Osteoblasts aggregates cultivated in a 3-dimensional culture environment rigorously respond to Porphyromonas gingivalis culture supernatants Amy Marie Riffel , University of Iowa Follow

[摘要] An exciting alternative to the current methods for bone regeneration is osseous tissue engineering. One such method focuses on enhancement of osteoblast differentiation through rotary cell culture techniques. The response of osteoblast aggregates to periodontal microorganisms and their by-products will ultimately be important in their success as a method of bone regeneration. In this study, I hypothesize that human embryonic palatal mesenchymal (HEPM, ATCC 1486) pre-osteoblast cells produce different cytokine responses depending upon whether they are grown in a 2-dimensional tissue culture flask or a 3-dimensional tissue culture vessel and whether they are exposed to an un-inoculated, sterile Porphyromonas gingivalis growth medium or exposed to a 24 hour, sterile, P. gingivalis culture supernatant. Objectives: My objectives were first to determine and compare the cytokine response of HEPM, ATCC 1486 pre-osteoblast cells depending upon whether they are grown in a 2-dimensional tissue culture flask or a 3-dimensional tissue culture vessel and whether they are exposed to an un-inoculated, sterile P. gingivalis growth medium or exposed to a 24 hour, sterile, P. gingivalis culture supernatant. Methods: In 3 experiments, 5 X 106 HEPM, ATCC 1486 cells were grown in a 2-dimensional tissue culture flask or a 3-dimensional tissue culture vessel and exposed to an un-inoculated, sterile P. gingivalis growth medium or exposed to a 24 hour, sterile, P. gingivalis culture supernatant and incubated for 72 hours in 5% CO2 at 37oC. Media was removed from the tissue culture flasks or rotary vessels at 0, 1, 2, 4, 8, 12, 24, 36, 48, 60, and 72 hours to determine cytokine concentrations in the Luminex 100 IS Instrument (Luminex®, Austin, TX). HEPM, ATCC 1486 pre-osteoblast cell morphology was assessed by light and scanning electron microscopy at 96 hours. Results: In experiment 1, there were increases in IL-6 and IL-8. The IL-6 response of cells grown in a 2-dimensional tissue culture flask was higher than that of cells grown in a 3-dimensional tissue culture vessel. The IL-8 responses of the cells grown in 2-dimensional, 3-dimensional tissue culture were nearly identical. In light and scanning electron microscopy cells appeared normal and HEPM, ATCC 1486 pre-osteoblast cell aggregates were similar to that previously reported. In experiment 2, there were also increases in IL-6 and IL-8. The IL-6 and IL-8 responses of HEPM, ATCC 1486 pre-osteoblast cells grown in a 3-dimensional tissue culture vessel exposed to a 24-hour, sterile, P. gingivalis culture supernatant were higher than cells exposed to un-inoculated, sterile P. gingivalis growth media. In experiment 3, HEPM, ATCC 1486 pre-osteoblast cells grown in 2-dimensional tissue culture flasks and 3-dimensional tissue culture vessel exposed to a 24 hour, sterile, P. gingivalis culture supernatant produced high levels of IL-6, IL-8, and VEGF. Again, in light and scanning electron microscopy, cells appeared normal. Conclusion: HEPM, ATCC 1486 pre-osteoblast cells display different cytokine profiles depending upon the type of vessel they are cultured in. They also rigorously respond to P. gingivalis culture supernatants suggesting that they may respond to the presence of microorganisms commonly found in the oral cavity and play an active role in immunity during their integration following bone regeneration.