip during experimentation The first male female luer parts we considered were 1 16 inner diameter barbed luer lock male and female tips Figure 22 capable of interfacing with luer slip syringes and silicone tubing The 1 16 inner diameter luer fittings is approximately 1 6mm which is as close to 1 4732 mm inner diameter of the silicone tubing as standard parts would allow The tight fit of the silicone tubing over the luer barb further aided in leak proofing the system The wider opening of the luer tip leading into the narrower inner diameter of the elastic tubing caused some problems with tube failure when distended past 20 during testing This gives the user the option to inflate the tube up to 120 of its original diameter in order to increase the strain magnitudes imparted on the tissue rings
104. thermore the movements of the solenoid are abrupt irregular and mav be abrasive to the silicone tubing J O Rourke personal communication October 2010 5 Motor and cam svstem The team s final conceptual design for the mechanical conditioning svstem involves a small motor Figure 10 1 that contains acam and cam collar 2 As the cam Spins it remains tangent with the head of a syringe plunger 3 that displaces a flow medium froma 1 Figure 10 Motor and cam system mL syringe 4 through a series of luer fittings 5 and into a flexible plastic tube 6 that houses the tissue rings The tube is secured at the distal end using a pinch clamp 7 The plunger is fitted with a compression spring 8 that makes it to retract stay in contact with the cam and allows for cyclic pumping of the syringe One highlight of this design is that the user may choose different cams or motors to customize the parameters of the mechanical conditioning For example a larger cam would displace a larger volume of medium through the syringe thereby causing a greater distension of the tissue rings A motor may be run at lower or higher voltages to increase or decrease the frequency of the conditioning 4 2 2 Tissue chamber All of the designs for the mechanical conditioning system involve the tissue samples being mounted ona flexible plastic tube that is inflated in order to achieve the desired distension The final assembly therefore req
105. tion svstem that distends the inner diameter 2mm of tissue engineered blood vessel rings and or tubes 1cm long by 10 1 at a frequency of 1 Hz This will be accomplished by means of cyclic circumferential strain which increases cellular alignment and ECM synthesis Multiple cartridges should allow for media regulation in a safe sterile leak proof in vitro setting that accurately simulates an in vivo environment Finally the device should be inexpensive and easy to manufacture and use With this enhanced definition of the problem we then entered the development stage which is completely outlined in the next chapter 31 Page Chapter 4 Alternative Designs Based on our initial client statement we developed constraints objectives functions and means by which to formulate design alternatives Using the air pressurization system designed by Ali 2009 Figure 5 as a template and the designs of other successful bioreactors as models the team evaluated a number of design alternatives Li AA NN M i l l i l Figure 5 Air pressurization svstem Ali et al 2009 After studving the Ali tissue chamber design outlined in vellow in Figure 5 we determined that the design had several faults The silicone tube fixed to the threaded cap was dangling within the BD Falcon conical tube Figure 4 A Without any effective means of fixing the free end of the silicone tube there was nothing to prevent it from curli
106. tioning the TEBVs through physiological cardiac loading while maintaining cell viability This response to physiological mechanical environments provides insight into how mechanical conditioning during culture could result in an improved graft If cells respond to in vivo mechanical manipulation they should also respond to in vitro loading assuming appropriate culturing techniques are maintained These outcomes acted as the inspiration for the development of the in vitro mechanical loading model presented in this paper The Rolle Lab at Worcester Polytechnic Institute WPI uses tissue engineered blood vessel rings to model the mechanical and physiological properties of complete blood vessels Rat aortic smooth muscle cells SMCs are grown in agarose molds over 8 days to form completely cell derived three dimensional rings with an inner diameter of 2 4 or 6 mm It has also been shown that rings cultured in close proximity for an extended period of time could fuse to form a single cohesive tube demonstrating possible future applications for in vivo studies Furthermore the rings relatively small size and short production time allowed us to experiment with greater sample sizes Gwyther et al 2011 The goal of this project was to design a bioreactor that would impart mechanical conditioning on 2 mm inner diameter tissue rings from the Rolle Lab The bioreactor also had to be capable of maintaining basic cell culture conditions It was then ou
107. tissue based grafts greatly improved on past technologies concerns and limitations remain This approach used SMCs to improve the graft s strength However while the addition of these cells increased the strength they limited the graft s ability to expand and contract Postoperative studies showed that while implanted in vivo remodeling of the graft resulted in higher levels of elastin L Heureux 2003 Elastin is a protein that acts like a rubber band as it contracts the vessel In order to overcome the stiffness resulting from high levels of SMCs and collagen it is vital to have enough elastin so that the vessel can return to the original diameter during pulsatile and pressurized loads The elastin network also contributes to the long term success of tissue engineered grafts If a graft is too stiff there will be a difference between the natural tissue and the graft causing the fluid dynamics to change thus impacting the stream of fluids as they pass through the engineered vessel The increased failure rate due to compliance differentials is seen with synthetic materials as well Mitchell et al 2003 An additional concern with the application of SMCs as a means of structural support is that it remains difficult to regulate their proliferation once implanted If the cells proliferate excessively the graft can actually occlude itself resulting in failure L Heureux 2003 Collagen networks are the natural means of vasculature support and can
