Figure 1. Cyclic Loading System. (A) 3D cultured constructs produced from human TMJ synovium-derived cells mixed with collagen gel and seeded onto a porous collagen disc (9 mm diameter, 4 mm thick). A round 3D collagen scaffold disc was set on the bottom of a centrifuge tube of the same diameter, to produce the porous collagen disc. Next, a cell suspension in a collagen solution was loaded onto the 3D collagen disc, and the tube was then spun at 1000 rpm for 5 min at 5°C. The cell-scaffold construct was incubated at 37°C for 3 hrs to allow gelation to form the 3D cell construct. Bar = 5 mm. (B,C) Cyclic load bioreactor, consisting of cylindrical loading pistons connected to weights, a moving stage that raises and drops the loading pistons onto the constructs, and a linear actuator that controls the motion of the moving stage (B). Because the pistons (and weights) are raised by the moving stage and then allowed to fall onto the 3D constructs without actually being attached to the moving stage, during loading, stimulation of each sample is subject to constant peak load, due to the weight on each piston (C). The weights on the top of each piston are exchangeable, so that the cyclic load bioreactor can apply a designated peak load to each 3D tissue in the culture wells (C). With the cyclic load bioreactor, a maximum of 12 specimens can be simultaneously subjected to dynamic compressive stimulation with a constant peak load in an ordinary CO₂ incubator. The loading pistons are constructed of stainless steel and can be removed and sterilized. Specimens were cultured in 48-well culture plates for loading of uni-axial unconfined compression. The loading experiments were conducted in a humidified incubator maintained at 37°C in 5% CO₂. (D–F) HE-stained sections after loading treatment (D, non-loaded construct; E, 5-kPa-loaded construct; F, 20-kPa-loaded construct). Bar = 100 µm. The cells in the 3D constructs were evenly encapsulated in the collagen gel, with no cell leakage after loading. There was no major mechanical breakage of the collagen sponge after cyclic compression. (G) The total DNA contents of the constructs. There was no statistically significant difference among the 3 test construct groups. (H) The ratio of apoptotic cells in the 3D constructs presented as percentages of apoptotic cells relative to the total cell count in 24 fields (40x mag.) of 2 sections per sample. There was no statistically significant difference among 3 test construct groups. Data represent the mean ± SD (n = 3). Statistical analysis was performed by one-way ANOVA (p < 0.01).