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Nitric Oxide Production by Menical Explants Following

1. 0 1000 2000 3000 4000 5000 6000 7000 8000 Time sec Figure 3 1 Representative data of Pressure vs Time displays the stress relaxation characteristics of meniscal explants Regression equations with R values are displayed for the 5 and 20 strain test The data shows significant relaxation within the first 1000 cycles of the 7200 cycle test The test frequency was 1 Hz for a duration of 2 hrs 53 Percent Strain vs Time For Load Control Animal 12 y 3 2441Ln x 7 9143 R 0 9781 0 1 Mpa 0 05 Mpa w Log 0 1 Mpa og 0 05 Mpa y 1 1771 Ln x 2 0485 R 0 8419 0 1000 2000 3000 4000 5000 6000 7000 8000 Time sec Figure 3 2 Representative plot of load control tests displayed creep characteristics with the plot of compressive Strain vs Time Regression equations and R values are displayed next to each data set These results show large increases in compressive displacement within the first 1000 cycles At the end of the test the 0 1 MPa test required 0 4 mm more compression than the 0 05 MPa test _____ Pressure MPa Strain Start End 596 0 166 0 108 0 038 0 010 Strain 10 1 141 0 103 0 046 0 010 Press MPa Start 15 2 185 0 827 0 035 0 026 0 05 2 6 0 53 11 6 1 36 20 3 548 0 429 0 128 0 020 0 10 3 0 0 12 201 1 45 A B Table 3 1 A Average and standard deviation p
2. 13 14 15 16 17 18 19 20 21 22 23 Place frame dish plunger cap and tools in the culture hood and sterilize Assemble the components into the frame as shown in Figure 1 and place the explant in the center of each well top side up Maintain order of explants in each well Well 1 is the well with alignment holes on each side Lower the plunger into contact with the explants keeping the cap suspended above with Alan wrench Fill each well with 400 ul of flow media using pipette Lower cap remove from frame and bring to bioreactor keeping it level and steady to prevent spilling from wells Attach the bottom pin first and then manually lower the actuator to match the top hole and attach top pin Turn the balance knob on channel 2 of the 2100 system until both light at the top of channel 2 are extinguished Click the R button on the tool bar to run the test When test is complete remove top pin raise actuator D 70000 and remove lower pin before bringing back to the culture hood Remove samples and media as desired and place in 24 well plates with post comp media 2 FBS e Explants need to be cut into superficial and deep zones using tweezers and scalpel e Weight each explant half with scale placed in incubator and put in separate wells in 24 well plate e Add 1 ml of flow media into each well e Place into incubator with CO supply Incubate 24 hours After incubation time immediately se
3. 2 3 2 Accuracy Evaluation of the System Frame alignment and machining of the parts determined how accurately the system produced even pressure on all six wells The length of each compression rod was measured using a micrometer with 2 54 um resolution Measurements of all of the wells were taken to ensure that they were all the same depth using a dial indicator with a resolution of 25 4 um The top surface of the dish was also measured using a dial indicator to prove that the top surface would be perpendicular to the axis of the load cell and actuator Once these measurements were taken to prove the geometry was correct ultra low pressure film Sensor Products Inc East Hanover NJ was used to measure well pressure during compression Pressure film analysis was done using Scion Image National Institute of Standards and Technology Gaithersburg MD to measure the density of the pressure 30 film samples When using the pressure film repeatability tests were performed to determine the precision of the pressure film The repeatability of the film was determined by loading the film in a materials testing machine Instron Corp Canton MA to 70 2 N target load This was repeated seven times The film was placed on top of a 13 66 mm diameter by 3 mm thick piece of rubber which was resting on the lower platen The upper platen 2 cm square was lowered to the surface of the film and compressed to the target load of 70 N corresponding to a pre
4. knees shock absorbing capacity by 20 1 2 In Vivo Loading Environment The meniscus experiences a complex loading due to its form and function as a weight bearing structure and joint stabilizer Their location between round femoral condyles and the tibial plateau creates compressive and tensile forces in the tissue matrix The loading is further complicated during joint flexion and extension as the tibia rotates and the knee locks during the screwed home process This occurs when the knee reaches full extension and is actually in a few degrees of hyperextension increasing the load on the anterior portion of the meniscus However the menisci experience the highest level of loading during 0 flexion 1 11 16 Overall the meniscus experiences up to four times body weight a range of 0 3000 N of compressive loading during walking 17 The applied forces generate both a horizontal and vertical component on the superior surface as previously described The vertical components of force on the superior surface are balanced by the vertical components on the inferior surface generated by the tibial plateau This balance of vertical forces causes compressive stress in the meniscus and holds it tightly between the femur and tibia during high levels of joint load The horizontal force component is created by the rounded femoral condyle and matching concave superior surface of the meniscus This force component acts to displace the meniscus radia
5. 131 p 279 87 Kenny C Radial displacement of the medial meniscus and Fairbank s signs Clin Orthop 1997 339 p 163 73 Ahmed A M and D L Burke In vitro measurement of static pressure distribution in synovial joints Part I Tibial surface of the knee Journal of Biomechanical Engineering 1983 105 3 p 216 225 Collier S and P Ghosh Effects of transforming growth factor beta on proteoglycan synthesis by cell and explant cultures derived from the knee joint meniscus Osteoarthritis Cartilage 1995 3 2 p 127 38 Bylski Austrow D I et al Displacements of the menisci under joint load An in vitro study in human knees Journal of Biomechanics 1994 27 4 p 421 431 Leslie B W et al Anisotropic response of the human knee joint meniscus to unconfined compression Proc Inst Mech Eng H 2000 214 6 p 631 5 Grana W A S Connor and S Hollingsworth Partial arthroscopic meniscectomy a preliminary report Clin Orthop Relat Res 1982 164 p 78 83 Maquet P G A J Van de Berg and J C Simonet Femorotibial weight bearing areas Experimental determination J Bone Joint Surg Am 1975 57 6 p 766 Fle Kurosawa H T Fukubayashi and H Nakajima Load bearing mode of the knee joint physical behavior of the knee with or without menisci Clinical Orthopaedics and Related Research 1980 149 p 283 290 Walker P S and M J Erkman The role of the menisci in force transmission across the knee Clinical Ort
6. Post compression 24 well microplate tweezers scalpel frame post compression media 100 1000 ul pipette and tips A Autoclave Sterilize 1 Autoclave as much of the equipment as possible following the autoclave protocol 2 Sterilize inside of culture hood using spray bottle of 75 isopropanol and wipe down Sterilize the remaining equipment by spraying with alcohol and placing in the culture hood B Make Growth and Flow Media Noe Growth Media 44 5 DMEM F 12 10 FBS 1 Penn Strep Post Comp Media 48 5 DMEM F 12 2 FBS 1 Penn Strep Place all ingredients into 37 C water bath for 15 min Mix media in culture hood e 20 ml growth media per animal e 40 ml post comp media per animal Make sure media is 37 C before use with any tissue 103 C Dissection and Explant Removal 1 Dissect fresh porcine knee in culture hood using sterilized tools only 2 Using cleans scalpel and tweezers remove medial and lateral meniscus and place ge el in dish containing sterile Phosphate Buffered Saline 1X PBS For explant removal place meniscus on clean surface Plexiglas Remove 6 outer and 6 inner explants from each meniscus e Place sharp dermal punch flat against top surface of meniscus and cut using turning motion e Push tissue from punch into microtome using Alan wrench e Using razor and sawing motion trim explants removing as little top surface as possible e Remove from microtome using tweezers Plac
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8. as well as the constant p 0 000 while the linear term was not significant p 0 459 55 NO Production bv Superficial zone with Displacement Control NO Production 225 5 30 66 Strain Level 1 972 Strain Level 2 800 Regression 95 CI 700 600 R Sq 43 5 500 P 0 000 400 300 200 NO Production uM g 100 Strain Level 9 0 Figure 3 3 Displacement control graph showing quadratic fit to NO produced bv superficial zone of explants with 9590 confidence interval displaved R 0 435 For 0 n 8 5 n 6 10 n 6 15 n 6 20 n 4 56 NO Production bv Deep zone with Displacement Control NO Production 234 6 19 40 Strain Level 1 158 Strain Level 2 500 Regression 95 CI 3 00 R Sq 21 3 P 0 040 300 2 t 3 B 200 i A 2 100 Strain Level 9 0 Fiqure 3 4 Displacement control graph showing NO production of deep zone of explants compared to a quadratic fit with 95 confidence interval R 0 213 0 n 8 5 n 6 10 n 6 15 n 6 20 n 4 Results from the 0 05 MPa and 0 1 MPa load controlled tests were not statistically significant 0 MPa p 0 898 0 05 MPa p 0 361 0 01 MPa p 0 252 for comparison of superficial to deep zones using paired t test There were also no significant terms in the regression equations for the data other than the constants The trends show higher NO production than the 15 strain control te
9. p 460 6 Roughley P J and R J White The dermatan sulfate proteoglycans of the adult human meniscus J Orthop Res 1992 10 5 p 631 7 Peters T J and I S Smillie Studies on the chemical composition of the menisci of the knee joint with special reference to the horizontal cleavage lesion Clin Orthop 1972 86 p 245 52 Ghadially F N J M Lalonde and J H Wedge Ultrastructure of normal and torn menisci of the human knee joint J Anat 1983 136 Pt 4 p 773 91 Alberts B et al Molecular Biology of the Cell Third ed ed M Robertson 1994 Garland Publishing Inc 1291 Bird M D and M B Sweet Canals in the semilunar meniscus brief report J Bone Joint Surg Br 1988 70 5 p 839 Duncan R L and C H Turner Mechanotransduction and the functional response of bone to mechanical strain Calcif Tissue Int 1995 57 5 p 344 58 Martinac B Mechanosensitive ion channels molecules of mechanotransduction J Cell Sci 2004 117 Pt 12 p 2449 60 Grodzinsky A J et al Cartilage tissue remodeling in response to mechanical forces Annu Rev Biomed Eng 2000 2 p 691 713 LeGrand A et al Interleukin 1 tumor necrosis factor alpha and interleukin 17 synergistically up regulate nitric oxide and prostaglandin E2 production in explants of human osteoarthritic knee menisci Arthritis Rheum 2001 44 9 p 2078 83 Cao M et al Generation of nitric oxide by lapine meniscal cells and its effect on matrix
10. 0 077 0 069 0 059 0 183 0 12 0 127 0 092 7 464539 11 36525 9 946809 8 173759 30 15957 18 98936 20 2305 14 02482 116 999 195 9526 179 2218 152 7805 407 5618 395 6117 325 7729 286 2209 0 0632 0 0717 0 08 0 051 0 0653 0 0724 0 0729 0 0675 0 11 0 094 0 074 0 066 0 068 0 109 0 066 0 068 17 21631 14 37943 10 83333 9 414894 9 769504 17 03901 9 414894 9 769504 272 41 200 55 135 4167 184 6058 149 6095 235 3454 129 1481 144 7334 0 0704 0 0534 0 0872 0 0475 0 0661 0 0605 0 068 0 058 0 095 0 073 0 094 0 078 0 112 0 122 0 082 0 069 14 55674 10 65603 14 37943 11 54255 17 57092 19 34397 12 25177 9 946809 206 7718 199 5511 164 9018 243 0011 265 8233 319 7351 180 1731 171 4967 0 059 0 073 0 0754 0 0546 0 057 0 0504 0 0592 0 0591 0 098 0 112 0 073 0 076 0 173 0 113 0 131 0 068 15 08865 17 57092 10 65603 11 18794 28 38652 17 74823 20 93972 9 769504 255 7399 240 6976 141 3266 204 9074 498 0092 352 1474 353 7114 165 3046 0 0631 0 0657 0 0652 0 0516 0 124 0 062 0 113 0 061 19 69858 8 705674 17 74823 8 528369 312 1804 132 5064 272 2121 165 2785 Table B 4 This table shows the weight of the explants in the top section then the microplate reading the calculated concentration using the calibration and last the concentration normalized by weight of the tissue This data corresponds to the data in Table B 3 113
11. 0326 0 0301 0 0487 0 0182 Avg 0 0207 0 0281 0 0347 0 0251 0 0466 0 0207 List of Figures Figure 2 1 A 2 Dimensional view of the assembly of the plunger dish and cap The attachments to the linear actuator and load cell and the feature of the cap are pictured Figure 2 2 A 2 Dimensional drawing of test frame shows the side and top view of the test system The orientation of the actuator load cell and support frame can be seen Figure 2 3 A picture of the 0 477 MPa pressure film samples are shown Figure 2 4 A picture 0 564 MPa pressure film samples are shown Figure 2 5 Pressure film calibration curve 39 Linear Actuator Quick Disconnect Pin Meniscal Explant Emm dia 5mm thick 1 e 6 Load Cell Baseplate Figure 2 1 Plunger Dish Cap assembly The linear actuator is attached to the plunger using a quick disconnect pin The dish is attached to the load cell in the same manner The cap improves alignment of the plunger by utilizing a linear bearing 40 Top plate Alignment Smart Motor Linear Actuator 30 cm Load Cell J Baseplate To signal conditioner PC Support Rod 2 54 cm dia oK Support Rod To signal conditioner 25 cm Top View Support Rod Support Rod Figure 2 2 Test Frame The test frame is composed of two al
12. 4ml vol 1 ul cal AM 4 ml PBS Wrap tube is aluminum foil to keep out light Sample Preparation 1 Using pipette fill 1 column of 96 well plate with PBS 300 ul per well 2 Using scalpel and tweezers cut a thin lt 1mm slice of meniscal tissue keeping my 6 track top and bottom of tissue Cut tissue into top and bottom halves and place wash in wells filled with PBS Transfer tissue into blank wells Cover each piece of tissue with stain 100 ul of each stain May require more stain depending on sample size Wrap plate in aluminum foil and incubate at 37 C for 30 60 min Fluorescent Detection 108 Following incubation place plate in insulated container along with tweezers and microscope slide 2 Wash place the tissue sample is clean PBS 3 Turn on fluorescent lamp computer and monitor Fluorescent lamp must remain on a minimum of 30 min 4 Open the desktop icons DP Controller and DP Manager 5 Place tissue on slide and align in microscope using either the 10x or 20x lens 6 Turn shutter switch on and view under red dead and green live fluorescence 500 nm for red setting 4 600 nm for green setting 3 Focus using red fluorescence first due to image clearity 7 Make sure slide bar on top right of microscope is halfway out allowing eyepiece and camera viewing 8 On DP controller under Capture tab far left click on the play button far left just next to the capture button camera ico
13. 5508 0 5426 0 0272 15 2409 3 0 5591 0 5857 0 5870 0 5718 0 5715 0 5441 0 5698 0 0163 7 8899 4 0 5756 0 5841 0 5818 0 5626 0 5571 0 5730 0 5724 0 0106 4 8354 5 0 5785 0 5548 0 5803 0 5762 0 5702 0 5457 0 5676 0 0142 6 3441 Table 2 3 Displacement accuracy using gap measurement Gap Measurement mm Well 1 Well2 Well3 Well4 Well 5 Well 6 Max Diff Avg Std Dev Test 1 3 0556 3 1013 3 1090 3 1166 3 0353 3 0582 0 0813 3 0793 0 0338 Test 2 3 0353 3 0785 3 0861 3 0734 3 0048 3 0353 0 0813 3 0522 0 0320 Test 3 3 3934 3 4519 3 4468 3 4417 3 3655 3 3858 0 0864 3 4142 0 0370 Test 4 3 4036 3 4493 3 4671 3 4315 3 3782 3 4036 0 0889 3 4222 0 0331 Test 5 3 3985 3 4493 3 4544 3 4519 3 3731 3 4036 0 0813 3 4218 0 0345 Avg Diff 0 0838 38 Table 2 4 Data showing how far each well was from the average for each test The average variation from average for each well is displaved in the last row Negative number show the well had a smaller gap than the average for each test positive values are gaps that are greater than the average Gap Variation Welli Well2 Well3 Well4 Well5 Well 6 Test 1 0 0237 0 0220 0 0296 0 0373 0 0440 0 0212 Test2 0 0169 0 0262 0 0339 0 0212 0 0474 0 0169 Test 3 0 0207 0 0377 0 0326 0 0275 0 0487 0 0284 Test 4 0 0186 0 0271 0 0449 0 0093 0 0440 0 0186 Test5 0 0233 0 0275 0
14. 8mm compression rod and in a 10 mm deep well All surfaces are machined to a smooth frictionless finish to ensure the sample is exposed to pure unconfined compression A verification test has been performed to prove that the system remains accurate in the incubator environment A 2 hour 1 Hz displacement controlled test was run with the incubator at 37 C The displacement accuracy did not change at any point during the testing in the incubator Additional tests were performed to prove that the plunger dish and cap assembly could maintain a sterile environment from the culture hood to the 35 incubator and back A practice run of the test protocol using only culture medium showed no sign of bacteria after four days of culture This is evidence that the system can remain sterile through the testing procedure of explants The bioreactor has some limitations that need to be compensated for This system can only perform unconfined compression As the sample is compressed the top and bottom surfaces of the sample can expand To keep explants from slipping to one side of the compression rods the top and bottom surface of the explant needs to be trimmed to be parallel The design is ideal for an explant that is approximately 6 mm in diameter This is because the compression rod is 8 mm in diameter so the smaller explant will stay under the compression rod as long as it is centered The placement of each sample in the well has to be exact to ensure t
