Bioprinting is a technology that produces artificial tissues/organs for medical purposes using bioinks containing cells and 3D printers, and is currently actively used in various regeneration, diagnosis, and emergency medical research, such as cancer mechanism research using artificial cancer models that simulate cancer tissues, as well as the production of artificial organs for transplantation. In particular, research is actively underway to improve the physical properties and biological properties of bioinks containing cells to produce the desired three-dimensional structure and/or to induce cell activity and differentiation.

- Fabrication of cell structures for muscle regeneration using bioinks derived from fish skin and bidirectional photo-crosslinked bioprinting system

Since bioinks are cell carriers and the basis for cell growth, they are mainly made of hydrogels, and in particular, bio-derived hydrogels such as collagen and decellularized extracellular matrix, which contain a lot of cell-active substances, should be used to promote cell activity and differentiation. In the case of currently used bio-derived hydrogels, most of them rely on collagen and decellularized extracellular matrix derived from mammals such as pigs. However, these mammalian-derived biomaterials have limitations such as high inflammatory response and low angiogenesis.

In order to overcome these limitations, the research team (1st author: SeoYul Cho) led by Professor Geun Hyung Kim in School of Medicine has produced a composite bioink derived from fish skin using an extracellular matrix derived from seawater fish and an extracellular matrix derived from freshwater fish. Fish skin accounts for most of the by-products of fisheries generated during the processing process, and Professor Kim's research team used the discarded fish skin as a biomaterial to produce bioink for effective tissue regeneration. In particular, seawater fish contains abundant omega-3 fatty acids, which not only play an important role in promoting vascularization and anti-inflammatory responses, but also are known to enhance the expression of myogenic differentiation factors of stem cells in the process of myogenesis. However, extracellular matrix derived from seawater fish has a low denaturation temperature and low processability, so this research team produced a bioink that can be photo-crosslinked using an extracellular matrix derived from freshwater fish with a relatively higher denaturation temperature. In addition, the cells were contained in the bioinks and uniformly aligned to mimic the aligned structure of muscle tissue by inducing the shear stress in the printing nozzle using bidirectional photo-crosslinking during the printing process.

As a result of applying the cell structure produced by applying a composite material-derived bioink to a bidirectional photo-crosslinked bioprinting system to an animal muscle damage model, it was confirmed that the efficacy of muscle tissue regeneration and muscle function recovery was improved compared to the existing mammalian-derived bioink, and the formation of neuromuscular junctions as well as blood vessels was also improved. On the other hand, the inflammatory response was found to be reduced.

Prof. Kim said, "These fish-derived composite-based bioinks can complement the problems of existing mammalian-derived biomaterials and can be used as functional bioinks that can induce excellent angiogenesis and low inflammatory response, because they contain abundant omega-3 fatty acids. In addition, it has shown an excellent muscle regeneration effect through an animal muscle injury model, and it is expected that it can be used for the regeneration of various tissues such as skin or bone. In particular, the produced bioink can be applied to various systems using bioprinters, such as bidirectional photo-crosslinked bioprinting systems, and it is expected that it can be used for the regeneration of various complex tissues. In addition, it is expected that economic and environmental benefits can be derived from recycling fish skin, which was considered waste, into biomaterials.".

Figure 1. Fish-derived composite-based bioink

- Development of porous collagen bioink with improved shape processability and bioprinting platform for bone tissue regeneration that mimics the hierarchy of bone microenvironment

Natural hydrogels such as collagen are limited in their application to bioprinting due to their lack of physical properties. In addition, for the smooth supply of oxygen and nutrients in artificial tissues containing cells, it is essential to have a porous structure that serves as a channel for the circulation of the culture medium containing nutrients and oxygen every 2~300 micrometers. To this end, hydrogel-based bioinks have been stacked in the form of a mesh using bioprinting technology, or air has been injected into the bioinks to have their own porous structures.

However, most of the existing manufacturing methods have clear limitations, such as limited cell activity or reduced physical properties due to the injected air, and in particular, they are limited in properly mimicking the microstructure of living tissues such as bone hierarchy and vascularization. To overcome these limitations, the research team (1st author: YoungWon Koo) led by Professor GeunHyung Kim has developed a collagen-based bioink with a micro-porous structure that greatly improves three-dimensional shape processability.

Professor Kim said, "The significance of this study is to overcome the limitations of bioinks in existing bioprinting technologies and to develop a new concept of bioink that balances the two most important properties of bioinks, namely processability and biological functions, and it is expected that it will be possible to depict the details of vascularized living organs and simulate their 3D structures, which were difficult in the past. In the future, it is also expected that it can be directly applied to various disease researches, including more various tissue regeneration studies and biochips that simulate the cancer development environment due to their excellent physical and biological properties.".

Figure 2. Porous Collagen Bioink

The results of the above research were supported by the Korea National Institute of Health research project and also supported by a grant from the Ministry of Trade, Industry & Energy (MOTIE, Korea) under Industrial Technology Innovation Program, and are scheduled to be published in an international journal, Applied Physics Reviews (1^st author: SeoYul Cho, IF=15.0)*, and published online on February 28 in Advanced Functional Materials (1^st author: YoungWon Koo, IF=19.0)**, respectively,.

*Research title: Bioengineered Cell-constructs Using Decellularized Fish Skin-Based Composite Bioink for Regenerating Muscle Tissue

**Research title: An Approach for Fabricating Hierarchically Porous Cell‐Laden Constructs Utilizing a Highly Porous Collagen‐Bioink