FIG. 3.

Structural analysis of multilayer scaffolds. (A) Schematic illustration technique for fabrication of multilayer poly(lactide-co-glycolide) (PLG) scaffolds (details in the Materials and Methods section). Vortexed mixtures of PLG microspheres and sieved salt (NaCl) particles for each layer were sequentially added to the cylindrical scaffold mold (step 1) and lightly compacted with a hammer (step 2). The mold contents were then compressed in a hydraulic press at 2.5 MPa for 4 min to produce a scaffold tablet (step 3). The tablets were then equilibrated with 800 psi CO₂ for 16 h in a high-pressure chamber, followed by rapid gas release to induce foaming of the PLG polymer around salt particles (step 4). To generate a porous scaffold structure, salt was removed from the scaffold by immersing the foamed scaffolds in distilled water, and scaffolds were then dried by fluid wicking (step 5). Before cell seeding, scaffolds were sterilized by bathing in 70% ethanol for 10 min, washed twice with phosphate-buffered saline, and serum coated in 100% fetal bovine serum for 30 min. (B) Representative photograph of the external surface of a three-layer scaffold, in which outermost layers were highlighted using a red dye, orcein. Inset image (top right) shows higher magnification detail and a standard US penny for size comparison. (C) Perspectives at 90° rotations from microcomputed tomography reconstruction of a five-layer scaffold, in which BaSO₄ was incorporated in the second and fourth layers. Color images available online at www.liebertpub.com/tea