15. Figure A 5 Pressure film impressions made during a repeatability test on the Instron material testing machine at a target pressure of 0 477 MPa 70 psi Pressure Film Repeatability Test 477 MPa Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Test 7 Max Diff Error Density 64 92 70 78 65 04 70 41 78 53 76 87 65 00 13 61 20 96 A Pressure Film Repeatability Test 477 MPa Applied Pressure Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Test 7 Average Std Dev Press 0 4776 0 4769 0 4776 0 4770 0 4775 _ 0 4771 0 4776 0 4773 0 0003 B Table A 2 A Data collected from the repeatability test in term of density measure using Scion Image B Result of the repeatability test using the calibration curve found in A 4 70 Figure A 6 Impressions made at a load level resulting in 0 477 MPa 70 psi 71 Figure A 7 Impressions made at a load level resulting in 0 564 MPa 82 psi 72 477Mpa Denstiv 0 255 Test Rod 1 Rod 2 Rod 3 Rod 4 Rod 5 Rod 6 Average Max Diff Error 1 67 78 74 05 69 24 68 79 73 98 72 37 71 04 6 27 9 25 2 72 01 75 51 72 11 74 36 74 96 68 07 72 84 7 44 10 93 3 67 14 73 10 71 74 70 38 71 35 68 72 70 41 5 96 8 88 4 67 33 73 13 71 22 73 25 75 78 73 33 72 34 8 45 12 55 5 63 55 69 32 71 56
16. Pathol 2003 54 4 p 335 8 Grodzinsky A J et al Cartilage tissue remodeling in response to mechanical forces Annu Rev Biomed Eng 2000 2 p 691 713 Spilker R L Donzelli P S A biphasic finite element model of the meniscus for stress strain analysis in Knee Meniscus Basic and Clinical Foundations V C Mow Arnoczky S P Jackson D W Editor 1992 Raven Press New York Spilker R L P S Donzelli and V C Mow A transversely isotropic biphasic finite element model of the meniscus Journal of Biomechanics 1992 25 9 p 1027 45 Zielinska 3D Finite Element Model of Medial Meniscus Meniscectomy Changes in Contact Behavior Journal of Biomechanical Engineering 2005 Frank E H et al A versatile shear and compression apparatus for mechanical stimulation of tissue culture explants J Biomech 2000 33 11 p 1523 7 45 CHAPTER THREE NITRIC OXIDE PRODUCTION Nitric Oxide Production bv Menical Explants Following Dvnamic Compression Jeffrey A McHenry and Tammy L Haut Donahue 3 1 Abstract This paper describes the dynamic compression of porcine meniscal explants using a custom built tissue compression bioreactor capable of various displacement and load control testing The goal was to better understand the relationship of mechanical stress to nitric oxide production in the meniscus during physiological conditions and determine the identity of nitric oxide producing cells Cyclic compression testing was conduct
17. The actuator remained at this position until the body filler hardened to a rigid body The assembly was removed at which point the body filler was measured using a micrometer this was repeated five times It was also necessary to verify the load program was reading accurately the load it was recording on the computer This was done first by calibration of the load cell by 68 incrementallv adding known weight while recording voltage read bv the oscilloscope and the encoder counts read through the SMI software Calibration curves were made to ensure linear relationships Figure A 4 and A 5 A sample load program was then run and monitored on the oscilloscope to determine if the peak voltage matched encoder counts and the target load Mean Densitv vs Pressure y 1E 06x 0 0002x 0 0132x 0 19 1 4 R 0 9622 0 8 Pressure MPa 0 6 0 4 0 2 0 T T T T T T 1 0 20 40 60 80 100 120 140 160 180 Mean Density Figure A 4 The calibration of the pressure film showing the relationship of mean density measured with Scion Image and pressure in MPa The regression equation is also displayed 69 Pressure MPa Densitv 0 2 3 03 0 4 15 95 0 54 55 18 0 703 100 39 0 757 115 23 0 822 118 52 0 92 116 87 1 04 124 97 1 211 132 69 1 29 150 31 1 48 150 06 1 64 153 Table A 1 Data collected from calibration of the pressure film used to create the calibration curve
18. The data collected required converting the microplate to nitrite concentration using the standard curve produced in the assay The data was then normalized by wet weight of the tissue samples and divided into categories of strain load level for data analysis The following tables show the explants tested the strain load levels the raw data and the conversion to normalized and arranged data 1 2 3 4 5 6 7 8 9 10 11 12 A 0 ALMT ASRL2T AILL2ZT AM4LL2T A2RLIT AALVET A4RM2T A2LM2T AQLLIT AQRLST B 5 AMLM2B A3RL2B AILL2B MAL2B A2RLIB ALMPB AMRMEB A2LMPB A9QLLIB A9RL3B C 10 AILM8T ASRL3T AILL3T A4L3IT A2RL2T AMALM8T AARMST A2M3T A9LL3T A9RL4T D 15 AILMBB A3RL3B AILL3iB AMALL3B A2RL2B AMHLMBB A4RM3B A2LMBB A9LL3B A9RLAB E 20 A2LMAT ASLIZT A2LL2T ASRMBT AIRL3T ASLLAT ASLM2T AIRL2T A9LM2T AQLL2T F 2 A2LMBB A3LI2B A2LL2B A3RMBB AIRL3B ASLLAB A3SLM2B AIRL2B A9LM2B A9LL2B G 30 A2LMST A2RM8T A2LL3T ASRMET AIRLAT ASLMAT ASLLST AIRL3T AMB A9LM6T H 35 A2LMSB A2RM3B A2LL3B ASRMEB AIRLAB ASLMAB ASLL3B AIRL3B AMST A9LM6B 1 2 3 4 5 6 7 8 9 10 11 12 A Std c c 10 10 15 15 5 5 20 0 1 B Std c c 1096 10 15 15 5 5 20 0 1 G Std c c 1096 10 15 15 5 5 20 0 1 D Std c c 1096 10 15 15 5 5 20 0 1 E Std c c 1096 10 5 15 5 5 0 05 c F Std c c 1096 10 5 15 5 5 0 05 c G Std c c 1096 10 15 15 5 5 0 05 c H Std c c 1096 10 15 15 5 5 0 05 c Table B 1 This table shows the arrangement of the first 96 well microplate that was set up for
19. a linear actuator Ultramotion Mattituck NY to compress 6 explants simultaneously in unconfined compression The system is contained within a CO incubator at 37 C Explants were centered in each well and covered with 50 400 ul of test medium 48 5 Dulbecco s modified Eagle s medium 48 5 Ham s F 12 2 Fetal Bovine Serum 1 penicillin streptomvcin The explants were transferred to the bioreactor and preloaded using the weight 132 05 grams of the plunger containing the compression rod Displacement tests were run at 0 n 8 5 n 6 10 n 6 15 n 6 and 20 n 4 strain calculated from the original height of the meniscal explants These explants were taken from both the anterior portion of the medial and lateral menisci of both the left and right knees The range of strains was chosen to encompass strains above and below physiological conditions experienced by an intact menicus The tests ran at 1 Hz in a sinusoidal fashion with time position and load recorded throughout the test using system software SmartMotor Interface Load control tests were run in a similar way to 0 00 MPa n 8 0 05 MPa n 3 and 0 1 MPa n 4 load levels Higher loads levels were investigated on two sets of samples 0 5 and 1 0 MPa however tissue integrity was compromised at the end of the two hour loading regime Again the weight of the plunger 132 05 grams was used as the preload and starting position Samples were loaded at 1 Hz for two ho
20. equivalent to that provided by conventional incubators and that its strain output was uniform and reproducible The system incorporates a linear actuator and load cell aligned together in a frame that is contained within an incubator The actuator has bi directional repeatability of 00762 mm and a uni directional repeatability of 00254 mm The actuator can thrust to 2225 N with speed up to 50 cm sec The load cell has a 8895 N capacity with a sensitivity of 2 225 N Explants Smm in height 6 mm in diameter are contained in a six well aluminum dish that is attached to the load cell A plunger with six Teflon filled Delrin compression rods is attached to the actuator which is rigidly suspended above the load cell System performance analysis showed that the 23 greatest difference in displacement between the wells was 0 0889 mm Out of five tests the maximum difference between each well ranged from 0 0813mm to 0 0889 mm with the same wells producing the greatest difference each time Since this error is consistent adjustments can be made to normalize meniscal explant test results We conclude that this device will be useful in determining the biochemical response of tissue culture explants to dynamic compression 2 2 Introduction Mechanical loading of the meniscus plays a crucial role in the metabolic activity of fibrochondrocvtes 1 5 It is not fully understood how biomechanical and biochemical events interact to produce changes i
21. or by the displacement of the actuator Displacement resolution for the actuator is 4 um because a 2500 count on the encoder is equal to 1 mm 28 Two dimensionallv identical strain gage load cells Interface Scottsdale AZ with two different load capacities were used This allows for a more flexible range of testing combining higher accuracv at low range testing and greater capacitv for high load applications The first had a 1334 N capacitv and the second has an 8896 N capacitv Tests that require loads near or above 1334 N will use the higher capacitv load cell to reduce defection and therefore error Tests run with the lower capacitv load cell will have the advantage of a sensitivity of 1 30 N and a more accurate signal The sensitivity of the higher capacity load cell is 2 17 N A 2100 series signal conditioner Vishay Intruments Raleigh NC was used to amplify the load cell signal to produce a 5 volt signal maximum allowable input voltage to the SmartMotor Interface at the maximum load The choice of load cell is critical because the movement of the actuator during testing needs to represent the displacement of the meniscal explant Excess deflection of the load cell will lead to inaccurate displacement reading through the motor The load cell is centered on a 2 54 cm thick aluminum plate that is the base of the system frame Fig 2 1 A stud with a shoulder turned onto it is threaded into the load cell This stud connects to the alu
22. regulate matrix metabolism In the case of the meniscus that response may be to increase or decrease the production of collagen proteoglycans or other matrix molecules 22 32 33 34 39 The biochemical factors produced by the mechanical stimulation of the meniscus are not fully understood nor are the interactions of signaling molecules Some of the biochemical factors that have been studied in the meniscus and articular cartilage are interleukin 1 IL 1 tumor necrosis factor a TNFa Prostaglandin E2 PGE2 and nitric oxide NO 7 36 40 These factors play a role in matrix metabolism and have been implicated in the onset of osteoarthritis 36 41 IL 1 and TNFa are proinflammatory cytokines that may induce production of the mediators NO and PGE These cytokine have also been associated with up regulation of genes responsible for possible matrix degradation and inflammation of cartilage such as inducible nitric oxide synthase NOS2 and cyclooxygenase 2 COX 2 Genes such as NOS2 and COX 2 are most likely responsible for production of NO and PGE respectively In the meniscus IL 1B significantly increases production of NO 36 41 and PGE 36 IL 17 and TNFa also increases NO production in the meniscus although to a lesser extent These cytokines also produce increased levels of COX 2 and NOS2 with IL 1 and IL 17 producing the greatest amount of NOS2 36 The highest levels of COX 2 are produced when meniscal tissue is incubated wi
23. the meniscus has the greatest compressive strength in the axial direction which is expected 1 4 Composition and Structure The meniscus is a biphasic material that includes an extra cellular matrix filled with interstitial fluid It is composed of approximately 75 water 20 collagen and 5 non collagenous substances such and proteoglycans lipids and cells 1 7 14 23 These components contribute to the specialized structure and function of the meniscus The fluid within the matrix experiences limited flow through the boundaries 24 As load is added to the meniscus the hydrostatic pressure increases within the meniscus making it strong in compression 14 This pressure decreases with time as fluid flows out of the meniscus compressing the matrix When load is removed the matrix returns and fluid flows back into the meniscus returning it to equilibrium This behavior makes the meniscus a natural load bearing and shock absorbing structure The extra cellular matrix is dominated by collagen specifically Type I with small amounts of Type II HI and IV 1 6 23 25 Numerous bundles of circumferentially oriented Type I collagen fibers are dispersed throughout the meniscus giving its highest tensile strength in that direction Figure 1 1 3 12 14 21 23 26 27 Some of these layers extend past the meniscus and form the horn attachments Other collagen fibers are oriented radially and woven into the circumferential bundles Fig
24. the total NO assav kit The top section shows the samples used and the bottom shows what strain load level was represented bv each sample 110 READINGS 1 2 3 4 5 6 7 8 9 10 11 12 0 003 0 039 0 064 0 025 0 03 0 03 0 039 0 039 0 031 0 097 0 072 0 0005 0 143 0 046 0 063 0 031 0 051 0 062 0 045 0 046 0 039 0 086 0 059 0 003 0 301 0 09 0 064 0 027 0 039 0 039 0 05 0 046 0 042 0 072 0 057 0 004 0 452 0 022 0 062 0 035 0 046 0 029 0 043 0 117 0 039 0 081 0 051 0 006 0 576 0 045 0 117 0 039 0 061 0 027 0 097 0 056 0 05 0 07 0 061 0 006 0 733 0 063 0 075 0 048 0 079 0 055 0 083 0 132 0 056 0 111 0 108 0 035 0 815 0 034 0 034 0 027 0 063 0 032 0 066 0 065 0 055 0 069 0 068 0 004 0 96 0 04 0 026 0 034 0 059 0 047 0 058 0 053 0 043 0 078 0 086 0 007 TOMNMOLODP 5 684982 13 01099 1 582418 3 047619 3 047619 5 684982 5 684982 3 340659 22 68132 15 35531 7 736264 12 71795 3 340659 9 201465 12 42491 7 443223 7 736264 5 684982 19 45788 11 54579 20 63004 13 01099 2 168498 5 684982 5 684982 8 908425 7 736264 6 564103 15 35531 10 95971 0 703297 12 42491 4 512821 7 736264 2 754579 6 857143 28 54212 5 684982 17 99267 9 201465 7 443223 28 54212 5 684982 12 13187 2 168498 22 68132 10 66667 8 908425 14 76923 12 13187 12 71795 16 23443 8 322344 17 40659 10 37363 18 57875 32 93773 10 66667 26 78388 25 90476 4 21978 4 21978 2 168498 12 71795 3 6337 13 59707 13 30403 10 37363 14 47619 14 18315 5 978022 1 875458 4 21978 11 54579 8 029304 11 25275 9 787546 6 857143 17 1
25. 0 5881 249 9541 340 9163 322 2558 51 44558 Table B 5 A This is the table for the strain data with the repeat for each meniscal location averaged together The data was used to create the regressions used in Chapter 2 B The is the table for the load data with the repeat for each location averaged to give the data used in Chapter 2 The control samples used for comparison are in B 5A which incorporated control sample take from animals used in both strain and load controlled tests 115
26. 1355 19 45788 Weight g 0 0689 0 0875 0 0562 0 0707 0 049 0 0553 0 0693 0 0658 0 0496 0 0605 0 077 0 044 0 0677 0 084 0 062 0 0473 0 0671 0 0602 0 0524 0 0552 0 0297 0 0742 0 06 0 0655 0 0717 0 0553 0 0612 0 0585 0 0595 0 0621 0 0931 0 032 0 0645 0 07 0 0711 0 0441 0 0711 0 071 0 0634 0 0525 0 0765 0 0618 0 0583 0 0829 0 0606 0 0464 0 0881 0 0523 0 0759 0 0455 0 0574 0 0424 0 063 0 0471 0 0752 0 0516 0 0726 0 0734 0 0516 0 0378 0 0828 0 0825 0 0631 0 0745 0 0655 0 071 0 0915 0 0606 0 0678 0 0862 0 0626 0 0774 0 0628 0 0563 0 0391 0 0515 0 0557 0 0752 0 0432 0 0639 uMg 82 51062 148 697 28 1569 43 10635 62 19631 102 8026 82 03437 50 7699 457 2847 253 8068 100 471 289 0443 49 34504 109 5413 200 4017 157 362 115 2945 94 43491 371 3335 209 1628 694 614 175 3503 36 14164 86 79361 79 28845 161 0927 126 4095 112 2069 258 0725 176 4848 7 554207 388 2784 69 96621 110 5181 38 74232 155 4908 401 4364 80 07016 283 7961 175 266 97 29704 461 8467 97 51255 146 3434 35 7838 488 8215 121 0745 170 3332 194 588 266 6345 221 5671 382 8876 132 1007 369 5667 137 9472 360 0534 453 6877 145 3224 519 0675 685 3112 50 96353 51 14885 34 36606 170 7107 55 47633 191 508 145 3992 171 182 213 5131 164 5377 95 49556 24 23072 67 19395 205 0762 205 353 218 4999 175 719 91 18541 396 1471 304 5051 Table B 2 The top section show the reading produced by the microplate reader The next portion is the concentration as determined by the standard curve produce by column 1 The next secti
27. 1992 Raven Press New York Kobayashi K et al Chondrocyte apoptosis and regional differential expression of nitric oxide in the medial meniscus following partial meniscectomy Journal of Orthopaedic Research 2001 19 p 802 808 Kobayashi K et al The suppressive effect of hyaluronan on nitric oxide production and cell apoptosis in the central region of meniscus following partial meniscectomy Iowa Orthop J 2002 22 p 39 41 McHenry J et al Nitric Oxide Production of Meniscal Explants Following Dynamic Compression Thesis 2005 Ch 2 p 25 45 64 APPENDIX SUPPLEMENTARX INFORMATION ON CHAPTER 2 VALIDATION OF BIOREACTOR 65 A 1 Description of Bioreactor Components and Features The bioreactor used in these experiments is a speciallv design unit with a unique combination of components The svstem is set in a dimensionallv critical frame that able to fit in an incubator The device utilizes a Smartmotor 1720 This is a belt driven linear actuator made by Ultramotion It is capable of thrusting to 500 lbs with bidirectional repeatability of 0 0003 in and a unidirectional repeatability of 0 0001 in at a maximum speed of 20 in sec Attached to the actuator are the compression surfaces contained by an aluminum cap with a linear bearing The plunger is attached to the actuator while an aluminum dish is attached to a load cell from interface Figure A 1 The load cell used was a 300 Ib capacity load cell to increase accurac
28. 2 3 4 13 14 15 Follow the protocol given in the Nitric Oxide assay kit instructions Turn the microplate reader on Switch on back right Log into the computer and open the SOFTmax PRO software on the desktop On the top tool bar that says READ click on the thermometer icon and set the temperature to 37 C Wait until the read out on the upper left reaches 37 C before performing the test On the top menu click on Assays then click on Set Folder Select Directory will pop up double click on SOFTmax PRO 4 0 and in that folder open Basic Protocols Again on the top menu click on Assays and open up Basic Endpoint Protocols In the Plate 1 section click on setup and open setup menu Turn the wavelength to 540 nm Turn automix on at the default 5 sec before first read Click OK in the lower right and close the setup menu Click on Template and open the menu Click and drag the sections of Standards Controls Unknowns and Blanks to each section of wells by the using the pull down menu in the upper left Set the Standards and Unknowns in Series to help with data graphs Close template Click READ and run the experiment 106 LOAD PROGRAM ADJUSTMENT 1 Determine the total Newton force that will be applied bv the SmartMotor Newtons per explant multiplied bv 6 2 Multiply the total Newton force by 0 8073 and then add 3 This puts the force in counts for the SmartMotor to read The addition of three acc
