Development of a 3-Dimensional Cell Culture Method for Osteocyte Cell Growth and Proliferation

Osteocytes are mature bone cells that are crucial for the makeup and maintenance of bone within the human body. Any dysfunction in the makeup and function of these cells can be detrimental to human health- therefore, it is important to understand how osteocytes function within the body. In order to better understand the growth and development of osteocyte cells, a 3-dimensional bone culture would help in the ability to study and treat bone disorders. Current osteocyte models use 2-dimensional cultures, which provide a less similar environment than in the natural structure of bone. Therefore, we have attempted to develop a 3-dimensional cell culture model in which we grew osteocyte cells from mouse models in 3-dimensional inserts. The goal of this experiment was to not only see growth of the cells 3-dimensionally, but to measure gene and protein expression of Cx43, E11, and SOST- all aspects that are critical for the maintenance and proliferation of osteocyte cells. We used collagen, hydroxyapatite, and a collagen-hydroxyapatite mix as different mediums for this experiment to test which would be the most successful in the growth and proliferation of the osteocyte cells over a 14 day growth period. Both collagen and hydroxyapatite are crucial for the makeup of the bone matrix, as well as support of the osteocyte cells. 

Our experiment results yielded success with the 3-dimensional growth of the osteocyte cells, and staining and imaging further proved that the growth of the cells was successful. We additionally had successful results with expression of both the E11 gene and Cx42 protein. The collagen medium displayed the highest levels of Cx43 expression, while the hydroxyapatite displayed the highest levels of E11 expression. However, the SOST gene, which primarily functions to mark fully differentiated osteocytes and maintain bone formation, was not expressed in any of the models.  Our results indicate that the mediums and methods used to grow the osteocyte cells in a 3-dimensional cell culture method were not entirely successful, as the osteocyte cells did not fully express the three main genes and proteins that mark mature and fully functioning bone relative to 2-dimensional controls. The ability to grow the cells into a 3-dimensional shape proves significance and hope for the future directions of this research, as different mediums and timeframes may be successful in the expression of all three genes.

For more information: gkmckinley@eckerd.edu or bullocklab.eckerd.edu

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