Fabrication and Evaluation of Electrospun, 3D-Bioplotted, and Combination of Electrospun/3D-Bioplotted Scaffolds for Tissue Engineering Applications

Abstract

Electrospun scaffolds provide a dense framework of nanofibers with pore sizes and fiber diameters that closely resemble the architecture of native extracellular matrix. However, it generates limited three-dimensional structures of relevant physiological thicknesses. 3D printing allows digitally controlled fabrication of three-dimensional single/multimaterial constructs with precisely ordered fiber and pore architecture in a single build. However, this approach generally lacks the ability to achieve submicron resolution features to mimic native tissue. The goal of this study was to fabricate and evaluate 3D printed, electrospun, and combination of 3D printed/electrospun scaffolds to mimic the native architecture of heterogeneous tissue. We assessed their ability to support viability and proliferation of human adipose derived stem cells (hASC). Cells had increased proliferation and high viability over 21 days on all scaffolds. We further tested implantation of stacked-electrospun scaffold versus combined electrospun/3D scaffold on a cadaveric pig knee model and found that stacked-electrospun scaffold easily delaminated during implantation while the combined scaffold was easier to implant. Our approach combining these two commonly used scaffold fabrication technologies allows for the creation of a scaffold with more close resemblance to heterogeneous tissue architecture, holding great potential for tissue engineering and regenerative medicine applications of osteochondral tissue and other heterogeneous tissues.

Figure 1

Fabrication of combined micro- and nanofibrous scaffold by sandwiching an electrospun layer between 3D-bioplotted layers. (a) Schematic of technical approach. (b) 3D-bioplotted scaffold with and without an electrospun layer (scale bars top = 2 mm; bottom = 500 μm). (c) Cross-sectional CAD representation of three different scaffolds created and evaluated. Top: microfibrous scaffold fabricated using 3D bioplotting technique only. Middle: nanofibrous scaffold fabricated using electrospinning only. Bottom: alternating micro- and nanosized fibers by combining 3D bioplotting and electrospinning techniques. Colors and textures for visualization purposes only.

Figure 2

Scanning electron microscopy of (a) 3D-bioplotted scaffold; (b) electrospun nanofibers; (c) combined 3D-bioplotted and electrospun scaffolds (electrospun layer in middle); (d) cells growing on 3D-bioplotted scaffold; (e) cells growing on electrospun nanofibers; and (f) cells growing on combined 3D and electrospun scaffold (scale bars (a), (c), (d) = 500 μm; (b) = 100 μm; (e) = 50 μm; (f) = 1 mm).

Figure 3

Cell viability and proliferation of human adipose-derived stem cells seeded on (a, c) 3D-bioplotted scaffolds and (b, d) electrospun scaffolds (green = live cells; red = dead cells).

Figure 4

Cell spreading and proliferation of human adipose-derived stem cells throughout scaffolds. Cells were cultured for 21 days in 3D-bioplotted, electrospun, or combination of 3D-bioplotted/electrospun scaffolds, fixed, and stained (actin = red; nuclei = blue). Superficial and cross-sectional views show cells present both on the surfaces of the scaffolds (superficial) and throughout the centers of the scaffolds on the 3D-bioplotted and combined scaffolds (cross-section). Human ASC exhibited steady proliferation over 21 days of culture on all scaffold types as indicated by AlamarBlue (% AB reduction). Bars indicate mean ± SEM (^∗∗∗p < 0.0001; ^∗∗p < 0.005).

Figure 5

Implantation technique of 3D-bioplotted, electrospun, and combined 3D-bioplotted/electrospun scaffolds into a cadaveric porcine knee. (a) A power reamer was used to create an osteochondral defect to a depth of 8 mm and 8 mm diameter. (b) The COR system was used to implant the scaffolds into the osteochondral defect. (c) View of 3D-bioplotted scaffold (left) and electrospun scaffold (right) after implantation. (d) Combined 3D-bioplotted/electrospun scaffold prior to implantation (black arrows indicate electrospun layer, scale bar = 1 mm). (e) Combined 3D-bioplotted/electrospun scaffold inserted into the COR system for implantation (bracket and arrow pointing at scaffold inside the device). (f) Combined 3D-bioplotted/electrospun scaffold after implantation.