29. 4 Validation Programs The following are programs written in SmartMotor programming language which were used in various wavs during validation of the bioreactor Check current load reading RUN UAI b UAA Rb END checkload sms This program is a simple code used to check the current load measured by the load cell Check load after one compressive displacement RUN O 0 MP A 1000 V 100000 D 500 Change value to increase or decrease compressive displacement G TWAIT UBI b UBA Rb P 0 G TWAIT END findload sms This program is used to check the load after a set displacement before returning to the starting location This program was used during validation due to the high repeatability of the displacement of the actuator I was also used when determining the resistance of samples to different displacements 80 Moves actuator until desire load is reached then returns RUN O 0 resets the preload position to the zero position A 1000 V 100000 je user input for cycles i 0 WHILE i lt j start of load cycling i i 1 C7 f 198 user input for force q 5 position increment value UAI b UAA WHILE b lt f run until voltage reaches user input for force UAI b UAA Deq position increment TWAIT LOOP once loop is ended the load will have been found TWAIT UAI b UAA Rb reports voltage where the max load occured user input for load LOOP P 0 moves actuator back to position where pr
30. 47 0 0671 0 0532 0 073 0 0586 0 0671 0 232 0 174 0 139 0 169 0 154 0 178 0 201 0 122 38 84752 28 56383 22 35816 27 6773 25 01773 29 27305 33 35106 19 34397 952 145 328 6977 475 7054 412 4785 470 2581 401 0007 569 1308 288 2857 0 0528 0 061 0 0803 0 0557 0 0514 0 0707 0 0537 0 0657 0 1 0 074 0 136 0 089 0 031 0 065 0 039 0 037 15 44326 10 83333 21 82624 13 49291 3 20922 9 237589 4 62766 4 27305 292 486 177 5956 271 8087 242 2425 62 43618 130 659 86 17616 65 03881 0 0551 0 0753 0 0713 0 06 0 0607 0 0723 0 0503 0 0847 0 118 0 109 0 095 0 089 0 099 0 127 0 081 0 075 18 63475 17 03901 14 55674 13 49291 15 26596 20 2305 12 07447 11 01064 338 1988 226 2816 204 1618 224 8818 251 4985 279 8132 240 0491 129 9957 0 0883 0 0565 0 0664 0 0683 0 0722 0 0686 0 0703 0 072 0 0607 0 0907 0 0682 0 071 0 0489 0 0671 0 0489 0 0622 0 0638 0 058 0 0555 0 0535 0 074 0 048 0 0621 0 049 microplate reading 0 16 0 09 0 111 0 067 0 131 0 096 0 079 0 075 26 08156 13 67021 17 39362 9 592199 20 93972 14 73404 11 71986 11 01064 295 3744 241 9507 261 9521 140 4421 290 0238 214 782 166 7121 152 9255 0 147 0 142 0 137 0 15 0 038 0 071 0 032 0 024 23 7766 22 89007 22 00355 24 30851 4 450355 10 30142 3 386525 1 968085 391 7067 252 3712 322 6326 342 3734 91 0093 153 5234 69 25409 31 64124 0 055
31. 76 93 68 83 72 05 70 37 13 38 21 05 564Mpa 1 113 40 114 17 113 138 13 138 107 99 113 79 112 60 6 18 5 72 2 110 20 108 44 102 70 114 67 98 25 109 59 107 31 16 42 16 71 3 111 08 115 26 1115 45 113 17 1138 12 1108 31 112 73 7 14 6 59 4 113 76 115 038 114 70 1111 67 110 74 1113 37 113 21 4 29 3 87 5 114 21 1110 32 111447 113 86 112 92 10862 112 40 5 85 5 39 A 477MPa Pressure MPa Test Rod 1 Rod 2 Rod 3 Rod 4 Rod 5 Rod 6 Average Std Dev Error 1 0 4773 0 4768 0 4771 0 4771 0 4768 0 4768 0 4770 0 0002 0 0912 2 0 4768 0 4769 0 4768 0 4768 0 4769 0 4772 0 4769 0 0001 0 0788 3 0 4773 0 4768 0 4769 0 4770 0 4769 0 4771 0 4770 0 0002 0 1108 4 0 4773 0 4768 0 4769 0 4768 0 4769 0 4768 0 4769 0 0002 _ 0 1056 5 0 4778 0 4771 0 4769 0 4771 10 4771 0 4768 0 4771 0 0003 0 1986 564 MPa 1 0 5732 0 5783 0 5715 0 5715 0 5425 0 5758 0 5688 0 0132 6 6006 2 0 5541 0 5447 0 5194 0 5816 0 5047 0 5508 0 5426 0 0272 15 2409 3 0 5591 0 5857 0 5870 0 5718 0 5715 0 5441 0 5698 0 0163 _ 7 8899 4 0 5756 0 5841 0 5818 0 5626 0 5571 0 5730 0 5724 0 0106 4 8354 5 0 5785 0 5548 0 5803 0 5762 0 5702 0 5457 0 5676 0 0142 6 3441 B Table A 3 A Density measurements using Scion Image for all the impressions made on ultra low pressure film with 255 representing a saturated sample The average density standard deviation and percent error for each test are calculated B The data from table
32. A is converted to pressure using calibration results from figure A 4 73 200 180 160 140 120 100 80 Encoder Counts 60 40 20 Load Cell Calibration y 0 8073x 3 7455 50 100 Load N 150 200 250 Figure A 8 Calibration of load cell relating encoder counts to known loads applied 74 Load vs Voltage 1000 900 y 3 8424x 4 8714 S E o D S gt 0 50 100 150 200 250 Load N Figure A 9 Calibration of the load cell relating known loads applied to voltage measured on the oscilloscope Load Ibs 0 4 0 0 1 4 4482 8 20 3 2 8 8964 11 37 5 3 13 3446 15 54 7 4 17 7928 18 71 9 10 44 482 38 167 20 88 964 76 363 30 133 446 110 525 40 177 928 149 713 50 222 41 183 831 Table A 4 Table showing data used to create calibration of load cell in A 5 and A 6 75 A 3 Validation Protocols LOAD CELL REPLACEMENT AND CALIBRATION PROTOCOL Equipment List 1 Weights 2 3 16 Alan wrench 3 Adjustable wrench 4 Oscilloscope Load Cell Replacement 1 Unscrew lower attachment post from load cell 2 Remove all alan bolts with 3 16 alan wrench 3 Disconnect load cell cable 4 Replace load cell with dimensionally similar load cell 5 Orient load cell to align wires and tighten down alan bolts evenly 6 Tightly screw in the lower attachment post 7 Assemble dish plunger and cap and secure on l
33. NITRIC OXIDE PRODUCTION OF MENICAL EXPLANTS FOLLOWING DYNAMIC COMPRESSION BY JEFFREY A MCHENRY B S M E Michigan Technological University 2005 A THESIS Submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE IN MECHANICAL ENGINEERING MICHIGAN TECHNOLOGICAL UNIVERSITY 2005 Jeffrey A McHenry NITRIC OXIDE PRODUCTION BY MENISCAL EXPLANTS FOLLOWING DYNAMIC COMPRESSION Jeffrey A McHenry Department of Mechanical Engineering and Engineering Mechanics Michigan Technological University 2005 ABSTRACT Meniscal fibrochondrocytes have been suspected of producing nitric oxide in response to dynamic compression The relationship of compressive strain and compressive stress to nitric oxide production in meniscal explants has not yet been characterized It may be true that physiological strain and pressure conditions may decrease nitric oxide production compared to the unloaded state thus reducing the harmful affects that nitric oxide has on matrix metabolism in the meniscus It may also be true that overloading as well as unloading may produce an up regulation of nitric oxide when compared to physiological conditions The identity of nitric oxide producing cells in the meniscus is also still unclear The chondrocytic cells found in the deep zones of the meniscus have been shown to produce nitric oxide in articular cartilage while the fibroblastic cells in the superficial zone have recently bee
34. RUN2 Run followed bv 7 enables program not to run when the motor is first powered up UBI opens the A D port containing the load cell signal v UBA this will be the voltage in counts from the load cell c9 s 0 user input for the preload ss 25 position increment value for finding the preload MP specifies position control A 100 V 1000000 TWAIT waits until actuator is done moving to continue running the program A 10000 V 1000000 At this point the preload has been found WAIT 8000 wait 2 seconds O 0 sets the zero position to the position where the preload exists CLK 0 resets the clock RP UAI b UAA Rb reports the position at start of test should alwavs be 0 RCLK reports the clock should always be 0 GOTO1 this tells the program to go to label C1 POSITION CONTORL IN THE NEXT PORTION OF CODE C1 C8 x 7200 user input for number of cycles a 0 WHILE a lt x a a 1 A 775 this is the needed acceleration to achieve 1Hz loading C7 V 44000 P 1250 VARIABLE SPEED DEPENDING ON USER DISPLACEMENT USER INPUT POSITION u P sets u equal to the user position input G TWAIT 34 RP reports position at the displaced value UAI b UAA RCLK reports clock at displaced value D u moves the opposite distance of what the user inputed for displacement TWAIT waits til actuator reaches the preload position RP reports position at the preload position UAI b UAA RCLK reports clock a
35. Standard Curve 1 2 y 0 0282x 0 0129 1 e e 3 e D Microplate Reading o BR 0 2 0 T T T T T T T 1 0 5 10 15 20 25 30 35 40 Concentration uM Figure B 3 This is the standard curve created for the second nitric oxide assay This was used to convert the microplate readings to concentration in uM 114 Control 388 5623 74 13028 162 0236 256 4978 215 5861 292 1962 185 8368 148 1104 AVG 215 3679 STD DEV 96 98882 Top B T Averages uMo 5 B 54 01258 58 90908 126 6209 158 5813 123 742 427 562 338 6613 98 038456 135 0769 203 55691 141 27 112 6963 494 9081 231 834 134 3905 148 8925 261 6536 261 0279 221 2761 174 3665 224 276 152 5822 199 5624 135 3564 78 5898 124 124 Averages uM g 05 Mpa Bottom 198 5333 222 8025 203 9133 192 5779 295 3675 366 2903 AVG 232 6047 260 5569 STD DEV 54 42073 92 80652 B 10 T B 32 14927 59 65563 65 9898 99 64733 64 4998 110 0297 158 5271 287 3214 74 30617 97 84888 228 3679 183 8537 104 04 139 7261 74 10579 82 92744 1 Mpa Top 519 6944 425 8603 139 3788 215 1458 325 0198 177 5781 Bottom 344 6432 258 726 190 0394 192 2144 246 4057 72 8383 15 70 74238 79 13945 324 9571 264 8534 249 7681 176 4807 194 3235 108 8958 119 572 181 3575 207 7721 222 3908 233 4162 146 9828 198 5819 53 54627 20 713 9252 222 9982 366 6674 415 3173 209 6365 327 5648 37
36. This will be done using Ultra Low pressure film which will be placed between each compression rod and each sample to be compressed The film will be analvzed using Scion Image to check the densitv The densitv values will be related to pressure using calibration and the results will determine the accuracv of even well pressure Pressure Film Calibration e Using a punch cut six identical cylindrical pieces of sample material rubber of constant thickness Measure dimensions of samples e Cut matching samples of Pressurex Ultra Low pressure film 28 psi 85 psi e Place the sample material on the lower platen of the Instron 8872 tensile testing hydraulic press and place the pressure film on top of the sample The dull sides of each layer of film have to be in contact e Change the display of the Instron to read load measured in Newtons Using the fine adjustment manually lower the upper platen and compress to a known load within the pressure range of the pressure film Immediately unload e Remove and discard translucent sheet of pressure film e Perform 12 compressions using different loads each time Load at increments of 5 psi going from 30 to 85 psi e Using Scion Image measure the mean density of each pressure film compression Use the results to create a calibration curve of density vs pressure 78 Testing Well Pressure Place sample material rubber in the bottom of each well Make sure each rubber sample is sitting fl
37. after exposure to diverse stimuli such as inflammatory cytokines NOS2 or iNOS is the inducible form of nitric oxide synthase that is responsible for producing NO in tissues such as the meniscus and articular cartilage 36 37 39 43 44 Nitric oxide is an important molecular messenger in mechanical signal transduction and has a very short half life of less than 10 seconds at which point it breaks down into stable 16 nitrite and nitrate 7 45 As a short acting signaling molecule NO requires a fast acting signaling pathway in order to produce cellular response Furthermore NO should only act on nearby cells and proteins How this molecule is produced and its interaction with cytokines cells and tissue are important in determining the affect it has on articular cartilage and the meniscus NO has been found in high levels in the synovial fluid of patients with osteoarthritis and rheumatoid arthritis 39 46 Osteoarthritic cartilage has been shown to produce NO spontaneously Healthy articular cartilage and meniscus have been studied to determine what loading and biochemical conditions produce an up regulation in nitric oxide production by cells 7 37 38 40 43 45 47 49 These studies in general have shown that both chondrocytes and fibrochondrocytes spontaneously produce NO Also dynamic compressive strain appears to increase NO production in both articular cartilage and meniscal explants It is still unknown what levels of physio
38. and how nitric oxide mediates matrix metabolism in the meniscus NO production should be measured during normal physiological loading and strain conditions as well as pathophysiological conditions 19 1 8 Hvpotheses and Specific Aims Osteoarthritis is a condition characterized bv the degradation of articular cartilage and is positivelv associated with the presence of knee meniscectomv The pathogenesis of osteoarthritis is not well understood however it is well accepted that the removal of meniscal tissue associated with the mensicetomv procedure serves to increases the forces experienced by the remaining meniscal tissue This increase in meniscal loading produced by the menisectomy has been postulated to underlie the etiology of this disorder Elevations in nitric oxide production have been positively correlated to joint inflammation matrix degradation and osteoarthritis progression 37 39 41 44 46 48 50 Evidence exists suggesting that mechanical compression up regulates NO production in meniscal explants 7 40 however neither the relationship between physiological loading conditions and meniscal nitric oxide production nor the identity of nitric oxide producing cells if present has been established The objective therefore of this study is to determine how loading influences meniscal nitric oxide production The working hypothesis for this study is that increased mensical loading will be associated with an augmented nitric oxide pr
39. are the most abundant form in the adult human meniscus 5 27 28 Proteoglycans that do not aggregate to hyaluronic acid are smaller contain dermatan sulfate and are either decorin or biglycan Decorin has one dermatan sulfate chain and biglycan has two Approxmimately 75 of the dermatan sulfate proteoglycans in the meniscus are decorin 28 Proteoglycans are woven into the collagen matrix and distributed in an inhomogeneous manner allowing fluid flow in the meniscus Therefore these molecules contribute in numerous ways to the compressive strength of the meniscus Some other elements present in small amounts in the meniscus are elastin uronic acids hexosamine and ash 29 These elements form very small amounts of the extra cellular matrix The cells in the meniscus which are responsible for proper maintenance of the matrix are called fibrochondrocytes 1 5 Cellularitv and Nutrition The meniscus is composed of two distinct species of fibrochondrocvtes that are generallv distributed through the extra cellular matrix in a homogenous manner 30 The first species resembles fibroblasts and is found toward the superior surface The second tvpe resembles chondrocvtes and is found closer to the inferior surface of the meniscus Each tvpe has a different phenotvpe function and distribution throughout the extra cellular matrix These fibrochondrocvtes produce the components needed to maintain the fibrous tissue structure Fibroblasts are a t
40. as 100 um Thus this system 27 is not feasible for larger scale testing on 5mm meniscal explants which require displacements of 0 5mm for 10 strain The goal of this study was to design a tissue engineering bioreactor that cyclically compresses meniscal explants to physiological stresses and strains The system had to meet the following criteria 1 apply and measure compressive load up to 350 N per explants 2 create a cyclic compression test using load or displacement control accurate to within 1 and 3 maintain explants in a physiological environment The subsequent sections describe the design of the system accuracy evaluation and application of the system to explant testing 2 3 Materials and Methods 2 3 1 Design of Bioreactor To create physiological loads the system was based on a belt driven linear actuator made by Ultramotion and Animatics The Smartmotor 1720 Ultramotion Mattituck NY is an actuator that is part of the Bug series of actuators by Ultramotion and utilizes a control package by Animatics The actuator has a maximum stroke length of 5 cm and can thrust to 2225 N It also has a maximum speed of 50 cm sec with bi directional repeatability of 00762 mm and a unidirectional repeatability of 00254 mm Motor control was achieved by using the SmartMotor Interface SMI programs written with SMI programming language This allows the motion of the actuator to be controlled by the signal generated by the load cell
41. at in the bottom of each well with some clearance around the edges Place pressure film on top of each sample Assemble the well plate plunger and lid and connect to load cell and actuator The load cell is connected first and then the actuator is lowered to meet the connection with the plunger Pick a target load within the range of the pressure film to compress the samples To move the actuator refer to the commands in chapter s 2 amp 3 of the SmartMotor user s manual Double Click the Marrow folder on the desktop and open Smartmotor Interface shortcut Open the file titled target load sms Change the voltage value for load to the desire amount To change pressure to voltage determine the number of Newton s on each sample multiply by 6 number of wells and then multiply by 46 This value goes into the f for the user input for force Click the T transmit button When the program is finished transmitting click R run to start the program When the program is finished raise the actuator back up and take out the dish and film cutouts Perform a total of 15 tests at three different loads Scan results and measure density of each well for each test Using calibration results find pressure by using density for each sample and each test Calculate differences in pressure between each well and determine percent error Compare target pressure with pressure found from film and using load from load cell 79 A
