Fabrication of scalable and structured tissue engineering scaffolds using water dissolvable sacrificial 3D printed moulds

Abstract

One of the major challenges in producing large scale engineered tissue is the lack of ability to create large highly perfused scaffolds in which cells can grow at a high cell density and viability. Here, we explore 3D printed polyvinyl alcohol (PVA) as a sacrificial mould in a polymer casting process. The PVA mould network defines the channels and is dissolved after curing the polymer casted around it. The printing parameters determined the PVA filament density in the sacrificial structure and this density resulted in different stiffness of the corresponding elastomer replica. It was possible to achieve 80% porosity corresponding to about 150 cm(2)/cm(3) surface to volume ratio. The process is easily scalable as demonstrated by fabricating a 75 cm(3) scaffold with about 16,000 interconnected channels (about 1m(2) surface area) and with a channel to channel distance of only 78 μm. To our knowledge this is the largest scaffold ever to be produced with such small feature sizes and with so many structured channels. The fabricated scaffolds were applied for in-vitro culturing of hepatocytes over a 12-day culture period. Smaller scaffolds (6×4 mm) were tested for cell culturing and could support homogeneous cell growth throughout the scaffold. Presumably, the diffusion of oxygen and nutrient throughout the channel network is rapid enough to support cell growth. In conclusion, the described process is scalable, compatible with cell culture, rapid, and inexpensive.

Fabrication of scalable tissue engineering scaffolds with dual-pore microarchitecture by combining 3D printing and particle leaching

“…Sacrificial moulds for both structured and dual-pore scaffolds were 3D printed using a Replicator 2X 3D filament deposition printer (MakerBot Industries, LLC, Brooklyn, NY, USA) as described previously [25]. In brief, a 3D cube (L 25 mm × W 25 mm × H 10 mm) was designed using SolidWorks 2013 3D CAD design software (Dassault Systems, SolidWorks Corp., Waltham, MA, USA).…”

“…The scaffolds were prepared according to the protocol published previously [25]. In brief, cylindrical PDMS scaffolds of each type (Ø 6 mm, height 6 mm) were used in the experiments.…”

“…The scaffolds were washed twice with cell culture tested phosphate buffered saline (PBS) to remove excess collagen and then incubated in culture medium at 37°C for 2 h until cell seeding. The cells were seeded into the scaffolds using a special 6 mm thick cell seeding tray with several holes (Ø 6 mm), fabricated of poly(methyl methacrylate) (PMMA) using a CO 2 laser ablation machine (Epilog Mini 18 Laser, CO 80403, USA) as described previously [25]. The seeding tray was sterilized by washing with 0.5 M sodium hydroxide solution followed by rinsing with sterile water.…”

“…The metabolic activity of the cells was measured using the alamarBlue® assay (Life Technologies Corporation, UK) as described previously [25]. In short, the scaffolds were rinsed with PBS and incubated in 1 ml of culture medium containing 10% alamarBlue® solution for 2 h in the incubator.…”

“…The 3D printing of a sacrificial PVA mould allows generation of structured pores with a high degree of control over the inner architecture designed according to the requirements of an application as we have described previously [25]. The parameters of the random pores, e.g., degree of porosity and pore size, are controlled by varying the size of the salt crystals used as the porogen.…”