Smooth muscle cell responses to poly(3-hydroxybutyrate) spherulites

Abstract

Event Abstract Back to Event Smooth muscle cell responses to poly(3-hydroxybutyrate) spherulites Jinbo Dou1, Juliana G. De Menezes1 and Shanfeng Wang1, 2 1 The University of Tennessee, Knoxville, Department of Materials Science and Engineering, United States 2 The University of Tennessee, Knoxville, Institute of Biomedical Engineering, United States Statement of Purpose: Semi-crystalline polymers such as poly(ε-caprolactone) can crystallize into spherulitic surfaces such as banded spherulites for investigating cell-substrate interactions[1]-[3]. Poly(3-hydroxybutyrate) (PHB) is a bioderived and biodegradable semi-crystalline polyester with a low Tg of 0 °C and a high Tm of 175 °C[3]. Here we report primary aortic smooth muscle cell (SMC) responses to different surface morphologies of PHB films prepared at different conditions such as crystallization temperature (Tc), film thickness, and cooling rate from the melt. These morphologies included flat, banded spherulitic, and non-banded spherulitic surfaces, and surfaces with or without cracks. Methods: PHB was purchased from Sigma-Aldrich and used as received and dissolved in CHCl3 (1 g/10 mL) at 60 °C for 12 h. About 100-200 μL of the PHB solution was drop-coated onto glass coverslips. After the PHB films were fully dried in a vacuum oven, they were melted at 190 °C for 10 min and quickly transferred onto a hot stage for isothermal crystallization at Tc of 45, 65, 80, and 90 °C. Flat PHB films as the control were prepared by compressing the melt between two glass slides and cooled down to room temperature. SMCs were cultured for 4 days and characterized on the prepared PHB films with different topographic features. Results: Banded spherulites were clearly observed on PHB crystallized at the higher temperatures of 80 and 90 °C. At the lower temperatures of 45 and 65 °C, non-banded structures were obtained (Figure 1b and 1c). The average ridge-to-ridge widths in the PHB banded spherulites were 11.3 ± 0.8, 17.5 ± 4.6, and 35.2 ± 9.7 μm for Tc of 65, 80, and 90°C, respectively. The size of the spherulites was affected by the film thickness. Thicker films made spherulite sizes smaller as well as the band widths, by decreasing to 14.3 ± 2.3 and 24.5 ± 5.2 μm at 80 and 90°C, respectively (Figure 1c). Cracks appeared on the PHB films as the result of the different radial and circumferential thermal expansion coefficients[4]. There were two different crack types: long random cracks appeared in cooling and can be minimized by lowering the cooling rate while circular cracks formed for big spherulites. As shown in Figure 1a, both SMC attachment and proliferation were better on the flat surfaces of the flat, compressed PHB films than on the spherulitic surfaces. The lowest SMC attachment and proliferation occurred on the banded spherulites. Instead of showing alignment along the concentric ridges, the cells grew along the circular cracks, while the random cracks did not affect cell alignment evidently (Figure 1c). The cell nuclei did not show any obvious circularity differences, and the cell areas did not show too much differences except for the ones aligned (Figure 1d). Figure 1. a) SMC attachment at 4 h and proliferation on PHB films at day 1, 2 and 4 post-seeding. *: p <0.05. b) POM images of the flat film and films crystallized at 45 and 65 °C, and the corresponding cell images on them. c) Merged images of SMCs at day 1 on PHB films crystallized at 80 and 90°C in the presence or absence of circular cracks. d) SMC nuclei circularities and cell areas at day 1. *: p <0.05. Conclusions: SMC attachment and proliferation were lower on the rougher spherulitic surfaces of PHB films than on the flat, compressed ones. SMCs were aligned by the circular cracks formed in big spherulites, not by the concentric ridges in the banded spherulites. After the circular cracks were minimized, SMCs spread randomly with a moderately higher cell number. NSF (DMR-11-06142).