42. atan sulfate proteoglycans of the adult human meniscus J Orthop Res 1992 10 5 p 631 7 Tanaka T K Fujii and Y Kumagae Comparison of biochemical characteristics of cultured fibrochondrocytes isolated from the inner and outer regions of human meniscus Knee Surg Sports Traumatol Arthrosc 1999 7 2 p 75 80 Leslie B W et al Anisotropic response of the human knee joint meniscus to unconfined compression Proc Inst Mech Eng H 2000 214 6 p 631 5 Proctor C S Schmidt M B Whipple R R Kelly M A Mow V C Material Properties of the normal medial bovine meniscus Journal of Orthopaedic Research 1989 7 6 p 771 782 Djurasovic M et al Knee joint immobilization decreases aggrecan gene expression in the meniscus Am J Sports Med 1998 26 3 p 460 6 Dowdy P A et al The effect of cast immobilization on meniscal healing An experimental study in the dog Am J Sports Med 1995 23 6 p 721 8 Fink C et al The effect of dynamic mechanical compression on nitric oxide production in the meniscus Osteoarthritis and Cartilage 2001 p 1 8 LeGrand A et al Interleukin 1 tumor necrosis factor alpha and interleukin 17 synergistically up regulate nitric oxide and prostaglandin E2 production in explants of human osteoarthritic knee menisci Arthritis Rheum 2001 44 9 p 2078 83 Cao M et al Generation of nitric oxide by lapine meniscal cells and its effect on matrix metabolism stimulation of c
43. ation of Even Well Pressure Validation of the bioreactor required collecting evidence that the svstem compressed all six explants to the same pressures and strains The first method involved placing pressure film between the surfaces used for compression To do this the pressure film was calibrated in the Instron materials testing machine to pressures from below to above the pressure range of the film Figure A 4 Table A 1 Also several samples were loaded to the same pressure to determine the repeatability of the film Figure A 5 Table A 2 Next the dish was measured using micrometers to make sure the top surface the bottom of the well and the compression rod surfaces were all parallel A 3 8 in thick uniform machined circular piece of steel was added onto the top of the dish At this point a uniform piece of rubber was placed on top of that followed by the pressure film The plunger was then pressed into the pressure film to a specific displacement That was repeated five times at two different displacements These were then compared to the calibration done on the Instron using Scion image to determine the pressure differences between wells A second technique was used to collect a physical measurement of the gap between the bottom of each compression rod and the bottom of each well To do this each well was filled with auto body filler and the plunger was quickly lowered into the aluminum dish until body filler surrounded each compression rod
44. condyle rotates further forward until the popliteal tendon tibial and fibular collateral ligaments are tight 4 20 At this point in extension the meniscal horns add further restriction since the anterior portions of the menisci are tightly wedged between the femur and tibia This is when the knee has screwed home To unlock the knee the popliteus muscle contracts and rotates the lateral femoral condyle posteriorly 20 This is approximately 18 of rotation which occurs in the first 30 of flexion 4 As the knee unlocks and moves in flexion the menisci move with the femoral condyles keeping a large contact area 9 This happens when the meniscofemoral ligaments pull the posterior section of the lateral meniscus in the medial direction The popliteus muscle also pulls the posterior section back over the tibial plateau The medial meniscus is pulled forward during flexion by the deep and superficial medial ligaments The motion of the meniscus as the knee flexes shifts load constantly through the knee The menisci shift constantly with the femur to keep contact area high and maintain their weight bearing function Constant compressive and tensile forces are present on the structure making the material properties particularly important for the menisci to function properly 1 3 Material Properties of the Meniscus The complex loading environment shows that the meniscus requires different compressive and tensile strengths The circ
45. customize each test During the test displacement and load are recorded at 2 Hz min and max of each cycle with a resolution of 0 44m and 1 30 N respectively This system can be utilized to produce useful test data about tissue response to physiological loading ACKNOWLEDGMENT The authors are grateful to the Whitaker Foundation for their financial support 37 Table 2 1 Precision data of ultra low pressure film Pressure Film Repeatablitv Test 477 MPa Applied Pressure Testi Test 2 Test 3 Test 4 Test 5 Test 6 Test7 Average Std Dev Press 0 4776 0 4769 0 4776 0 4770 0 4775 0 4771 0 4776 0 4773 0 0003 Table 2 2 Results of pressure film verification at two different loads 477MPa Pressure MPa Test Rod 1 Rod2 Rod3 Rod4 Rod5 Rod6 Average Std Dev Error 1 0 4773 0 4768 0 4771 0 4771 0 4768 0 4768 0 4770 0 0002 0 0912 2 0 4768 0 4769 0 4768 0 4768 0 4769 0 4772 0 4769 0 0001 0 0788 3 0 4773 0 4768 0 4769 0 4770 0 4769 0 4771 0 4770 0 0002 0 1108 4 0 4773 0 4768 0 4769 0 4768 0 4769 0 4768 0 4769 0 0002 0 1056 5 0 4778 0 4771 0 4769 0 4771 0 4771 0 4768 0 4771 0 0003 0 1986 564MPa 1 0 5732 0 5783 0 5715 0 5715 0 5425 0 5758 0 5688 0 0132 6 6006 2 0 5541 0 5447 0 5194 0 5816 0 5047 0
46. d Figures 2 3 and 2 4 There was 0 0912 to 0 1986 and 4 83 to 15 24 percent error for 0 477 MPa and 0 564 MPa respectively Table 2 2 At higher loads there was an average difference in pressure of approximately 8 2 percent but at lower loads this error showed an average difference in pressure of approximately 0 18 Due to the variability of the pressure film at higher pressures these results alone were inconclusive in determining the accuracy of the system 2 4 2 Determination of Displacement Accuracy The micrometer measurements from the first samples of body filler showed that the greatest difference between any of the wells was 0813 mm with well 5 having the smallest gap The second and fifth tests gave the same results as the first test Test three showed 0864mm and test four showed 0889mm with both showing well 5 to have the smallest gap All five tests were run with the same plunger dish orientation and all tests showed compression rod 5 to produce a smaller gap The percent error can be calculated from the amount of displacement that will be run during each test If a test is run with a maximum displacement of 0 5 mm then there is approximately 16 76 difference in compression on average For a 0 5 mm target compression all the explants would be compressed between 0 4581 mm and 0 5419mm A 1 mm displacement test would only see an 8 38 difference in compression on average For a target of Imm compression 33 all explants would be co
47. d Understanding this mechanism is crucial to improving medical treatment to common meniscal injurv Acknowledgement The authors are grateful to the Whitaker foundation for their financial support 62 REFERENCES 1 2 10 11 12 13 14 15 16 17 Aagaard H and R Verdonk Function of the normal meniscus and consequences of meniscal resection Scand J Med Sci Sports 1999 9 3 p 134 40 Ahluwalia S et al Distribution of smooth muscle actin containing cells in the human meniscus J Orthop Res 2001 19 4 p 659 64 Collier S and P Ghosh Effects of transforming growth factor beta on proteoglycan synthesis by cell and explant cultures derived from the knee joint meniscus Osteoarthritis Cartilage 1995 3 2 p 127 38 Gershuni D H A R Hargens and L A Danzig Regional nutrition and cellularity of the meniscus Implications for tear and repair Sports Med 1988 5 5 p 322 7 Ghadially F N J M Lalonde and J H Wedge Ultrastructure of normal and torn menisci of the human knee joint J Anat 1983 136 Pt 4 p 773 91 Peters T J and I S Smillie Studies on the chemical composition of the menisci of the knee joint with special reference to the horizontal cleavage lesion Clin Orthop 1972 86 p 245 52 Roughley P J et al The presence of a cartilage like proteoglycan in the adult human meniscus Biochem J 1981 197 1 p 77 83 Roughley P J and R J White The derm
48. dered normal activity actually decreases NO production in the meniscus The data also shows that overstrain as experienced by meniscectomized tissue up regulates NO above both the zero load strain condition and physiological levels of strain These findings challenge the conclusion of others that dynamic compression increases NO production 14 19 The 5 and 10 strain levels are physiological and appear to reduce NO production by meniscal explants compared to 0 strain If this is in fact the case for the meniscus moderate levels of NO may be required for maintenance of normal healthy tissue NO is typically linked to tissue degradation but its role in matrix metabolism is not completely understood It is possible that NO modulates matrix resorbtion to allow for the addition of newly remodeled matrix constituents by 60 fibrochondrocvtes If this were the case excessive NO production would lead to an unbalance of matrix metabolism in favor of resorbtion and result in tissue degradation Differences in response of superficial and deep zone to compression were not demonstrated in this studv Previous studies showed that fibroblastic cells produced higher NO levels than the deep chondrocytic cells 14 19 In this study the trends produced by both regions were similar Further investigation is required to reconcile differences between this investigation and previous There were several limitations to this study that prevent further defining t
49. dies employing 24 hours of cyclic compression at a load resulting in 10 strain have shown that increasing compressive strain in the meniscus leads to an up regulation of NO 14 19 Jn vivo partial mensicectomy results in elevated strain levels in the meniscus 23 24 and has been shown to lead to osteoarthritis OA Portions of the inner two thirds of the tissue is often removed leaving the remaining tissue to carry increased load Strain increases from approximately 10 for an intact meniscus to up to 30 strain for a partial meniscectomy where 60 of the inside tissue is removed This increased strain may be partially responsible for the high level of NO found in the osteoarthritic knee Indeed in vivo experimental osteoarthritis models including partial meniscectomy and ACL transection have been shown to result in increased NO liberation in the meniscus 25 21 In addition Kobayashi et al 2001 showed that following a partial medial meniscectomy in rabbits there was a spatial variation in NO production with the tissue adjacent to the location of the meniscectomy producing significantly more NO than the peripheral meniscal tissue 25 Similar spatial trends were seen with iNOS expression 25 48 The source of NO in the meniscus is fibrochondrocvtes However these cells are represented bv two distinct populations separated bv laver The superficial laver is composed of cells that appear and behave more like fibroblasts while the de
50. e accuracy of the system involved measuring the gap between the bottom of the compression rods and the bottom of the wells while the system was assembled into the bioreactor This was done using auto body filler and a cream hardener that when mixed together harden to form a rigid body The actuator was used to compress the body filler until the gap between the bottom of the compression rod and well bottom was filled The actuator remained at this position until the body filler hardened completely The plunger was then removed along with the pieces of body filler A micrometer 2 54 um resolution was then used to measure the thickness of the body filler This process was repeated 5 times with the same plunger and dish orientation 2 4 Results 2 4 1 Accuracy Evaluation of the System The greatest difference in length between any of the compression rods was 0381 mm All of the well depths were within 0 0254 mm of each other when measured with a dial indicator The well depths were measured from the top surface of the aluminum dish which was flat to within 0 0254 mm 32 The densities produced for all the pressure film were compared to the calibration of the pressure film to determine the pressure The results of the repeatability test showed there was an average of 0 4773 MPa with a standard deviation of 0 0003 MPa Table 2 1 The pressure film from both the 0 477 MPa and 0 564 MPa tests appear to show equal pressure in each well for each loa
51. e in 96 well microplate keeping top surface up Fill wells with 300 ul of growth media Label plates and place in incubator for 48 hours After 24 hrs remove old media with pipette and refill with fresh growth media D Compression Testing Compression Program 1 2 3 8 On PC open marrow folder on desktop and double click on SmartMotor Interface Icon Go to File Open and select compression program out of C Program File Program Editor The following displacement control files are available for use 5 disp sms 10 disp sms 15 disp Sms 20 disp sms The load control programs are 1 Mpa sms 5 Mpa sms 1 Mpa sms Make sure j equals the number of cycles desired and u equals the displacement desired e 2500 counts Imm is the downward direction e For load 300 Ib load cell f is the load variable and 0 8073 counts 1 Newton Turn the motor on the switch on the power unit on top of the PC and transmit the program by clicking the T button on the tool bar Turn on the 2100 System power by the switch on the lower right below the channel dial Gain should be set to 200x with the dial at 1 35 Channel selector should be on channel 2 Excit switch on channel 2 should be on Bridge volts on meter should read 10 volts For help use 2100 system manual The test is now ready to run after samples are loaded Loading Samples 9 Make sure post comp media is at 37 C 104 10 11 12
52. ed on 6mm diameter explants 5mm in height at a frequency of 1 Hz for two hours Compression magnitudes included 5 10 15 20 strain as well as 0 05 MPa and 0 1 MPa tests compared to a 0 strain OMPa control representing an unloaded state These magnitudes were chosen to cover the range of stress and strain experienced in the normal meniscus and to investigate how unloading and overload affects nitric oxide production Result from testing showed 5 and 10 strain produced less nitric oxide than control samples in both the surface and deep zones of the explants The 15 strain testing showed comparable results to control while the 20 strain testing produced the greatest amount of nitric oxide in both zones Statistical analysis showed a significant 46 quadratic relationship p 0 000 for both zones and no significant difference between means of surface and deep Results from load control provided inconclusive data These findings suggest a complicated relationship between mechanical stress and nitric oxide production Physiological strain levels and durations may reduce nitric oxide produced by meniscal fibrochondocytes 3 2 Introduction The menisci are specialized structures that are vital to normal function of the knee In addition to distributing load from the femoral condyles to the tibial plateau the meniscal attachments aid in maintaining knee joint stability and congruency Meniscal tissue is approximately 75 water Fibrochondocy
53. eload occured unloads sample G END target load sms This program is used to compress as sample to a desired load and then return to the starting location This program was used during validation to apply repeated load to multiple pressure film samples 81 APPENDIX B SUPPLEMENTARX INFORMATION ON CHAPTER 3 NITRIC OXIDE PRODUCTION 82 B 1 Compression Programming Load and displacement control programs were written in SmartMotor programming language The displacement programs compresses to a target displacement and then returns to the starting location The velocity and acceleration are adjusted to achieve 1 Hz motion At the peak and valley of each cycle the SmartMotor Interface SMI software on the PC records load position and time This repeats for a desired number of cycles at which point the actuator returns to the starting position Load control works by displacing a distance determine by the size of the error signal of the difference from the current load to the target load The larger this error signal is the greater the displacement Following each movement the program checks the load signal and moves again a smaller distance for a smaller error signal This loop continues until the target load is reached and then returns to the starting location of the test This program also records the load position and time at the peak and valley of each cycle An example of these programs can be seen below 83 10 Strain
54. emical signals require a pathway such as blood synovial fluid and gap junctions to create a healing response from distant cells Without this pathway the tissue is not capable of regeneration 1 6 Mechanotransduction Mechanotransduction is the mechanism that presumably leads to remodeling in several types of tissue In this process a mechanical signal creates a change in the environment of a cell which produces a biochemical response These chemical 12 responses are carried throughout the tissue serving as paracrine and autocrine signals to produce changes in cellular behavior throughout the tissue This mechanism is responsible for the maintenance of matrix metabolism and the remodeling of many types of tissue The process requires sensor cells a pathway for signal transduction and effector cells to respond to the signaling Mechanocoupling is the transduction of mechanical forces to a form that can be detected by cells 33 Physical stimuli include factors such as tension shear hydrostatic pressure fluid flow and the frequently studied physical condition in the meniscus compression As these physical stimuli are imposed on tissue the extracellular matrix deforms transmitting the mechanical energy to the cells Sensor cells respond to stimuli with various chemical signals with mechanisms that are not completely understood Theory suggests there are multiple ways a cell can sense physical change One way the cell may detect chan
