Event Abstract Back to Event 3D printed scaffold architecture controls stem cell differentiation Murat Guvendiren1*, Carmelo De Maria2, Francesca Montemurro2, Giovanni Vozzi2* and Joachim Kohn1 1 Rutgers University, New Jersey Center for Biomaterials, United States 2 University of Pisa, Department of Ingegneria dell'Informazione (DII), Italy Introduction: Chemistry, mechanics, and topography of a biomaterial strongly regulate the behavior of cells on it, such as attachment, proliferation and differentiation. On 2D substrates cell area and aspect ratio have been shown to control stem cell differentiation[1],[2]. The surface geometry of the substrate has been shown to determine spatial patterning of cell fate[3],[4]. However, the majority of studies have focused on 2D substrates and the use of 3D scaffolds is rather limited. In this study, we used 3D printed scaffolds with a range of designs composed of square, hexagonal or octagonal grids to investigate stem cell response to 3D architecture. Methods: 3D scaffolds were printed using a pressure-activated microsyringe system. Scaffolds were fabricated from poly(tyrosol carbonate) (PTyC) or poly(L-Lactic acid) (PLLA) (10wt% in THF). Each scaffold was composed of three layers with identical design (square, hexagonal and octagonal). Each layer was shifted by one half unit with respect to the previous layer (Fig. 1 insets) to create a 3D porous structure composed of three layers. Human mesenchymal stem cells (hMSCs, from Texas A&M) at passage 3 were seeded (6.5x103 cells/cm2). Alamar Blue assay was performed at day 1, 7 and 14 to evaluate cellular activity. One sample from each group was fixed to evaluate cell morphology. For differentiation studies, hMSCs were cultured 1 day in growth media followed by 14 days in mixed adipogenic/osteogenic induction media. Cells were fixed and stained for F-actin (rhodamine phalloidin), nuclei (Hoechst), and alkaline phosphatase (Fast Blue RR/napthol solution). All measurements were done using NIH ImageJ. Results and Discussion: These adipogenic cells represent a cell population with AR < 2 and cell area < 4000 mm2. The cellular morphology for osteogenic population was significantly different for the square pattern scaffolds (AR < 2 and mean cell area = 8000 mm2) when compared to hexagonal AR =3.7 and cell area = 5500 mm2) and octagonal (AR = 3.5, cell area = 5300 mm2) pattern scaffolds. This indicates an increase in cellular elongation on curved design (in the case of hexagonal and octagonal) when compared to linear design. The percentage of osteogenic cells was significantly higher on scaffolds with hexagonal and octagonal patterns (~80%) as compared to those with square patterns (~65%). These adipogenic cells represent cell population with AR < 2 and cell area < 4000 mm2. The cellular morphology for osteogenic population was significantly different for square scaffolds (AR < 2 and mean cell area = 8000 mm2) when compared to hexagonal AR =3.7 and cell area = 5500 mm2) and octagonal (AR = 3.5, cell area = 5300 mm2) scaffolds. This indicates an increase in cellular elongation on curved design (in the case of hexagonal and octagonal) when compared to linear design. The percentage of osteogenic cells was significantly higher on hexagonal and octagonal scaffolds (~80%) as compared to linear (~65%). Conclusions: Our results show that hMSC cell spreading (area and aspect ratio) can be controlled by 3D scaffold architecture. Cells were highly elongated on hexagonal and octagonal scaffolds leading to a significantly higher osteogenic phenotype as compared to square scaffolds. This study shows the importance of scaffold architecture on stem cell differentiation, and can aid as a first step to develop scaffolds that promote osteogenic differentiation by controlling design. This work was supported by Award Number P41EB001046 from the National Institute of Biomedical Imaging and Bioengineering.