ABSTRACT: The high discrepancies between adverse effects of chemicals in animal and human have led to the development of alternative in-vitro human tissue models to improve the reliability and accuracy of toxicology testing. 3D bioprinting technology has emerged as an advanced platform that facilitates simultaneous and highly-specific patterning of multiple types of cells and biomaterials, which is lacking in conventional tissue engineering approaches. The goal of this review is to highlight the achievements in skin bioprinting and present the future outlook on standardization of in-vitro human tissue models for toxicology testing.

KEYWORDS: 3D printing, 3D bioprinting, skin tissue engineering, human tissue models, toxicology testing

1. Toxicology testing

Toxicology testing is performed to identify the potential adverse effects a chemical poses to an individual and its surrounding environment (Hartung, 2009b). The different types of chemicals include active pharmaceutical drugs, cosmetics ingredients, household and industrial chemicals. An estimated number of 2,000 new chemicals are produced annually for various applications; routine toxicology tests are conducted on increasing number of new chemicals on a daily basis to ensure its safety to potential consumers. The global market for in-vitro toxicology testing market has been estimated to be ~USD 13 billion in 2016 and it is projected to reach USD 20.8 billion by the end of 2021 (MarketsandMarkets, 2016). An ideal study to evaluate the toxicity of a chemical/substance to humans would require an extremely large number of human subjects who are representative of the diversity of humans, which is unrealistic and unethical. As such, the use of animal models provides preliminary safety data to satisfy the conservative regulatory requirements. The crucial issue is the extent to which these animal models can predict the human responses in an accurate and reliable manner. It is clearly evident that the use of animal models has several caveats: the differences in the absorption or distribution of the chemicals/substances; the way the substances are metabolized and the short duration of animal lifespan (to accurately monitor disease development). As such, the use of animal models remains highly controversial as there are significant discrepancies between adverse effects of chemicals in humans and animals (Lilienblum et al., 2008). Furthermore, a complete ban on animal testing for cosmetics ingredients in 2013 has necessitates the development of alternative in-vitro skin models (W. L. Ng, Wang, Yeong, & Naing, 2016). A paradigm shift in the testing models has occurred over the last few years; the implementation of non-animal testing strategy has spurred the development of numerous 3D in-vitro testing methods (Burden, Sewell, & Chapman, 2015; Hartung, 2009a). In this review, we present and discuss the recent progress in skin bioprinting for potential toxicology applications.

3D BIOPRINTING OF SKIN CONSTRUCTS FOR TOXICOLOGY TESTING

WEI LONG NG1, CHEE KAI CHUA1

1Singapore Centre for3D Printing (SC3DP), School of Mechanical & Aerospace Engineering, Nanyang Technological University (NTU), 50 Nanyang Avenue, Singapore 639798, Singapore

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Proc. Of the 3rd Intl. Conf. on Progress in Additive Manufacturing (Pro-AM 2018) Edited by Chee Kai Chua, Wai Yee Yeong, Ming Jen Tan, Erjia Liu and Shu Beng TorCopyright © 2018 by Nanyang Technological UniversityPublished by Nanyang Technological University ISSN: 2424-8967 :: https://doi.org/10.25341/D4RC72

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3.1. Microvalve-based bioprinting Multi-layered collagen constructs containing human keratinocyte and fibroblast cell lines were deposited on a non-planar surface via layer-by-layer manufacturing approach (W. Lee et al., 2009) and greater cell viability in the 3D constructs with fluidic channels (85% viability) as compared to the ones without any channels (60% viability) was reported (W. Lee et al., 2010). A recent work emulated the native cellular density of different skin cells (HFF-1 fibroblasts and HaCaT keratinocytes) within the 3D bioprinted skin constructs using contactless microvalve print-heads (V. Lee et al., 2013) but the use of human skin cell lines resulted in poor stratification and keratinization of the printed keratinocyte layers. As such, recent works on skin bioprinting utilized primary human stem cells and skin cells. The amniotic fluid-derived stem cells (AFSCs at a cell density of 16.6 x 106 cells/ml) were encapsulated in the fibrinogen/collagen solution at 00C. Microvalve-based print-heads were used to print the fibrinogen/collagen solution and its cross-linker, thrombin, over the full-thickness skin wounds (2 cm x 2 cm) of nu/nu mice (Skardal et al., 2012). Although the AFSCs only remained transiently in wound sites, the secretion of important growth factors from AFSCs expedited wound closure rates and angiogenesis. Furthermore, the feasibility of in-situ printing was also explored on full thickness large wounds (10 cm x 10 cm) of nude mice (Binder et al., 2010). A single layer of fibrinogen/collagen hydrogel precursor containing fibroblasts (1.0 x 105 cells/cm2) was first cross-linked by nebulized thrombin to form fibrin/collagen hydrogel, followed by bioprinting another layer of keratinocyte cells (1.0 x 107 cells/cm2) above the fibroblast-populated fibrin/collagen matrix. Although complete re-epithelialization of the large wound was achieved after 8 weeks, it is highly challenging to deposit cells on areas of significant curvature. Notably, a recent study has reported the fabrication of pigmented human skin constructs (W. L. Ng, Tan, Yeong, & Naing, 2018). A two-step bioprinting strategy was implemented; hierarchical porous collagen-fibroblast dermal matrices were first fabricated using macromolecular crowding (W. L. Ng, Goh, Yeong, & Naing, 2018), followed by pre-defined patterning of the primary human keratinocytes and melanocytes in highly-specific arrangement found in native human skin. The 3D bioprinted human skin constructs were then matured under optimal culture conditions to eventually achieve the 3D pigmented human skin constructs. The 3D printed human skin constructs in most studies showed high resemblance to native skin structure and they can serve as potential in-vitro tissue models for toxicological testing.

3.2. Laser-based bioprinting A recent in-vitro study deposited 20 layers of fibroblasts (mouse NIH-3T3) and subsequent 20 layers of keratinocytes (human HaCaT) embedded in collagen gel onto a sheet of Matriderm®

(decellularized dermal matrix) (Lothar Koch et al., 2012). The presence of cadherins and connexin 43 (Cx 43) in the epidermis indicated tissue morphogenesis and cohesion. Another study reported good graft-take of printed skin construct with the surrounding tissue and angiogenesis from the wound bed was observed after 11 days of transplantation (Michael et al., 2013).

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4. Future Outlook One of the greatest challenges in toxicological testing will be the need to standardize the existing testing models. Currently, many researchers are developing 3D organotypic cultures that closely mimic the skin structure and function (Visk, 2015). As such, it is important to evaluate and assess the current in-vitro skin models and their limitations. Furthermore, it is also important to evaluate the inter-laboratory reproducibility of such in-vitro skin models. The implementation of 3D printing technology can offer a highly-automated fabrication process for standardization of in-vitro testing models. To date, no single testing approach is perfect and it is important to combine different testing approaches and integrate them into a holistic testing strategy to enhance the accuracy and reliability of toxicological testing.

5. Concluding Remarks The bioprinting platform offers high spatial control over the cellular and material deposition which is critical for cell-cell and cell-matrix interactions. To date, several researchers have demonstrated the fabrication of biomimetic skin constructs using different bioprinting techniques and the implementation of 3D bioprinting techniques not only helps to standardize the fabrication of in-vitro skin models but also improves the inter-laboratory reproducibility of in-vitro skin models. REFERENCES Binder, K. W., Zhao, W. X., Aboushwareb, T., Dice, D., Atala, A., & Yoo, J. J. (2010). In situ bioprinting of

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