55. ep zones are composed of cells that are more like chondrocytes Fink et al 2001 showed that following mechanical stimulation the surface zone of the meniscus produced higher levels of NO when compared to the deep zone Recently these same research group added IL 1 a proinflammatory cytokine secreted by cells to the media during compression and found a synergistic increase in NO compared to compression alone 19 However in each of these studies only one level of pressure was investigated Interestingly in contrast to mechanical stimulation data direct stimulation with a chemical signal IL 1 in the absence of mechanical loading demonstrated increase NO production in deep zone cells compared to little or no production from the surface zone 16 Currently there is a lack of data relating short periods of physiological strain that may result from walking or exercise to NO production It is our goal to determine how NO production changes through a range of compressive strain that covers physiological levels seen in the intact meniscus and following meniscectomy for periods of normal activity Comparing these results to results with 0 strain will determine how activity compares to inactivity in terms of NO production and therefore meniscal health Another goal is to show how strain relates to load during unconfined compression of meniscal explants Investigating meniscal compression using load and displacement control will accomplish these g
56. ere used to determine the fit of the regression P lt 0 05 was considered significant NO production from the superficial and deep zones was compared for a given load or strain level using paired t tests 3 4 Results Meniscal Compression Data collected from each test confirmed the bioreactor reached target compressive displacement 0 001 mm or load 0 01 MPa during each cycle Displacement controlled tests showed a rapid drop in load within the first 1000 cycles with little change 52 following 4000 cycles Figure 1 For displacement tests below 15 strain load settled at or below 0 05 MPa while the 20 strain test remained above 0 1 MPa throughout the duration of the test The difference of load measured from start to finish can be seen in Table 1A Load controlled tests Figure 2 showed a rapid increase in compressive displacement within the first 1000 cycles The 0 05 MPa load level reached maximum displacement near 3000 cycles and remained at that level to the end of the tests The 0 1 MPa tests reached 18 strain near 3000 cycles but steadily increased to 20 7 strain by the final cycle These differences in strain from start to finish of these tests can be seen in Table 1B Pressure vs Time for Displacement Control Tests 15 20 10 5 e Power 20 e Power 5 Pressure MPa o a y 2 5527 x 8505 R 0 9018 y 0 1784x 01977 R 0 5113
57. f the structure The components necessary for synthesis of the extracellular matrix are provided by the blood and by 11 svnovial fluid There is a larger population of cells near the surface of the meniscus with a decreasing population of cells towards the interior The interior region is only nourished by diffusion of the blood from the periphery and diffusion of synovial fluid from the exterior Cells that do not receive blood supply directly depend on fluid flow within the tissue The nutrients are believed to diffuse through the tissue if the molecules are small enough 24 Fluid is able to move through the articulating surface through canals 10 200 um in diameter 1 32 These canals may play a role in nourishing the tissue even though they are not filled by the blood supply Fluid motion associated with mechanical loading aids in nutrition by creating a greater flow of nutrition to cells The limited nutrient supply to the meniscus is believed to be the reason for its poor healing characteristics 24 The inner two thirds of the meniscus heals poorly and is therefore frequently removed when torn The vascularized portion is usually repaired because the vascularization increases the chances of healing Sutures are often used to close tears which occur in the outer one third of the structure This region is capable of cell proliferation and remodeling The ability of a tissue to remodel depends on the chemical signaling between cells These ch
58. ges is through the activation of mechanosensitive MS ion channels 34 These are gated channels that are found in the membrane of all types of living cells The two basic types are stretch activated and stretch inactivated ion channels 34 both are used for electrical and or chemical intracellular signaling Stretch activated MS channels are controlled by gates that respond to mechanical forces The bilayer model and the tethered model are two theories used to describe gating of these channels 34 In the bilayer model mechanical forces produce tension in the lipid bilayer of the cell membrane which directly gates the MS channels 34 In the tethered model there are direct connections between the cytoskeleton and MS channels 34 Gating occurs when mechanical forces deform the cell and displace the channel gate relative to the cytoskeleton 13 The cvtoskeleton potentiallv plavs a larger role in mechanotransduction than gating It forms a network connecting the extracellular matrix to the nucleus and other organelles found within the cytoplasm Glycoproteins called integrins extend from the actin of the cytoskeleton through the membrane to the extacellular matrix 33 This allows for mechanical signals to be rapidly transmitted from the extracellular matrix to the nucleus possibly altering gene expression Recent studies have shown that deformation of tissue by compression brings distinct changes in cell and nucleus shape 35 Static comp
59. he relationship between compression and NO production These tests were performed using unconfined compression which may not be physiological The results show that physiological strain produces sub physiological stresses experienced in the meniscus throughout the test This may be caused by excessive fluid flow out of the explant due to its unconfined treatment To date only 6 animals have been used for each level of strain n 8 for control 0 and n 4 for 20 and only 3 animals for load levels Recommedations Meniscal location lateral or medial and explant location from anterior to posterior are other factors that may respond differently to compression Other compressions studies using confined compression would be beneficial in determining how compression affects NO production However to completely understand the mechanism of NO production and its role in matrix metabolism chemical factors must be investigated along with mechanical factors One chemical factor of particular interest is interleukin 1 IL 1 for its apparent link to NO IL 1 is a proinflammatory cytokine that is believed to induce NO production Blocking this cytokine during compression may 61 reduce NO production during phvsiological strain for normal activitv periods Such a result could change treatment of meniscal lesions and improve tissue healing In order to create the best healing response bv the meniscus chemical as well as mechanical treatment mav be require
60. he sample stays under the compression rod Once the cap is put on and the test starts running the samples cannot be viewed to determine if they are being compressed correctly The only indication of this is the orientation of the explants when the test is completed and when they are removed Another limitation is the machining of each component within the system The most accurate machining procedures used can create a part within 0 0254mm This is accurate enough for most applications but requires extra compensation for the bioreactor Since the displacements in this system are so small the machine error has to be measured and accounted for The gap measurement using the body filler provides a method for compensation The results show that well 5 has the smallest gap and should therefore produce different results than the other wells Despite the limitation described above the explant compression system has features that are advantageous to tissue compression experimentation The system 36 maintains a sterile environment throughout the assembly and testing procedures The components in direct contact with the tissue sample are made of material that can be autoclaved or rinsed with alcohol Six tissue explants are exposed to uniaxial unconfined compression simultaneously The system can compress these samples using load or displacement control settings Waveforms can be manually programmed into the linear actuator using the interface software to
61. hopedics 1975 109 p 184 92 Seedhom B B and D J Hargreaves Transmission of the load in the knee joint with special reference to the role of the menisci Engineering in Medicine 1979 8 p 220 228 22 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 Welsh R P Knee joint structure and function Clin Orthop Relat Res 1980 147 p 7 14 Tissakht M and A M Ahmed Tensile stress strain characteristics of the human meniscal material Journal of Biomechanics 1995 28 4 p 411 422 Fithian D C et al Exponential law representation of tensile properties of human menisci IMechE 1989 p 85 90 Proctor C S Schmidt M B Whipple R R Kelly M A Mow V C Material Properties of the normal medial bovine meniscus Journal of Orthopaedic Research 1989 7 6 p 771 782 Gershuni D H A R Hargens and L A Danzig Regional nutrition and cellularity of the meniscus Implications for tear and repair Sports Med 1988 5 5 p 322 7 Upton M L et al Differential effects of static and dynamic compression on meniscal cell gene expression J Orthop Res 2003 21 6 p 963 9 Ahluwalia S et al Distribution of smooth muscle actin containing cells in the human meniscus J Orthop Res 2001 19 4 p 659 64 Djurasovic M et al Knee joint immobilization decreases aggrecan gene expression in the meniscus Am J Sports Med 1998 26 3
62. howing arrangement of fixture during sample loading and unloading 89 90 91 92 Wao ee ONIMVIQ FIOS LON Od 1909 IV NO1 LVE 319345 Se e SATONV s Wmi D30 SNOI LOVES 33UV SIONVEIIOL SIHONI NI JYV SNOISN2AIG 03131934S 3SEIMM3HIO SS3INA i Sidvd NOILdI89S30 BO ON SNIASTINIGI FL BMW ONA ON JO lid 93 KEE IT TE IET es JIN eee OL Sv TENS tv ae re EEI 033031 ll USHR Hi ESTATE AI wD 1 8 l DNIMVUQ 31WIS 10N 00 31G S IWAOSdaV gt 400 Xxx z aur z 10 nxx 3 S3I0N STYMID3O SNOILOVU3 1S11 Sidvd NO114180530 40 UNL JON ZMON 1909 IY wiagivn 94 133HS A ISSA 3215 WID J9MOJ SUL a E Za JUSHI a HOW 349f TGW p wa SWAQBdAW Ia 1909 IV ISIT SisSVva ROTIATBOS3U BO 38N 1V JONI ON SIM IJ3IO SNOJJOWUA 138W S3JNVJJIOL S3HONI NI 3UV SNOISNI IQ IWY 3 NOX 03141933S 3SIMU3HiO SS31NO ON DNIAJIINZOI W IBWA 6038 410 Es LET 95 NOLIVJI 31234S WISJIVA NOIIJIBJSJJ B BUT WTONJ ON n JUSHOW 459 nay T zuma 3WIS LON A 100 xxx 10 Xxx SINY STWAIDIO Sovis 138V S3IJNVHIIOL S3HONI NI 3WY SNOISNJ IQ 031419395 SSIWA 96 ml lej pj OL UNIS ee Se eee esog Jayan Paar ae 8 y3ND ied MAL GNIAVEG 31WIS LON 00 SIWAY SIW ID3O SNOJJIVIS 138W SINAL SHON NI JUV SNOLSNI IG 031 4193dS_ISIMYJHLO SSJINA 1511 Sidvd i NOIIVIIJIJIHS NOTIATUNSIO BO ON ONTASTINIOT f 0034 11071 1909 WV
63. ircumferential The elastic modulus ranges from 2 MPa to 23 MPa depending on the region layer and location of the tissue 21 On average the middle layer has the lowest elastic modulus with the proximal and distal layer being the highest The posterior region has the highest modulus at the proximal and distal layers while the anterior seems to have the lowest at those layers The compressive strength of the meniscus depends largely on the strain rate at which the tissue is tested 3 Krause 1976 et al tested percent energy reduction in compressed canine menisci at 3 different deformation rates 2 12x107 4 23x107 and 21 16x10 m sec The resulting percent reduction in energy was 46 8 18 6 42 3 20 3 and 32 2 1 6 respectively This data shows that a greater amount of energy is required to compress the meniscus at higher strain rates The high water content of the tissue creates a hydrostatic pressure that provides the compressive strength As load is added to the tissue hydrostatic pressure increases and then decreases as fluid flows out of the tissue The compressive strength has also been shown to increase exponentially with level of strain 14 At 0 2 strain the compressive elastic modulus for the circumferential radial and axial direction is 10 MPa 13 MPa and 19 MPa respectively At 0 8 strain the compressive elastic modulus for the same three directions are 288 MPa 287 MPa and 299 MPa respectively This shows that
64. is balanced by the reaction force of the tibial plateau 4 The horizontal force is opposed by the hoop stress that forms in the circumferential direction of the meniscus 3 9 Following partial and full meniscectomy changes occur in the knee due to a loss in the weight bearing capacity of the menisci Narrowing of the joint space formation of an osteophytic ridge between the femoral condyles flattening of the femoral articular surface and osteoarthritis are svmptoms seen in meniscectomized knees 1 3 4 10 Osteoarthritis OA characterized as the loss of articular cartilage has been investigated and is thought to be triggered by meniscectomy 1 11 OA following meniscectomy is hypothesized to be a result of the increased contact pressure between the femoral condyles and the tibial plateau resulting in overstraining and degeneration of the articular cartilage Thus this demostrates the vital role the meniscus plays in the weight bearing function of the knee joint The meniscus also provides stability between the femur and tibial plateau The semicircular shape and the meniscal attachments help keep the femoral chondyles in the correct location by providing resistance This aids the other ligaments in the stability of the joint by reducing motion The movement of each meniscus is restricted by the ligamentous anterior and posterior horns connecting the meniscus substance to the tibial plateau The circumferential matrix fibers of the me
65. lly outward Opposing this force is the circumferential Type I collagen bundles that continue into the menical attachments and connect to the tibia These fiber bundles provide tension that resist stretching and displacement of the tissue The tibia provides the anchor point for resisting radial displacement During joint flexion the central portion of the meniscus does displace slightly outward 9 However the anterior and posterior regions move inward to produce a more compressed C shape meniscus than during the unloaded state The loading conditions on the meniscus also change as the knee moves from full flexion to full extension 1 9 13 18 This range of motion is approximately 140 4 16 As the knee moves from 30 of flexion to full extension 18 of internal rotation of the femur occurs with respect to the femur 4 11 During flexion the distance between the femoral condyle increases and the radius of curvature increases This keeps the contact area high and pushes the menisci away from the center 9 As the knee moves to extension the radii of the femoral condyles increase and the distance between them decreases When load is applied during extension the menisci deform anteroposteriorly 9 During full extension the femoral condyles slide posteriorly as they contact the anterior horns of the menisci 4 18 19 This motion tightens the anterior cruciate ligament and stops the extension of the lateral femoral condyle The medial
66. logical load and strain produce harmful amounts Stimulation with IL 1f and lipopolysaccarides LPS an endotoxin that activates iNOS also increases NO in the meniscus suggesting high levels in osteoarthritic knees may be due to other factors in addition to mechanical stimulation In the meniscus cytokines appear to play a major role in the up regulation of nitric oxide Inflammatory mediators IL 1 IL 17 and TNFa have all shown to increase NO production in meniscal explants 36 37 40 41 On the other hand hyaluronan HA has been shown to suppress NO production in the meniscus 45 HA is glycosaminoglycan which is often injected into the knee to slow osteoarthritic progression HA is known to inhibit the release of glycosaminoglycans in articular 17 cartilage delav degradation and reduce inflammation N monomethyl L arginine L NMA a commonly used inhibitor of nitric oxide sythase was also found to strongly inhibit NO production in meniscal cell cultures 37 Studies by Cao et al 1998 showed that meniscal explants did not produce nitric oxide in response to cytokine stimulation if only fibroblastic cells were present However enzymatic digestion of fresh meniscal fragments containing both fibroblastic and chondrocytic cells produced large quantities of NO in response to cytokine stimulation This suggests that perhaps chondrocytes are a large source of nitric oxide in the meniscus Since both articular caritlage and meniscu
67. metabolism stimulation of collagen production by arginine J Orthop Res 1998 16 1 p 104 11 Maneiro E et al Aceclofenac increases the synthesis of interleukin 1 receptor antagonist and decreases the production of nitric oxide in human articular chondrocytes J Rheumatol 2001 28 12 p 2692 9 23 39 40 41 42 43 44 45 46 47 48 49 50 Murrell G A et al Nitric oxide an important articular free radical J Bone Joint Surg Am 1996 78 2 p 265 74 Shin S J et al Regulation of matrix turnover in meniscal explants role of mechanical stress interleukin 1 and nitric oxide J Appl Physiol 2003 95 1 p 308 13 Hashimoto S et al Nitric oxide production and apoptosis in cells of the meniscus during experimental osteoarthritis Arthritis Rheum 1999 42 10 p 2123 31 Dowdy P A et al The effect of cast immobilization on meniscal healing An experimental study in the dog Am J Sports Med 1995 23 6 p 721 8 Hayashi T et al Nitric oxide production by superficial and deep articular chondrocytes Arthritis Rheum 1997 40 2 p 261 9 Kobayashi K et al Chondrocyte apoptosis and regional differential expression of nitric oxide in the medial meniscus following partial meniscectomy Journal of Orthopaedic Research 2001 19 p 802 808 Takahashi K et al Hyaluronan suppressed nitric oxide production in the meniscus and synovium of rabbit osteoarthritis m
