Discipline: Biological Sciences
Subcategory: Biomedical Engineering
Guleid Awale - University of Connecticut
Co-Author(s): Kevin W.-H. Lo, University of Connecticut, CT; Cato T. Laurencin, University of Connecticut, CT
Traumatic bone fractures have emerged as a growing epidemic in the United States, due to its high societal cost and the downsides of conventional treatment options such as autografts and allografts. Regenerative engineering is an innovative approach towards the formation of complex tissue and organ systems through the convergence of advanced material science, stem cell science, physics, developmental biology, and clinical translation. We have previously identified the capability of a short-term treatment scheme (24h) of the small molecule cAMP analogue, 8-Br-cAMP, to promote in vitro osteoblastic differentiation of osteoprogenitor MC3T3-E1 cells, and well as in vitro angiogenesis through the cell-based production of VEGF. In order to further the understanding of the mechanistic action of 8-Br-cAMP, we propose the development of an in vitro MC3T3/HUVECs co-culture system. By constructing the co-culture environment using a transwell system, we hypothesize that the short-term treatment of 8-Br-cAMP will promote the synergetic, dual effect of osteoblastic differentiation and VEGF production of MC3T3 cells and subsequently, the angiogenic differentiation of HUVEC cells. A transwell system equipped with 0.4 µm polyester membrane inserts was used to serve as the dual-chamber co-culture system, where the MC3T3 cells treated with 100 µM of 8-Br-cAMP were cultured within the top inserts, and the HUVEC cells were positioned in the adjacent bottom wells. This type of configuration allowed for adequate transport of the secreted VEGF from the MC3T3 cells in the top insert towards the HUVEC cells, thus enhancing its rate of angiogenesis. To determine the optimal ratio of growth media in the co-culture system, a MTS cell proliferation assay was conducted on MC3T3 and HUVEC cells. After the optimal culture conditions were determined, an ELISA test was performed to quantify the level of VEGF production of the MC3T3 cells in the surrounding cellular medium, and was followed up by RT-PCR to quantify the gene expression of the angiogenic markers: VEGF-A, ICAM-1, and VCAM-1. Statistical analysis between the untreated and treated experimental groups was accomplished using a Student’s t-test, where a p value < 0.05 indicated statistical significance. Our observations showed a significant increase in VEGF production and angiogenic gene expression within the 8-Br-cAMP treated co-culture system, thus illustrating its prospective usage in bone regenerative engineering. Future studies will include the testing of osteogenic differentiation markers, as well as the fabrication of a co-culture, bone organ model geared towards drug discovery applications. References: Lo, K. W.-H., Kan, H. M., Gagnon, K. A. & Laurencin, C. T. One-day Treatment of Small Molecule 8-Bromo-cyclic AMP Analogue Induces Cell-based VEGF production for In Vitro Angiogenesis and Osteoblastic Differentiation. J. Tissue Eng. Regen. Med. 10, 867–875 (2016).Not Submitted
Funder Acknowledgement(s): The authors gratefully acknowledge the funding from the Northeast LSAMP Bridge to the Doctorate (BD) fellowship program (1400382), the Raymond and Beverly Sackler Center for Biomedical, Biological, Physical, and Engineering Sciences, and NIH DP1 AR068147.
Faculty Advisor: Cato Laurencin, email@example.com
Role: I conducted the majority of the experimental planning and execution, such as the MTS, ELISA, RT-PCR, and other accompanying cell culture procedures.