108. tor 20 Carefully lift the tray by its handles with the syringes pointing away from you and transport it to the incubator 21 Position the tray within the tray holder with syringes on top of the syringe holders in the incubator 22 Depress the syringe plunger so that it s flush with the cam and snap the syringe s into place 23 Close the incubator doors and switch on the mechanically conditioned tissue chambers 107 Page Appendix F Bill of Materials 0 D G Q fo EE D af Fr T GA bb bt Gan ST LC LK TE SF Bore DEN el JEJOL D D TO GEL HE hp cT FFT crs Fr Fe SETTI SOL OLK DFL D 8 g9 96E aud Amuenp Ui ei LA Hi Hi L L L d L T F L T T T T T T FI L L FI SQ2 SULSSIU Y N MPULIBISA Jaqa M IDBJNUEJA jjemads UDIJEJOAJO UEJA 7 GHUDMMSJ JISNOJ JIEJ SSISEJADA JIEJ SSISEJADA pasg UDIEWOJNV SJIISEJ4 ANJEA SIIJSBI4 anes JIEJ SSISEJDA JJEJ SSISEJADA JIEJ SSISEJADA JIEJ SSISEJADA pag uonewony JJEJ SSISEJADA JIEJ SSISEJADA sisijepads wn 35EJJA JENLIA JJEJ SSISEJADA JIEJ SSISEJADA JIEJ SSISEJAA OD LONG VIN VIN d il 908 0 TOO000 06F800 LEQ TETEL iz 8 TOSELId 709 TLTA SD6 T1 696 I DTIL D THIMBTIL TENSOSTO ETLOTZZ GEREGELT BINGOErL TITS EEVEJE fewores TI MEZ TS4d STT O8 TO0 TSTH OL8 ISO rg CESSES JIQLUNH Weg fWaqj JAE anjq swe JESUS Jd SPM JIQWELWI SNSSIL DOT JO XOQ ajpaau apqeuIejag s85 X GT
109. ue chamber wall 11 Depress the syringe plunger until the compression spring is flush with the plunger head and syringe barrel 104 Page 12 Use a pair of forceps to hold a pinch clamp in one hand use the other hand to push the free end of the silicone tube through the pinch clamp opening 13 Hold the pinch clamp sidewavs onto the base of the tissue chamber with a pair of forceps with one hand and with vour other dominant hand grip the inside of the silicone tube using narrow tipped forceps and pull or slide the silicone tube bevond the clamping point Be verv careful not to damage the tubing or pull it more than half a centimeter past the clamping point 14 Place the pinch clamp onto the steel dowel at the base of the tissue chamber 105 Page 15 Use one hand to pressurize the svringe plunger down to 0 2 mL and the second hand to press the pinch clamp closed using a pair of forceps 16 Fill the tissue chamber with an additional 35 mL of complete culture medium with FBS and P S added 17 Cover the tissue chamber with a sterile lid 18 Position the tissue chamber within the trav with the clamp end nearest to the plastic screws and secure the tissue chamber into the base bv hand tightening the screw can further tighten once removed from the biosafetv cabinet 106 Page 19 Repeat steps 2 19 until all tissue chambers are secured and ready for transport from the biosafety cabinet to the incuba
110. uires a unit made from biocompatible materials that safely houses the tubing and cells prevents contamination allows gas exchange between the cells and the environment and interfaces effectively with the mechanical conditioning system 37 Page The current design used in the Rolle lab in conjunction with the air pressurization svstem contains a number of flaws The design fails to secure the silicone tubing at both ends Fixing the silicone tube at both ends and keeping it taut prevents the tube from moving and ensures that the displacement of water through the tube leads to inflation and distension Additionallv the current design offers no outlets or means for waste removal and media exchange 1 Double threaded cartridge The first design alternative was based on the tissue chamber from the air pressurization design which uses a series of polvstvrene Falcon conical tubes Because these units are small and create isolated environments for each tube and set of cells there are Figure 11 Double threaded cartridge manv possibilities for a high throughput assembiv The design consists of a hollow tube Figure 11 1 with threaded caps on each end 2 One cap would permanentiv house the distal end the silicone tube 3 The second cap would function much like the current design as it would be interfaced with a fitting that leads to the pump svstem 4 A lock and kev pin mechanism 5 fixes the proximal end of the silicone tube to thi
111. week and 4 weeks total Mauck 2000 found a sixfold increase in equilibrium aggregate modulus over controls and matrix elaboration due to heightened ECM synthesis over time for conditioned samples Syedain 2009 created tissue engineered heart valves TEHVs by entrapping dermal fibroblasts into molded fibrin gels left to statically incubate for 2 weeks These TEHVs were then strained incrementally at 5 1 week 10 275 week and 15 3 week over a 3 week period in a custom built bioreactor at a frequency of 0 5 Hz Syedain found that after cyclic stretching collagen and cellular alignment was primarily circumferential which correlates with native TEHVs Collagen also appeared more organized into mature bundles at a density 86 greater than static TEHVs Tensile strength and elastic modulus were also improved by over 97 and 77 respectively compared to static TEHVs Mechanical properties of tissue engineered constructs such as tensile strength elastic modulus and vessel stiffness have been improved by mechanical conditioning Hahn et al 2007 Diamantouros et al 2009 Freed et al 2006 Fujiwara et al 2003 Gimbronejr et al 1999 Isenberg et al 2006 Isenberg et al 2003 Konig et al 2009 Ku et al 2006 Niklason et al 1999 Schutte et al 2010 Hahn 2007 used mouse smooth muscle progenitor cells within poly ethyleneglycol based hydrogels with adhesive ligands and a collagenase degradable sequence to cre

Chapter 1 - Worcester Polytechnic Institute

Contents

Download Pdf Manuals

Related Search

Related Contents