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Effects of Compressive Loading on Human Synovium-derived Cells
Y. Muroi1,*,
K. Kakudo1 and
K. Nakata2
1 Second Department of Oral and Maxillofacial Surgery, Osaka Dental University, 1-5-17, Otemae, Chuo-ku, Osaka, 540-0008, Japan; and
2 Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University
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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).
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Figure 2. RT-PCR analysis. RT-PCR products were subjected to electrophoresis through 2% agarose gels with ethidium bromide staining. Gels were digitally photographed under ultraviolet transillumination. The mRNA expression levels of (A) MMP-1, (B) MMP-2, (C) MMP-3, (D) TIMP-1, (E) ADAMTS-4, (F) ADAMTS-5, (G) IL-8, and (H) G3PDH. In each panel, a representative analysis is shown above a graph indicating the normalization relative to G3PDH, as determined by Scion Image analysis. The results are expressed as the % of control (control = average of the non-loaded constructs). Data represent the mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA and the LSD test for multiple comparisons between individual groups (*p < 0.05).
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Figure 3. Protein expression of 3D constructs. (A,B) Photomicrographs of representative histological sections after immunohistochemical analysis for MMP-1 (A) and MMP-3 (B). The inset images show partial magnification. Bar = 100 µm. Intense dark brown staining of MMP-1 and MMP-3 is evident in all constructs. More immunopositive cells were observed in the 20-kPa-loaded constructs than in the non-loaded constructs. (C) Western blotting for the active form of MMP-2 in the 3D constructs. The expressions of 72-kDa and 62-kDa bands were higher in the 20-kPa-loaded blots than in other blots. As a positive control, the culture medium of HT 1080 fibrosarcoma cells was used. (D) Western blotting for MMP-9 in the 3D constructs. The expressions of 92- and 68-kDa bands were not detected. As a positive control, the culture medium of HT 1080 fibrosarcoma cells was used. (E) Gelatin zymography for pro-MMP-9, pro-MMP-2, and MMP-2 in the culture supernatants of 3D constructs. Three bands of gelatinolytic activity corresponding to molecular weights of 92, 72, and 62 kDa were detected in the culture medium of HT 1080 fibrosarcoma cells as the positive control, and 2 bands of gelatinolytic activity corresponding to molecular weightsec of 72 and 62 kDa were detected in the constructs.
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Journal of Dental Research, Vol. 86, No. 8,
786-791 (2007)
DOI: 10.1177/154405910708600819
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