68. minum dish via a quick disconnect pin The dish has six 10 mm deep wells equally spaced in a circular orientation Teflon filled Delrin compression rods diameter 8mm for each well are press fit into a plunger which attaches to the actuator via a quick disconnect pin The plunger also features two press fit aluminum pins that slide into matching holes in the dish This keeps the compression rods centered in each well and only allows for one plunger dish orientation To enclose the plunger and dish an aluminum cap rests on the shoulder of the dish and houses a linear bearing that is press fit into the cap Along the resting edge of the cap four 29 shallow grooves were machined to allow carbon dioxide supplv to the explants during testing The linear bearing allows the plunger to move up and down within the cap and restricts the plunger to vertical motion The frame is the most critical component to maintaining equal well pressure in all 6 wells An even well pressure will ensure all six explants experience the same mechanical stimulation The frame is rigid to maintain alignment during handling or assembly The frame is built out of two one inch thick parallel aluminum plates separated by one inch diameter aluminum support rods Figure 2 2 Centered on the bottom plate is the load cell with the six well dish attached The cylinder of the actuator is recessed into the top plate and a collar holds the actuator tight and perpendicular to the plate
69. mpressed between 0 9581mm and 1 0419 mm With this data the results from testing of explants can be normalized for the difference in wells The results from the above gap testing can be seen in Table 2 3 For each test the micrometer measurement is displaved for each well along with the maximum difference and average Tests 3 4 and 5 have higher values because the target displacement of the actuator was changed This was done to show that the difference in the wells would stav the same regardless of the target displacement The average maximum gap difference value is displayed below Table 2 3 2 5 Discussion The explant compression system meets the criteria necessary to obtain a realistic representation of physiological forces present in the knee joint This system is able to apply known pressures to six explants at once which is important when trying to gather data for hypothesis testing It is capable of applying physiological levels of load and displacement and has the ability to test in load or displacement control SMI programming allows for flexibility in frequency duration amplitude and waveform The system is small enough to fit in a standard incubator and is made of materials that can endure autoclaving and alcohol An important feature to this system is the ability to keep the explants and media sterile from the culture hood to the incubator The plunger dish and cap form an enclosure that allow easy transport without allowing o
70. n 9 Decrease the pixel size to 1360x1024 in the drop down box next to the capture button 10 Lower the intensity under the Intensity tab by moving the arrow to the right on graph far left until the image is clear 11 Under capture tab click on the capture button picture of a camera to record the picture on the screen 12 The image appears on the DP manager for saving to different directories Image Merging 1 Go to Start and open My Computer 2 Open Local C and click on the SPOTCam folder 3 Double Click on the SPOT32 icon 4 Go to File Open Image File and open the first image you want to merge 5 Go to Edit Merge Images 6 Check the red and green channels Whatever color the initial image is select CurrentlyOpen on that channel For the other channel select Image File and click the button Select the file and Click OK to merge the two files 109 B 4 Nitric Oxide Production Raw Data Nitric oxide produced bv the meniscal explants was measured using a total nitric oxide assav bv Cavman Chemical Ann Arbor MI The media samples were filtered and loaded into 96 well plates according to the protocol supplied bv the assav kit Wells were organized and recorded using abbreviations for animal location and laver tested For example A6LL2T represents Animal 6 Left knee Lateral 2nd explant from anterior Top of explant Al12RM3B represents Animal 12 Right knee Medial 3 explant from anterior Bottom of explant
71. n those in the interior layers These superficial layers have a more homogenous extra cellular matrix and appear to be more hyaline like This zone is a high density multilayer of fibroblastic cells that is surrounded by a large amount of Type I collagen Cells in lower density surrounded by less Type I collagen will appear and behave like chondrocytes In the deeper zones and closer to the inferior surface there is a lower cell density of round or polygonal shaped chondrocytic cells This subtype of cells synthesizes a large amount of sulfated proteoglycans and does not produce Type I collagen This is a major component of articular cartilage that provides compressive strength which is also found in the meniscus The lowest cell density is located in the central region of the meniscus 24 30 It is believed that cell density may be correlated to the supply of nutrition in the meniscus The nutrient supply to the meniscal cells depends on two main sources blood supply and synovial fluid Only the peripheral 10 to 30 of the adult meniscus is vascularized making the meniscus a relatively avascular structure Figure 1 Blood is the main source of nutrients necessary to keep the fibrochondrocytes alive and the tissue healthy This blood supply comes from the inferior superior and middle genicular arteries that run together in a capillary plexus on the periphery of the meniscus 1 Radial branches penetrate and spread into the peripheral one third o
72. n investigated To further understand these relationships the goals of the current project were to 1 validate a specially designed tissue compression bioreactor capable of a wide range of accurate displacement and load control 2 determine the relationship of strain pressure to nitric oxide production in both superficial and deep zones of meniscal explants il ACKNOWLEDGEMENTS First I would like to thank mv advisor Dr Tammv Haut Donahue for her support direction and patience through this project Her help and encouragement have been verv important to me and to the success and completion of this work I would also like to thank the Whitaker foundation that has provided funding for this research allowing the use of proper equipment and supplies needed for these experiments My committee members Dr Seth Donahue Dr Jeff Burl and Dr Eric Blough have provided critique of my work and have directed me in my writing and experimentation which is much appreciated I want to thank them for this advice and also for their patience I am also grateful for the assistance of my lab group members Tumul Basia and Jason They have provided much needed assistance while I was off campus and have provided a great working environment in the lab I would also like to thank Jesse Nordeng for dedication to providing professional quality machining work that was necessary for the accurate function of the bioreactor used in these experiments I want to thank m
73. n response to mechanical stimulation compared to superficial zones 21 REFERENCES 1 2 10 11 12 13 14 15 16 17 18 19 Aagaard H and R Verdonk Function of the normal meniscus and consequences of meniscal resection Scand J Med Sci Sports 1999 9 3 p 134 40 Fithian D C et al Human meniscus tensile properties Regional variation and biochemical correlation Trans ORS 1989 35 p 205 Krause W R et al Mechanical changes in the knee after meniscectomy J Bone Joint Surg Am 1976 58 5 p 599 604 McBride I D and J G Reid Biomechanical considerations of the menisci of the knee Can J Sport Sci 1988 13 4 p 175 87 Roughley P J et al The presence of a cartilage like proteoglycan in the adult human meniscus Biochem J 1981 197 1 p 77 83 Tanaka T K Fujii and Y Kumagae Comparison of biochemical characteristics of cultured fibrochondrocytes isolated from the inner and outer regions of human meniscus Knee Surg Sports Traumatol Arthrosc 1999 7 2 p 75 80 Fink C et al The effect of dynamic mechanical compression on nitric oxide production in the meniscus Osteoarthritis and Cartilage 2001 p 1 8 Voloshin A S and J Wosk Shock absorption of meniscectomized and painful knees a comparative in vivo study J Biomed Eng 1983 5 2 p 157 61 Shrive N G O C JJ and J W Goodfellow Load bearing in the knee joint Clinical Orthopedics 1978
74. n the extracellular matrix Recreating the physiological forces in vitro using tissue explants while measuring the biological response provides one method for observing the effect of mechanical stress on the meniscus 4 6 however the majority of commercially available bioreactors may not be suitable for application to meniscal loading studies Tissue explant culture studies allow control of loading and biochemical conditions For these studies to be an accurate in vivo representation the conditions within the body must be reproduced within the testing system A meniscal explant compression bioreactor must meet the following criteria to ensure successful experimentation Explants must remain sterile throughout the entire procedure thus all testing equipment and tools must be able to be sterilized by autoclave or alcohol before coming in contact with the tissue Culture media and incubation 5 CO 37 C used with fresh tissue is necessary for the biological response to resemble the 26 in vivo response To best create an in vivo response mechanicallv loading explants requires the tissue to experience pressures that the meniscus would experience in the knee Pressures up to 10 MPa and strains ranging from 2 to 20 are seen in the meniscus in vivo 7 9 The anterior central and posterior regions experience different strain levels making it necessary to test explants from all three regions Testing 6 explants at once makes it possible to tes
75. niscus extend to the intercondylar area to secure the meniscus The lateral meniscus is radially smaller than the medial and attaches centrally along the intercondylar eminence 1 12 The larger medial meniscus connects more on the anterior and posterior portion of the intercondylar area The deep medial ligament and posterior portion of the superficial medial ligament also fix the medial meniscus to the femur The lateral meniscus attachments are less firm allowing greater posterior displacement of the meniscus as the tibia rotates during flexion 1 13 The medial meniscus has been found to move a few millimeters while the lateral meniscus can move at least a centimeter 4 12 These attachments allow the meniscus to move slightly along the tibial plateau as the knee flexes The meniscus also serves as a limited shock absorbing medium 1 8 14 and aids in lubrication of the joint 4 15 These functions come from the composition of the meniscus and the abilitv of the tissue to allow fluid flow through the extra cellular matrix The smooth surface of the meniscus in the presence of the svnovial fluid is nearly frictionless allowing unrestricted motion in the knee Permeability of the tissue allows fluid to leave during compression reducing the hydrostatic pressure within the matrix This mechanism allows the meniscus to be a natural shock absorber The study performed by Voloshin et al 1980 concluded that removal of the meniscus reduces the
76. oals as well as demonstrate the creep properties of meniscal explants 49 These goals will be evaluated for the superior and deep zones to determine how each unique population of fibrochodrocvte responds to compression 3 3 Methods and Materials Meniscus Samples Meniscal samples were obtained from porcine knees typically harvested from 4 month old female pigs within 24 hours of death Left and right knees from 12 animals were dissected aseptically to retrieve the medial and lateral menisci Six explants were removed from each meniscus using a 6 mm diameter biopsy punch FRAY Products Corp Buffalo NY Samples were cut parallel to the superior surface to maximize the amount of superior tissue saved The explants were then transferred to a microtome and trimmed to achieve parallel top and bottom surfaces at a height of 5mm To allow for full recovery of the tissue samples were then incubated for 48 hours in culture medium 44 5 Dulbecco s modified Eagle s medium 44 5 Ham s F 12 10 Fetal Bovine Serium and 1 penicillin streptomycin at 37 C with 5 CO and 95 air The media was changed after the first 24 hours of this 48 hour incubation Meniscal Compression Explants were compressed for 2 hrs at 1 Hz to simulate physiological conditions equivalent to two hours of walking Tests were performed in a custom designed bioreactor previously described 27 Briefly the system is capable of both load and displacement control and utilizes
77. odel J Orthop Res 2001 19 3 p 500 3 Farrell A J et al Increased concentrations of nitrite in synovial fluid and serum samples suggest increased nitric oxide synthesis in rheumatic diseases Ann Rheum Dis 1992 51 11 p 1219 22 Sah R L et al Biosynthetic response of cartilage explants to dynamic compression Journal of Orthopaedic Research 1989 7 p 619 636 Taskiran D et al Nitric oxide mediates suppression of cartilage proteoglycan synthesis by interleukin 1 Biochem Biophys Res Commun 1994 200 1 p 142 8 Wiseman M et al Dynamic compressive strain inhibits nitric oxide synthesis by equine chondrocytes isolated from different areas of the cartilage surface Equine Vet J 2003 35 5 p 451 6 Stefanovic Racic M et al Nitric oxide and proteoglycan turnover in rabbit articular cartilage J Orthop Res 1997 15 3 p 442 9 24 CHAPTER TWO VALIDATION OF BIOREACTOR A Tissue Engineering Bioreactor for Dvnamicallv Compressing Meniscal Explants with Load or Displacement Control Capabilities Jeffrey A McHenry and Tammy L Haut Donahue 2 1 Abstract Motivated by our interest in examining meniscal mechanotransduction processes we report on the validation of a new tissue engineering bioreactor This paper describes the design and performance capabilities of a tissue engineering bioreactor for cyclic compression of meniscal explants We showed that the system maintains a cell culture environment
78. oduction To test this hypothesis and accomplish the objective of this study we will pursue the following two specific aims I To establish the relationship between meniscal strain and meniscal nitric oxide production Explants n 6 will undergo unconfined compression to 0 5 10 15 and 20 strain at a frequency of 1 Hz for 2 hrs Meniscal load will be calculated and correlated to meniscal nitric oxide production 20 Hypothesis 1 The meniscus produces low levels of nitric oxide without additional stimulation from cytokines or compression This suggests that low levels of nitric oxide are present in the meniscus without unhealthy effects We hypothesize that both overloading and underloading the meniscus results in increased NO production compared to the physiological levels of loading II To determine the identity of nitric oxide producing cells Following mechanical compression explants will be cut into superficial and deep zones with each zone representing a different cell phenotype Nitric oxide production from each zone quantified to establish the relationship between cell phenotype and NO production Hypothesis 2 The meniscus contains fibroblastic cells that are prominent in the superior zone and chondrocytic cells that reside in the deep zone Since chondrocytes have been shown to produce high levels of NO in articular cartilage following compression we hypothesize that cells from the deep zones will produce more NO i
79. ollagen production by arginine J Orthop Res 1998 16 1 p 104 11 Maneiro E et al Aceclofenac increases the synthesis of interleukin 1 receptor antagonist and decreases the production of nitric oxide in human articular chondrocytes J Rheumatol 2001 28 12 p 2692 9 63 18 19 20 21 22 23 24 23 26 27 Murrell G A et al Nitric oxide an important articular free radical J Bone Joint Surg Am 1996 78 2 p 265 74 Shin S J et al Regulation of matrix turnover in meniscal explants role of mechanical stress interleukin 1 and nitric oxide J Appl Physiol 2003 95 1 p 308 13 Taskiran D et al Nitric oxide mediates suppression of cartilage proteoglycan synthesis by interleukin 1 Biochem Biophys Res Commun 1994 200 1 p 142 8 Hashimoto S et al Nitric oxide production and apoptosis in cells of the meniscus during experimental osteoarthritis Arthritis Rheum 1999 42 10 p 2123 31 Upton M L et al Differential effects of static and dynamic compression on meniscal cell gene expression J Orthop Res 2003 21 6 p 963 9 Zielinska 3D Finite Element Model of Medial Meniscus Meniscectomy Changes in Contact Behavior Journal of Biomechanical Engineering 2005 Spilker R L Donzelli P S A biphasic finite element model of the meniscus for stress strain analysis in Knee Meniscus Basic and Clinical Foundations V C Mow Arnoczky S P Jackson D W Editor
