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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.

Wheat glutenin, the highly crosslinked protein from wheat, was electrospun into scaffolds with ultrafine fibers oriented randomly and evenly in three dimensions to simulate native extracellular matrices of soft tissues. The scaffolds were intrinsically water-stable without using any external crosslinkers and could support proliferation and differentiation of adipose-derived mesenchymal stem cells for soft tissue engineering. Regeneration of soft tissue favored water-stable fibrous protein scaffolds with three-dimensional arrangement and large volumes, which could be difficult to obtain via electrospinning. Wheat glutenin is an intrinsically water-stable protein due to the 2% cysteine in its amino acid composition. In this research, the disulfide crosslinks in wheat glutenin were cleaved while the backbones were preserved. The treated wheat glutenin was dissolved in aqueous solvent with an anionic surfactant and then electrospun into bulky scaffolds composed of ultrafine fibers oriented randomly in three dimensions. The scaffolds could maintain their fibrous structures after incubated in PBS for up to 35 days. In vitro study indicated that the three-dimensional wheat glutenin scaffolds well supported uniform distribution and adipogenic differentiation of adipose derived mesenchymal stem cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Intrinsically water-stable scaffolds composed of ultrafine keratin fibers oriented randomly and evenly in three dimensions were electrospun for cartilage tissue engineering. Keratin has been recognized as a biomaterial that could substantially support the growth and development of multiple cell lines. Besides, three-dimensional (3D) ultrafine fibrous structures were preferred in tissue engineering due to their structural similarity to native extracellular matrices in soft tissues. Recently, we have developed a nontraditional approach to developing 3D fibrous scaffolds from alcohol-soluble corn protein, zein, and verified their structural advantages in tissue engineering. However, keratin with highly cross-linked molecular structures could not be readily dissolved in common solvents for fiber spinning, which required the remarkable drawability of solution. So far, 3D fibrous scaffolds from pure keratin for biomedical applications have not been reported. In this research, the highly cross-linked keratin from chicken feathers was de-cross-linked and disentangled into linear and aligned molecules with preserved molecular weights, forming highly stretchable spinning dope. The solution was readily electrospun into scaffolds with ultrafine keratin fibers oriented randomly in three dimensions. Due to the highly cross-linked molecular structures, keratin scaffolds showed intrinsic water stability. Adipose-derived mesenchymal stem cells could penetrate much deeper, proliferate, and chondrogenically differentiate remarkably better on the 3D keratin scaffolds than on 2D PLA fibrous scaffolds, 3D soy protein fibrous scaffolds, or 3D commercial nonfibrous scaffolds. In summary, the electrospun 3D ultrafine fibrous scaffolds from keratin could be promising candidates for cartilage tissue engineering.

In this work, novel electrospun scaffolds with fibers oriented randomly and evenly in three dimensions (3D) including in the thickness direction were developed based on the principle of electrostatic repulsion. This unique structure is different from most electrospun scaffolds with fibers oriented mainly in one direction. The structure of novel 3D scaffolds could more closely mimic the 3D randomly oriented fibrous architectures in many native extracellular matrices (ECMs). The cell culture results of this study indicated that, instead of becoming flattened cells when cultured in conventional electrospun scaffolds, the cells cultured on novel 3D scaffolds could develop into stereoscopic topographies, which highly simulated in vivo 3D cellular morphologies and are believed to be of vital importance for cells to function and differentiate appropriately. Also, due to the randomly oriented fibrous structure, improvement of nearly 5 times in cell proliferation could be observed when comparing our 3D scaffolds with 2D counterparts after 7 days of cell culture, while most currently reported 3D scaffolds only showed 1.5- to 2.5-fold improvement for the similar comparison. One mechanism of this fabrication process has also been proposed and showed that the rapid delivery of electrons on the fibers was the crucial factor for formation of 3D architectures.