2.1. Preparation of Plasma Modified 2D and 3D PCL Scaffolds

Scaffolds of PCL in 2- and 3-dimensional (2D and 3D) forms were obtained from the National Metal and Materials Technology (MTEC, Bangkok, Thailand). Fabrication and oxygen plasma surface modifications were performed as previously described by Kosorn et al. (2012) [19]. Briefly, the 2D PCL scaffolds were prepared by hydrolysis of PCL pellets (Sigma-Aldrich, St. Louis, MO, USA) in 6 N of NaOH, at 50 °C, for 5 h, followed by soaking in deionized water and freeze-drying overnight. For the 3D scaffold preparation, the PCL pellets were loaded into a cylindrical vessel, heated at 60 °C for 10 min, filled with CO₂ at 15 MPa, and then soaked in deionized water for 3 h. Both 2D and 3D PCL scaffolds were then treated with pure oxygen plasma, using a low-pressure radiofrequency discharge plasma cleaner (model PDC-002, Harrick, Ithaca, NY, USA) at 30 W for 30 min. In this study, plasma-treated 2D and 3D PCL scaffolds are abbreviated as 2D-TP and 3D-TP, while untreated 2D and 3D PCL scaffolds are termed 2D-NP and 3D-NP, respectively. All types of scaffolds were finely cut into small circular pieces that were 6 mm in diameter for each well of 96-well plate (Figure 1). The 2D scaffold was similar to a flat sheet with a thickness of 0.5 mm (Figure 1a), whilst the 3D looked like a sponge disc with a thickness of 2.0 mm (Figure 1b). To sterilize the scaffolds for cell culture, each side was exposed for 15 min to standard UV light in a biosafety cabinet. The sterile circular piece was then immersed into the bottom of a well in a 96-well plate, washed by 1X phosphate buffer saline (PBS) twice, and then filled up with the complete medium mixture, for at least 30 min. This immersion steps aimed to avoid scaffold floating from the bottom of the culture plate since the air bubbles in the scaffold were fully replaced with the complete medium. After that, the scaffold was ‘ready to use’ for further cell culture experiment. The protocol diagram is provided in Figure 2a.

The appearance of finely cut circular polycaprolactone (PCL) scaffolds of (a) the 2-dimensional (2D) flat sheet and (b) the 3-dimensional (3D) sponge disc.

Simple diagrams exhibiting a protocol of each experiment performed in this research. (a) Polycaprolactone (PCL) scaffold preparation; (b) Initial cell attachment assay; (c) Cell proliferation and attachment assays by enzyme-linked immunosorbent assay (ELISA); (d) Observation of cellular attachment under scanning electron microscopy (SEM); (e) Gene expression by semi-quantitative polymerase chain reaction (PCR).