80. on shows the weight g of each sample for normalization and final concentration in UM g 111 Calibration Curve y 0 0273x 0 0196 Microplate reading 0 5 10 15 20 25 30 35 40 Concentration pM Figure B 2 The calibration curve used for the first NO assay to convert microplate reading to concentration of nitrite 6 7 8 9 10 11 12 A OABLLIT ALLIT ASLLIT AGRLIT A AVIT AQRVBT ATIRLIT AHLLST A2RLIT A1AM2T B SABLLIB ALLIB ASLLIB AGRLIB A RMIB A9RVBB AHRLIB ATILL3B A12RL1B A12LM2B Cc 10 A8LL2T ALLT AL T ABRVET APRVET ASAVAT ATIRL2T ALLST A12R2T M2LM8T D 15 A828 ALB ASLL2B ABRVEB A7RVEB ASAVAB ATIR28 ALLSB A12RL2B A12AM3B E 20 ASLMIT AZLMIT ABLLST AGLMBT AMAT ALLIT A MRMIT MALLIT A12RVAT F 25 ABSLMIB AAMIB A6LL3B ABLIVBB AMAB ALB A RMIB M2LLIB A2RVB G SOASLVET AAMT ASULAT ASMAT AR2T AM LLZT A RVET MaLLZT A2RVAT H 35 ABLMEB A LMPB ASAB AGLMB A RI2B AlB ATIRVEB M2AL2B A12RWB 20 5 15 15 LPS C 06 Cc 05 C 20 5 15 15 LPS je 06 C 05 C 20 5 15 15 LPS Cc 05 Cc 05 C 20 5 15 15 LPS Cc 05 C 05 C al 10 5 10 15 20 4 20 4 1 10 5 10 15 20 4 20 4 1 10 5 10 15 20 A 20 4 A 10 5 10 15 20 4 20 4 Table B 3 This table shows the arrangement on the microplate and strain load levels of the samples used for the second total NO assav 112 weight g conc uM standards 0 0 007 5 0 158 10 0 3 15 0 437 20 0 58 25 0 727 30 0 822 35 1 022 calc conc uM Conc uM g 0 0408 0 0869 0 0
81. ounts for the intial reading when both lights are extinguished on the 2100 svstem 3 Change the f value in the load program code to the value found in step 2 Highlighted below C8 j 7200 user input for cycles i 0 WHILE i lt j start of load cycling i i 1 C7 f 27 user input for force q 10 position increment value r 100 y 200 UAI b UAA A 10000 V 1000000 ll f 3 mme f 2 ee CLK WHILE b lt f run until voltage reaches user input for force UAI b UAA IF bell D y ELSEIF bemm D r ELSE D q ENDIF Save program with pressure as file name transmit T and run R the program 107 LIVE DEAD ASSAY PROTOCOL Sample Prep 10min Incubation 30 min Fluor Detect 30 min Clean up 10 min Total 80 min Equipment List 1 Live Dead assay kit w fluorescent dye L3224 Molecular Probes 2 Fluorescent microscope 3 Microscope slides 4 Tweezers 5 Scalpel 6 96 well plate 7 Phosphate Saline Buffer PBS 8 100 1000 ul pipette w tips 9 Aluminum foil 10 10 ml centrifuge tubes 11 Insulated container Stain Solutions 1 2 3 4 Allow staining chemicals to warm to room temperature before use Stain is photosensitive keep staining chemicals and mixtures out of direct light Turn off light in culture hood In two 10 ml centrifuge tubes mix chemicals with sterile PBS to the following concentrations uM calcein AM 8 uM ethidium homodimer 1 e 4ml vol 16 ul eth H 1 4 ml PBS e
82. ower attachment pin with quick disconnect pin 8 Turn on the power module to the motor and open the SmartMotor Interface in the marrow folder 9 Manually lower the actuator into the top of the plunger Code example MP A 1000 V 100000 D 60000 G For distance D 2500 Imm Down is neg 10 Loosen nut on actuator and turn end piece until holes line up Slide second quick disconnect pin into plunger actuator 11 Retighten actuator nut 12 Attach load cell cable Load Cell Calibration 1 Turn on 2100 system switch in lower right hand corner 2 Power up motor and SMI as previously described 3 Go to File Open and open the file checkload sms in the C Program Files Program Editor folder and click open 4 Make sure the cursor is in the program window and click T on the tool bar to transmit the program The actuator may need to be raised to provide room to add weights 5 Attach the aluminum dish to the lower attachment post for a weight platform 6 On channel 2 of the 2100 system turn the balance dial until both lights are extinguished 76 OO 10 11 12 13 14 15 16 17 Click R on the tool bar and note the encoder counts in the command window One number should show up after the RUN command This value is the initial offset of the load reading Attach an oscilloscope to the output of the 2100 system Hit the Cursors button and use the dial to the left of it to align the Y1 cursor at the starting poin
83. parate media and tissue samples into clean labeled 1 5 ml centrifuge tubes and immediately put on ice e Use parafilm on centrifuge tubes containing media samples Immediately bring samples to 80 C freezer Place media sample directly into freezer Tissue sample must be frozen in liquid Nitrogen tank for 24 hrs prior to being placed into freezer Testing Data 24 25 26 27 28 29 Place the cursor at the bottom of the data sting in the smart motor command window Highlight the data by moving to the top of the data string and pressing SHIFT Left Mouse Button Go to Edit Copy and then paste in Excel spreadsheet Go to File Open go to local C open Excel Macro folder and open Macrotest to open the Excel spreadsheet that contains the macro to separate the data into columns Minimize that window Highlight the first cell of the data string Go to Tools Macro Macros highlight dispwload and click Run This will separate the data into time load and displacement columns Divide the time column by 4000 A1 4000 to get time in seconds Divide the displacement by 2500 to convert to millimeters 105 kol The time column is the column with the greatest values The displacement column contain negative numbers representing compression 30 For load subtract the initial load reading from all of the value to start at zero load Then divide the data bv 0 8073 to get load in Newtons Nitric Oxide assay 1
84. pen air and bacteria to infect the sample Since the cap incorporates a linear bearing it does not need to be removed for testing Bacteria can kill cells and alter the chemical response leading 34 to inaccurate data Utilizing the svstem features and designing the correct protocol will help maintain a sterile environment This bioreactor is capable of creating higher loads and greater displacements than previous systems used for compressing explantsil 3 4 10 The Biopress system Flexcell International Hillsborough NC is not capable of pressures higher than 0 1 MPa since the pressure is applied by air into a flexible bottom Since our system applies load using a linear actuator loads up to 2225 N can be added In Frank et al 2000 a biaxial tissue loading device can load 12 explants in shear and compression 10 An advantage our system has is that it can create displacements over 10 mm with a resolution of 0 4 um The Frank et al 2000 creates displacements up to 100um Our actuator also has a bi directional repeatability of 7 62um compared to the 25um used in Sah et al 2003 In addition the present system is capable of 1 Hz cyclic compression in a sinusoidal type wave using displacement or load control The flexibility of the Smartmotor Interface will allow various alterations of test programs Frequency amplitude and number of cycles can be easily changed The bioreactor can be used to compress any tissue that fit under an
85. re the max load occured user input for load RCLK reports clock where max load occured dd nn ee WAIT 2000 dd A 10000 V 500000 P 0 moves actuator back to position where preload occured unloads sample G aa CLK 87 cc aa ee WAIT 4000 cc waits 0 5 seconds before loading may need to be changed if 1Hz is critical RP UAI b UAA a Rb reports voltage at preload RCLK reports clock at preload position LOOP END 88 B 2 Design Drawings Experiments performed in this research required some additional fixture and equipment to be made to increase accuracv and improve setup procedures The fixtures made include a microtome for trimming explants and a fixture for loading and unloading explants The first design for trimming the explants was not made These drawings can be seen from pages 83 89 The simple form of the microtome used just a rectangular aluminum post with a cvlinder cut to hold the explant while razors were used to trim both sides These drawing can be seen in pages 90 and 91 The fixture for loading and unloading the explants was a frame that set in the culture hood The components were made of aluminum and feature a centering hole for the aluminum dish and a rod used to suspend the plunger and cap above the dish during loading preloading and addition of media The drawing of this fixture can be seen in pages 92 95 Figure B 1 A picture of the setup frame assembled with the dish plunger and cap s
86. ression can alter the morphology of other organelles found in the cell thereby altering the location and activity of intacellular enzymes Compression is frequently studied in both articular cartilage and meniscus and is believed to play a major role in tissue homeostasis In articular cartilage compression alters the morphology and structure of the gogli apparatus and rough endoplasmic reticulum 35 which is believed to produce new matrix molecules with altered form and function Biochemical coupling is the mechanism of converting the physical stimuli sensed through mechanoncoupling into a biochemical signal 33 Though not fully understood the theory is that mechanical energy is transmitted to sensor cell through one of the mechanisms described above This produces a change in the normal behavior of the cell leading to altered gene expression enzyme production and signaling These factors produce autocrine and paracrine signaling that changes the function of the sensor cells and the effector cells An effector cell receives the biochemical signal produced by the sensor cell which alters the effector cells behavior This type of signaling paracrine require a pathway such as gap junctions or interstitial fluid Blood is a major pathway for biochemical signaling in many tissues Fluid either blood or interstitial fluid carry 14 cytokines to and from cells creating a reaction The result of such signaling is a response by cells that acts to
87. ressure at start and end of displacement control test at all tested strain levels B Average of displacements at the start and end of load control test for both tested load levels 54 Nitric Oxide Production Nitric oxide NO production was not significantiv influenced bv location within the explant There was no significant difference between NO production of the superficial and deep zones of the explants 090 p 0 898 590 p 0 443 10 p 0 176 15 p 0 978 20 p 0 351 using a paired t test However a distinct trend did appear with both displacement and load controlled tests For displacement control the 20 strain level produced the greatest amount of NO with 15 being lower and comparable to the 0 strain control samples The 5 strain produced the next lowest amount of NO while the 10 strain level produced the least amount of NO out of all strain levels The relationship of NO production to strain level fits the quadratic model NO Production 225 5 30 66 Strain Level 1 972 Strain Level for the superficial zone with R 0 435 Figure 3 The quadratic term was significant p 0 000 as well as the constant p 0 001 while the linear term was not found to be significant p 0 100 The deep zone showed a similar trend with 10 strain producing the least NO The quadratic NO 234 6 19 4 Strain Level 1 158 Strain Level fit the data with R 213 Figure 4 The quadratic term was statistically significant p 0 016
88. s contain chondrocytic cells NO studies performed on articular cartilage can be useful in understanding NO production in the meniscus Nitric oxide may decrease the synthesis of extracellular matrix increase degradation of the matrix and lead to cell apoptosis Cao et al 1998 found that in the meniscus NO inhibits collagen and proteoglycan 48 synthesis yet protects proteoglycans from the catabolic effects of IL 1 40 Nitric oxide is also believed to cause extracellular matrix degradation due to its high concentrations in osteoarthritic joints NO acts to break down collagen and proteoglycans through metalloproteases 39 Matrix degradation may also be a result of fibrochondrocyte apoptosis Hashimoto et al 1999 reported a high occurrence of apoptotic cell death associated with high levels of NO in the osteoarthritic knee This suggest that NO may play a part in apoptosis which would result in the calcification and loss of the cells pericellular matrix 41 Nitric oxide is suspected in playing a major role in the matrix metabolism of both the menisci and articular cartilage The high concentration of NO in the osteoarthritic knee implys that it plays a role in tissue inflammation and matrix degradation Whether 18 this cellular messenger is up regulated primarilv bv other cvtokines or mechanical stress is yet to be determined The amount of NO present in the meniscus during healthy loading is also unknown In order to better underst
89. ssembly Load signal connection to bioreactor Image of bioreactor setup in incubator Calibration curve for pressure film Presure film impression of repeatability test Repeat impression of 0 477 MPa test Repeat impression of 0 564 MPa test Calibration curve relating load applied to encoder count Calibration curve relating load applied to voltage Image of setup frame vii Figure B 2 Calibration curve for first NO assay es 112 Figure B 3 Calibration curve for second NO assay sss 114 vili CHAPTER ONE INTRODUCTION 1 1 Functions of the Meniscus The menisci are specialized fibrocartilaginous structures that plav a crucial role in the maintenance of knee stabilitv load distribution joint lubrication and shock absorption 1 8 They have a semicircular shape with a wedge shaped cross section that adapts the curvature of the femoral condyles to the flatter tibial plateau The tibial surface of the meniscus is flat while the femoral surface is convex Their shape increases the tibial plateau contact area thereby decreasing the contact stresses significantly in the knee It has been shown that between 30 and 65 of the total knee joint load is transmitted through the meniscus reducing the compressive stress on the articular cartilage and subchondral bone 3 9 During compressive loading of the knee pressure is added to the superior surfaces of the menisci that has both a horizontal and vertical component The vertical component
90. ssure of 0 477 MPa Once the target load was reached the upper platen was immediately raised from the surface of the pressure film Calibration of the pressure film was also done using the Instron and included loading pieces of pressure film ranging from 0 2 MPa to 1 64 MPa All film samples were scanned and analyzed using Scion Image with the density scale for this program set at a range of 0 255 with 255 being completely saturated Film was compressed between the platens and a piece of rubber similar to the rubber used for testing well pressure To determine well pressure in the bioreactor a machined plate was set on top of the dish with a 3 mm thick piece of uniform rubber Pressure film was placed on top of the rubber and the plunger was lowered near the surface of the film Two different load settings of 24 N and 28 N on an area of 50 27 mm corresponding to pressures of approximately 0 477 MPa and 0 564 MPa were tested with five tests per load The 24 N and 28 N loads were the loads on each compression rod and each rod had a radius of 4 mm These loads covered the upper end of the spectrum for the pressure film The film from the bioreactor was analyzed and density measured to determine the difference 31 between each compression rod The difference in film densitv and the maximum percentage error was determined to demonstrate the accuracv of the svstem 2 3 3 Determination of Displacement Accuracv A second technique for determining th
91. st and less then the 20 strain The superficial zone showed a quadratic increase from zero load condition Figure 5 while the NO production for the deep zone increased for 0 05 MPa and decreased again for 0 1 MPa Figure 6 57 NO Production bv Superficial zone with Load Control NO Production 215 4 407 Pressure 15036 Pressure 2 Regression 500 95 CI ma R Sq 16 5 D 400 z P 0 338 ku S fr 300 e 9 200 100 0 00 0 05 0 10 Pressure Mpa Figure 3 5 Load control graph showing quadratic regression with 95 confidence interval for superficial zone of explants R 0 165 0 MPa n 8 0 05 MPa n 3 0 1 MPa n 4 58 NO Production bv Deep zone with Load Control NO Production 224 3 1230 Pressure 10086 Pressure 2 500 Regression 95 CI g 400 R Sq 2 0 3 P 0 885 300 2 3 B 200 iL a 2 100 0 00 0 05 0 10 Pressure Mpa Figure 3 6 Load Control graph showing quadratic regression with 95 confidence interval for deep zone of explants R 0 02 0 MPa N 8 0 05 MPa N 3 0 1 MPa N 4 3 5 Discussion The findings of this research suggest a relationship between mechanical compression and nitric oxide production by meniscus The experimental setup 1 Hz 2 hrs simulates a short period of activity for comparison to inactivity in an attempt to determine how this affects NO production and ultimately meniscal health The data suggests
92. t how tissue from each region responds to the same level of loading or strain If order to create repeatable results and to show significance in data all six explants must experience the same compression Explants should receive the same strain within 5 error of each other throughout each test For a 5mm explant the displacement range has to be at least 0 1mm 2 5um to 1 0 mm 2 5um to achieve accurate displacement for strain levels ranging from 2 strain to 20 respectively Strains are relatively low in the normal healthy meniscus but these strain levels increase with a partial menisectomy 9 We hypothesized that higher strains would lead to degeneration of the meniscus by increased levels of nitric oxide Current systems for meniscal explant compression apply pressure near or below 1 MPa The Biopress system Flexcell International Hillsborough NC uses air pressure applied to a flexible bottom under each well It has been used to apply pressures of 0 1 MPa in previous studies done on meniscal explants 1 3 4 noting stain levels of approximately 10 due to the state of unconfined compression Another biaxial tissue loading device previously used to compress articular cartilage explants is able to create a maximum 400 N axial force on as many as 12 explants at once 10 This device also has the ability to create rotational motion with a resolution of 0005 and can only apply a sine wave with amplitudes as low as 10 um and as large
