Development of pigmented human skin constructs via 3D drop-on-demand bioprinting

• Main Author: Ng, Wei Long
• Other Authors: Yeong Wai Yee
• Format: Theses and Dissertations
• Language: English
• Published: 2018
• Subjects: DRNTU::Engineering::Bioengineering
• Online Access: http://hdl.handle.net/10356/73156
• Institution: Nanyang Technological University

As a proof-of-concept, a two-step bioprinting strategy is implemented to fabricate the 3D pigmented human skin constructs. The first step involves the development and optimization of a suitable polyvinylpyrrolidone (PVP)-based bio-ink for printing of cells with enhanced viability and homogeneity. The experimental results have highlighted the importance of bio-ink properties (represented by Z values) on printed cells; the printed cells that are cultured over a period of 96-hours do not show significant printing-induced damage when the Z values of the PVP-based bio-inks are below 9.30. This critical step facilitates the patterning of epidermal melanin units (EMUs, cell-cell interactions between the keratinocytes and melanocytes) at an optimal ratio and density. The second step involves the engineering the complex 3D microstructures in the collagen-fibroblast matrices using macromolecular crowding. An in-depth investigation is performed to first understand the synergistic effect of macromolecular crowding (MMC) on the complex 3D collagen-fibroblast matrices. The MMC can be used to alter the 3D microstructures (pore size and porosity) within the collagen-fibroblast matrices; the pore size of the 3D collagen-fibroblast matrices plays a critical role in regulating the cell-matrix remodeling process and cellular behavior. The study highlights the importance of hierarchical pore sizes within the 3D dermal skin constructs and provides critical insights (optimal pore size range for each region of the proposed tri-zone dermal constructs) for the development of improved dermal skin constructs using MMC. As such, a drop-on-demand bioprinting-based strategy is implemented to facilitate the precise deposition of PVP-based bio-ink at desired positions within each printed layer of collagen to manipulate the pore size within each printed region and eventually fabricate 3D hierarchical porous collagen-based structures. The 3D hierarchical porous collagen-fibroblast matrices serve as the dermal skin constructs for patterning of EMUs. Lastly, the feasibility of fabricating pigmented human skin constructs with uniform skin pigmentation (using 3 different skin cells from 3 different skin donors) is demonstrated. The histological analysis of the 3D bioprinted pigmented human skin constructs has revealed the similar morphological appearance to the native skin and the immunochemical analysis has indicated the presence of some important biomarkers in native skin. Although 3D bioprinting is an advanced manufacturing platform, it is critical to note that a holistic approach of combining bioprinting-based strategy with other important strategies such as MMC and co-culture techniques has facilitated the fabrication of 3D pigmented human skin constructs with uniform skin pigmentation.