INTRODUCTION 7 11 12 15 16 2 3 in vivo in vitro 9 15 7 15 13 18 in vivo 18 μ 8 10 18 The objectives of this study were (1) to design and build a practical, cost-effective device for applying homogeneous strains to tissue explants, and (2) to utilize the system to overload and underload meniscal tissue and measure the biochemical output. In this communication, we show that the ensuing device is a simple biocompatible design that applies accurate and reproducible strains and is made of components that can be sterilized. We utilized the bioreactor to apply both load and displacement controlled dynamic compression tests. Dynamic strain compression tests showed PG breakdown following overloading of meniscal tissue. No significant changes in concentration of PG released to the conditioned media was measured following various levels of dynamic compressive stress. A comparison between stress controlled dynamic compression versus strain controlled dynamic compression was also made. MATERIALS AND METHODS Design of Bioreactor 1 μ FIGURE 1. A 2-dimensional view of the assembly of the plunger, dish, and cap. The test frame is composed of two aluminum 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.  A strain gage load cell (Model 1210AF, Interface, Scottsdale, AZ) with a load capacity of 1334 N (sensitivity of ∼1.3 N) was utilized. For tests that require loads near or above 1334 N, a dimensionally identical load cell with 8896 N (sensitivity ∼2.17 N) (Interface, Scottsdale, AZ, USA) capacity is interchangeable with the current load cell. This design feature of interchangeable load cells allows for a larger range of loads to be accurately measured. A 2100 series signal conditioner (Vishay Instruments, Raleigh, NC, USA) amplifies the load cell signal to produce a 5 V signal at the maximum load. 2 2 FIGURE 2. A 2-dimensional drawing of the test frame shows the side and top view of the test system. 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.  Accuracy Evaluation of the System Ultra-low pressure film (Sensor Products Inc., East Hanover, NJ, USA) was used to measure well pressure during compression. First, the repeatability of the pressure film was tested by loading the film (seven times) between flat platens in a tensile testing machine (Model 8872, Instron Corp., Canton, MA, USA) to a 70 N target load, corresponding to a pressure of 0.477 MPa for the given indentor size. Calibration of the pressure film was also done using the tensile testing machine and included loading pieces of pressure film ranging from 0.2 to 1.64 MPa. Pressure film analysis was completed using commercial software (Scion Image, National Institute of Standards and Technology, Gaithersburg, MD, USA) to measure the density of the pressure film samples. 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. Four tests were conducted, each to the same pressure (0.477 MPa) to determine the repeatability of the bioreactor in load control. The film from the bioreactor was analyzed and density measured to determine the difference between each compression rod. The difference in film density and the maximum percentage error was determined to demonstrate the accuracy of the system. Determination of Displacement Repeatability μ Compliance of the System The compliance of the system was determined by placing a flat stainless steel plate over the wells and running a load-deformation test in the absence of menisci. The test was repeated three times and the load-deformation data recorded. Application of the System 3 FIGURE 3. a – Meniscus following biopsy, b – Maximum axial strain following different partial meniscectomy simulated by FE analysis. Various percentages (5%, 10%, 30% and 60%) removed from different portion of the medial meniscus (A – anterior, C – central, P – posterior). Knee loaded with 1200 N axial force.  2 μ 18 3 4 2 FIGURE 4. Cross-section of the meniscus with the direction of the cut and explants showing superior and deep zones.  5 6 Data Analysis R 2 p RESULTS Accuracy Evaluation of the System 5 1 FIGURE 5. Pressure film impressions at 0.477 MPa under the six bioreactor compression rods.  TABLE 1. Results of pressure film verification. 0.477 MPa Pressure (MPa) Test # Rod 1 Rod 2 Rod 3 Rod 4 Rod 5 Rod 6 Average Std. Dev. 1 0.4773 0.4768 0.4771 0.4771 0.4768 0.4768 0.4770 0.0002 2 0.4768 0.4769 0.4768 0.4768 0.4769 0.4772 0.4769 0.0001 3 0.4773 0.4768 0.4769 0.4770 0.4769 0.4771 0.4770 0.0002 4 0.4773 0.4768 0.4769 0.4768 0.4769 0.4768 0.4769 0.0002 Determination of Displacement Repeatability The micrometer measurements from the body filler showed that the bioreactor was extremely repeatable. Well 3 had the largest standard deviation in height, 3.4 ± 0.01 mm, whereas well 1 had the lowest standard deviation 3.4 ± 0.0015. Compliance of the System R 2 Application of the System 6 FIGURE 6. n n  7 8 FIGURE 7. n n p  FIGURE 8. n  9 2 10 3 FIGURE 9. Stress vs. time for four representative displacement control tests. Only peak values during each cycle are plotted. Stress was calculated by dividing the peak load by the initial cross-sectional area.  Table 2. Change in pressures over the duration of the stress-relaxation tests. Strain Pressure (MPa) Start End 5% 0.166 ± 0.108 *# 0.038 ± 0.010 # 10% 1.141 ± 0.103 # 0.046 ± 0.010 # 15% 2.185 ± 0.827 0.035 ± 0.026 # 20% 3.548 ± 0.429 0.128 ± 0.020 n p p FIGURE 10. Strain vs. time for two representative load control tests. Only peak strain values during each loading cycle are plotted. Strain was calculated by dividing the peak displacement by the original height of the explant.  Table 3. Change in strains over the duration of the creep tests. Pressure (MPa) Strain (%) Start End 0.05 2.6 ± 0.53 11.6 ± 1.36 0.1 3.0 ± 0.12 20.7 ± 1.45* n p 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 and supraphysiological 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 allows easy transport between the culture hood and incubator without allowing open air and bacteria to infect the sample. Since the cap incorporates a linear bearing it does not need to be removed for testing. Utilizing the system features and designing the correct protocol will help maintain a physiological loading sterile environment. 7 8 15 et al μ 8 μ μ et al 11 μ 14 10 et al 1 et al 12 It was surprising that explants tested at 0.05 MPa showed greater PG breakdown than explants tested at 0.1 MPa for the superficial zone. One possible reason for this result might be related to the cell viability. A compression of 0.1 MPa may induce more cell death than 0.05 MPa of compression and hence fewer cells may be available for production of metalloproteases that may contribute to the breakdown of PG. Current studies are underway to document the degree of cell death in the explants. 9 10 4 18 18 in vitro versus 14 17 In summary, this simple and practical experimental system allows for reproducible application and quantification of homogeneous stresses or strains to explants tissues, thereby providing a systemic and quantitative method for correlating external mechanical stimuli to cellular and molecular mechanisms of mechanotransduction.