93. t of the load signal The RUN STOP button should be green and the signal should be rolling across the screen If not hit the MAIN DELAYED button and select ROLL Also hit the ACQUIRE button and select AVERAGING Add a weight on top of the aluminum dish Click R on the tool bar and note the resulting encoder counts On the oscilloscope hit the CURSORS button and select the Y2 cursor and move it to the new location of the signal using the dial to the left or the CURSORS button Note the AY value Repeat steps 10 13 for several different weight increments To increase or decrease amplification turn the GAIN dial directly below the BALANCE dial until the desired amplification is reached The voltage output of the 2100 system should exceed 5V since the SmartMotor and only read a maximum of 5V For larger changes in amplification turn the multiplier switch next to the GAIN to 2x 20x or 200x amplification Plot results of load vs counts and load vs voltage to determine linearity Use a linear regression trend line to determine relationship between load and encoder counts e For 300 lb load cell GAIN should be at 200x with the dial set at 1 35 gt 3 84 mV 1 Newton 0 8073 counts 1 Newton e For 2000 Ib load cell Calibrate to 500 Ib gt 225 mV 1 Newton 0 46 counts 1 Newton 77 VALIDATION PROTOCOL FOR EVEN WELL PRESSURE Objective To determine the greatest difference in pressure between all six well of the bioreactor
94. t the preload position LOOP end of position cycle END 85 0 1 MPa Load Control RUN UAI opens the AID convert port containing the load cell signal b UAA this will be the voltage in counts from the load cell cg s 2 user input for the preload ss 25 position increment value uu 25 MP specifies position control A 1000 V 1000000 WHILE b s i UAI run until voltage reaches user input for preload f b UAA IF b gt s D uu position increment j ELSEIF b lt s D ss ELSE b s D 0 ENDIF G i TWAIT LOOP WAIT 8000_ waits at the preload position for 2 seconds O 0 resets the preload position to the zero position CLK 0 resets the clock RP reports position at start UAI sets A pin to input b UAA set variable b to analog of pin A Rb reports voltage from pin A RCLK reports clock A 10000 V 1000000 GOTO1 tells program to go to the label C1 C1 END OF PRELOAD BEGINING OF LOAD CONTROL C8 86 j 7200 user input for cycles i 0 WHILE i lt j start of load cycling i i 1 C7 f 18 user input for force 10 position increment value r 100 y 200 UAI b UAA A 10000 V 1000000 Il 1 8 mm f 2 ee CLK WHILE b lt f run until voltage reaches user input for force UAI b UAA IF bell Dev ELSEIF bemm D r ELSE Deq ENDIF G TWAIT LOOP once loop is ended the load will have been found TWAIT nn CLK RP reports position at max load UAI b UAA Rb reports voltage whe
95. tes maintain an extra cellular matrix containing proteoglycans and Type I collagen 1 11 Mechanical stimuli are believed to contribute to maintaining meniscal matrix metabolism however it is yet unclear how these signals are propagated 3 10 12 17 19 21 22 A better understanding of the relationship between mechanical loading and biochemical response could aid in understanding the poor healing characteristics of the meniscus and it role in the onset of osteoarthritis OA The meniscus has been shown to be a mechanically sensitive tissue with specific loading conditions resulting in various biosynthetic responses Unloading the meniscus has been shown to result in a decrease in production of matrix molecules such as aggrecan and collagen 12 13 Conversly extended periods of dynamic compressive stress 0 1 MPa 0 5 Hz 24hr increase gene expression of cyclooxygenase COX 2 and 47 inducible nitric oxide synthase iNOS causing an increase in mediators prostaglandin E2 PGE and nitric oxide NO respectively 14 19 NO is a gaseous free radical that acts as a messenger and is believed to regulate matrix metabolism by inducing the release of proteoglycans from the matrix decreasing collagen production by fibrochondrocytes and possibly causing cell apoptosis 16 18 20 22 Since meniscal tissue produces NO spontaneously baseline levels may be responsible for balancing the remodeling process of fibrochondrocytes Previous stu
96. th IL 1 and the NOS2 inhibitor 1400W 36 Taken together these studies show the effect cytokines and gene expression have on production of matrix metabolism regulating factors such as PGE and NO 15 Although the signaling pathwavs in the meniscus are not fullv understood compression is believe to play a role in maintaining tissue metabolism through mechanontransduction Unloading has been shown to decrease aggrecan 27 and collagen 42 in the meniscus While conversely dynamic compression has been shown to increase proteoglycan release rates from meniscal explants as well as increased NO and PGE production 40 The amount of proteoglycan release seems to be dependent on NO production Also the amount of NO produced seems to be dependent on the presence of IL 1 as well as compression Although complex understanding these mechanotransduction pathways is important because signaling molecules such as NO may play an important role in meniscal health and the onset of osteoarthritis 22 32 34 39 46 1 7 Nitric Oxide Nitric Oxide NO is a gaseous free radical that acts as an intercellular and intracellular messenger in several different tissues 39 It is a free radical that is synthesized from the conversion of L arginine to L citrulline and NO by a family of enzymes called nitric oxide sythases NOS There are three isoforms in this family of enzymes NOS1 NOS2 and NOS3 NOSI and NOS3 are calcium dependent while NOS2 is expressed
97. that inactivity 0 pressure strain as well as overstrain produces high amount of NO in comparison to physiological strain levels The results of this study also show that physiological pressures 1 MPa applied to meniscal explants in unconfined compression do not produce equivalent physiological strains Load controlled tests targeting 1 MPa produced approximately 30 strain and 0 1 MPa tests produced 99 approximately 20 9 strain It requires much lower pressure lt 0 05 MPa to achieve physiological strain 5 10 during unconfined compression The data also suggests that NO production is not dependent on location within each explant Nitric oxide is linked to inflammation and tissue degradation in articular cartilage and meniscus and is believed to be a direct result of compression 14 19 Previous studies have reported that dynamic compression of meniscal explants results in an up regulation of NO 14 19 However these experiments were performed for an extended period of time and at only one strain level which would not be considered normal activity Some studies have also reported that meniscal explants produce NO spontaneously without compression 14 16 19 21 25 26 The results from our study also show that NO is produced spontaneously but do not support the simple relationship that compression up regulates NO production in the meniscus Our data suggest that physiological strain levels for short periods of time that could be consi
98. tion Table 3 1 Stess relaxation data Table A 1 Data collected for calibration of pressure film Table A 2A Repeatability data in terms of density on Scion Image Table A 2B Repeatability data in terms of pressure Table A 3A Density values measured for validation Table A 3B Pressure values measure for validation Table A 4 Calibration data for load cell Table B 1 Microplate setup for first NO assay Table B 2 Data collected from first NO assay Table B 3 Microplate setup for second NO assay Table B 4 Data collected from second NO assay Table B 5A Averaged NO values for strain tests Table B 5B Averaged NO values for load tests Vi Figure 1 1 Figure 2 1 Figure 2 2 Figure 2 3 Figure 2 4 Figure 2 5 Figure 3 1 Figure 3 2 Figure 3 3 Fiqure 3 4 Figure 3 5 Figure 3 6 Figure A 1 Figure A 2 Figure A 3 Figure A 4 Figure A 5 Figure A 6 Figure A 7 Figure A 8 Figure A 9 Figure B 1 LIST OF FIGURES View of meniscus interior Plunger Dish Cap assembly Test Frame Pressure film impressions at 0 477 MPa pressure Pressure film impressions at 0 564 MPa pressure Calibration curve for pressure film Pressure vs Time for displacement control Strain vs Time for load control NO produced by superficial during displacement control NO produced by deep during displacement control NO produced by superficial during load control NO produced by deep during load control Image of dish load cell a
99. umferential direction must have high tensile strength to resist the hoop stress generated by the radial force component during joint load The meniscus must be strong enough in tension in the radial direction to keep the tissue from tearing under normal loading condition There also has to be a high compressive strength to distribute load from the femoral condvles The circumferential direction has shown to have the highest tensile strength 2 21 23 Tests performed on the meniscus have characterized the elastic modulus of the anterior central and posterior regions for both the lateral and medial meniscus The results from Fithian 1989 show the anterior region to have an average elastic modulus in the circumferential of approximately 160 MPa for both menisci Lateral 159 07 47 4 Medial 159 58 26 2 The central region showed 228 79 51 4 MPa for the lateral and 93 18 52 14 MPa for the medial The posterior region showed 294 14 90 4 MPa for the lateral and 110 23 40 7 MPa for the medial Tissakht 1994 documented changes through the depth of the tissue proximal middle and distal circumferential tensile elastic modulus On average the middle portion had the lowest elastic modulus with proximal and distal being close to one another Their results also showed the lateral meniscus had a higher elastic modulus than the medial meniscus for all regions The tensile modulus of the meniscus in the radial direction is much smaller than c
100. uminum plates supported by aluminum rods The actuator is positioned in a centered hole in the top plate and tighten into alignment with an adjustable collar 41 Figure 2 3 Pressure film impressions at 0 477 MPa pressure 42 Figure 2 4 Pressure film impressions at 0 564 MPa pressure 43 Figure 2 5 Calibration curve for pressure film correlating densitv of film to applied pressure Mean Densitv vs Pressure y 1E 06x 0 0002x 0 0132x 0 19 R 0 9622 0 8 Pressure MPa 0 6 0 4 0 2 0 r r r r 1 r 0 20 40 60 80 100 120 140 160 180 Mean Density 44 REFERENCES 1 2 10 Fink C et al The effect of dynamic mechanical compression on nitric oxide production in the meniscus Osteoarthritis and Cartilage 2001 p l 8 Sah R L et al Biosynthetic response of cartilage explants to dynamic compression Journal of Orthopaedic Research 1989 7 p 619 636 Shin S J et al Regulation of matrix turnover in meniscal explants role of mechanical stress interleukin 1 and nitric oxide J Appl Physiol 2003 95 1 p 308 13 Upton M L et al Differential effects of static and dynamic compression on meniscal cell gene expression J Orthop Res 2003 21 6 p 963 9 van Griensven M et al Cyclic mechanical strain induces NO production in human patellar tendon fibroblasts a possible role for remodelling and pathological transformation Exp Toxicol
101. ure 1 There are also some superficial fibers with random orientation creating a mesh like matrix on the femoral articular surface Type I collagen is a fibrous component that is strong in tension This collagen arrangement is ideal when resisting the hoop stress created during normal loading conditions Circumferential AD Fis fibers ilij i i Chondrocytes www orthoteers co uk Nrujp ij33 1m orthkneemenisc htm Figure 1 1 A cross section of the meniscus showing the radial and circumferential collagen fiber orientation Also shown are blood vessels penetrating the peripheral one third of the tissue and location of chondrocytes Proteoglvcans are another important component within the meniscus that add resilience and strength to the structure during compressive loading 1 27 Much of the noncollagenous portion of the extracellular matix is proteoglycans termed aggrecan decorin and biglycan with aggrecan being the major type These are large molecules with a core protein and a repeating sugar chain that is electronegative These hydrophilic molecules can entrain 50 times their weight in free solution The charge charge repulsion force stiffly extends the proteoglycans in the matrix making them naturally resistant to compression Aggrecan is a type of proteoglycan that aggregates to hyaluronic acid to form a large molecule like those found in articular hyaline cartilage These cartilage like proteoglycans
102. urs while time load and position were recorded Upon completion samples were removed from the dish and cut into superior and deep zones The wet weight of each half of the explant was determined prior to incubation for 24 hrs in test media 48 5 Dulbecco s modified Eagles medium 48 5 Ham s F 12 2 Fetal Bovine Serum 1 penicillin streptomycin A preliminary study that tested NO production following 15 strain with post incubation times of 24 48 and 51 72 hours showed maximal expression at 24 hours Following post incubation the media was immediately stored at 80 C until NO quantification NO Quantification Nitric oxide was measured from each explant by using a total NO assay as detailed by the manufacturer Cayman Chemical Ann Arbor MI The assay measured the stable form of NO nitrite and nitrate by converting all nitrate to nitrite using the Greiss reaction The reaction produces a colored azo dye that absorbs light at 540 nm which can be read in a microplate reader The resulting absorptivity was converted to concentration using a standard curve created from known concentrations of nitrite The resulting concentrations were then normalized by the weight grams of each tissue sample Statistical Analysis Regression analysis was used to determine the relationship between strain level and nitric oxide production Data points with quadratic regression lines and 95 confidence intervals were plotted R squared and P values w
103. vpe of connective tissue cell that secretes extra cellular matrix that is rich in Type I collagen These cells are capable of differentiating into several different types of more specialized cells 31 Fibroblasts can convert into chondrocytes osteocytes fat cells and smooth muscle cells 31 It appears that the conversion from fibroblast to chondrocyte is reversible The differentiation of these cells seems to be influenced by the extra cellular matrix through physical and chemical effects An example is chondrocytes that are cultured in low density as a monolayer Under these conditions the chondrocytes lose their rounded shape flatten and stop producing collagen matrix 31 Instead the cells stop producing Type II collagen and start producing Type I collagen taking on the appearance of fibroblasts 31 This helps explain why the cells within the meniscus appear as fibroblasts in some regions and chondrocytes in others Since the superficial region of meniscus has such a large amount of Type I collagen the cells take on the fibroblast form and produce Type I collagen The cells in the deep regions of the meniscus are surrounded by more proteoglycans and 10 small amounts of Type II collagen These cells are chondrocytic and function to maintain the pericellular matrix The cells of the meniscus are set in well defined lacunae and can be individual or paired 26 The lacunae in the superficial layer are more compressed and fusiform tha
104. y and resolution above a previously used 2000Ib capacity load cell with similar dimensions The signal from the force transducer connects directly to a 2100 series signal condition and amplifier Vishay This unit allow for easy adjustment of signal balancing and amplification That conditioned signal connects directly to the actuator to provide a continuous load reading Figure A 2 The connection supplies a voltage to the input pins which is read through the Smartmotor as an analog signal that is converted to an encoder count The actuator is then connected to the PC which uses an Animatics control system and Smartmotor SMI interface 66 Figure A 1 The dish and load cell assembly Figure A 2 The amplified load cell signal connection to of the bioreactor the SmartMotor actuator white cable to input pins The system stands 50 cm tall and 25 cm in length and width allowing it to be contained in an incubator Figure A 3 The l inch thick aluminum plates Al 6061 at the top and bottom of the bioreactor are supported by 1 inch aluminum rods The plunger is also made of aluminum and has six Teflon filled Delrin compression rods 8mm in diameter The dish is also machined out of aluminum allowing the compression surface assembly plunger and dish to be sterilized by alcohol or autoclave Figure A 3 The bioreactor frame allows the system to fit in an incubator and contains many components that can be sterilized 67 A 2 Valid
105. y family for their encouragement of me taking on and completing this project Their support had helped push me to finish work that I can be proud of and I greatly appreciate it ill TABLE OF CONTENTS List of Tables List of Figures CHAPTER ONE Introduction 1 1 12 1 3 1 4 LS 1 6 1 7 1 8 Functions of the Meniscus In Vivo Loading Environment Material Properties of the Meniscus Composition and Structure Cellularity and Nutrition Mechanotransduction Nitric Oxide Hypothesis and Aims References CHAPTER TWO Validation of Bioreactor 2 1 2 2 2 3 2 4 2 5 Abstract Introduction Materials and Methods Results Discussion References iv CHAPTER THREE Nitric Oxide Production 3 1 Abstract 3 1 Introduction 3 3 Methods and Materials 3 4 Result 3 5 Discussion Recommendation References APPENDIX A Supplementarv Information on Chapter Two A l Description of Bioreactor Components and Features A 2 Validation of Even Well Pressure A 3 Validation Protocols A 4 Validation Programs APPENDIX B Supplementary Information on Chapter Three B 1 Compression Programming B 2 Design Drawings B 3 Experiment Protocols B 4 Nitric Oxide Production Raw Data LIST OF TABLES Table 2 1 Precision data of ultra low pressure film Table 2 2 Results of pressure film verification at two different loads Table 2 3 Displacement accuracv using gap measurement Table 2 4 Gap varia

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