Development of a Multi-Layer Skin Substitute Using Human Hair Keratinic Extract-Based Hybrid 3D Printing

Abstract

Large-sized or deep skin wounds require skin substitutes for proper healing without scar formation. Therefore, multi-layered skin substitutes that mimic the genuine skin anatomy of multiple layers have attracted attention as suitable skin substitutes. In this study, a novel skin substitute was developed by combining the multi-layer skin tissue reconstruction method with the combination of a human-derived keratinic extract-loaded nano- and micro-fiber using electrospinning and a support structure using 3D printing. A polycaprolactone PCL/keratin electrospun scaffold showed better cell adhesion and proliferation than the keratin-free PCL scaffold, and keratinocytes and fibroblasts showed better survival, adhesion, and proliferation in the PCL/keratin electrospun nanofiber scaffold and microfiber scaffold, respectively. In a co-culture of keratinocytes and fibroblasts using a multi-layered scaffold, the two cells formed the epidermis and dermal layer on the PCL/keratin scaffold without territorial invasion. In the animal study, the PCL/keratin scaffold caused a faster regeneration of new skin without scar formation compared to the PCL scaffold. Our study showed that PCL/keratin scaffolds co-cultured with keratinocytes and fibroblasts promoted the regeneration of the epidermal and dermal layers in deep skin defects. Such finding suggests a new possibility for artificial skin production using multiple cells.

Novel polycaprolactone (PCL)‐type I collagen core‐shell electrospun nanofibers for wound healing applications

“…[4][5][6][7][8] Nevertheless, this process may be limited by factors, such as the size and depth of the wound, which delay tissue regeneration. [9][10][11] Tissue engineering has focused on the production of structures that can promote the formation of new tissues, supporting epithelial cell proliferation from biomaterials with structures close to those of the extracellular matrix (ECM) of the skin. 12,13 Nonetheless, the ECM of skin has a complex fibrillary structure, being a challenge for tissue engineering.…”

Biomaterials as a Vital Frontier for Stem Cell-Based Tissue Regeneration

“…Skin represents another example of complicated multilayer structure. Bioprinting and electrospinning provide a solution through the preparation of a multilayer scaffold that would support the attachment and survival of both keratinocytes and fibroblast to represent the epidermal and dermal components ( Choi et al, 2021 ). The multilayer scaffold should take into consideration the physical properties of each biological layer.…”

Electrospinning and Additive Manufacturing: Adding Three-Dimensionality to Electrospun Scaffolds for Tissue Engineering

“…Additional research has further explored the co-existence of electrospun fibers (usually randomly distributed) and 3D printed elements within the same scaffold to influence cellular behavior ( Mota et al, 2011 ; Yang et al, 2015 ). Multi-layer skin substitutes have been developed by depositing electrospun polycaprolactone (PCL) and keratin fibers on to the two surfaces of a PCL 3D printed scaffold ( Choi et al, 2021 ), aiming at mimicking the histological structure of skin. The top electrospun layer (100 µm thick) consisted of nanofibers ( ∼ 0.7 µm average diameter) to support the growth of human immortalized keratinocytes (HaCaT); while the bottom layer (300 µm thick) was made of microfibers (∼ 1.7 µm average diameter) to enable the proliferation of normal human dermal fibroblasts (NHDF).…”