Over the years, the field of bioprinting has attracted attention for its highly automated 16 fabrication system that enables the precise patterning of living cells and biomaterials at pre-17 defined positions for enhanced cell-matrix and cell-cell interactions. Notably, vat polymerization 18 (VP)-based bioprinting is an emerging bioprinting technique for various tissue engineering 19 applications due to its high fabrication accuracy. Particularly, different photo-initiators (PIs) are 20 utilized during the bioprinting process to facilitate the crosslinking mechanism for fabrication of 21 high-resolution complex tissue constructs. The advancements in VP-based printing have led to a 22 paradigm shift in fabrication of tissue constructs from cell-seeding of tissue scaffolds (non-23 biocompatible fabrication process) to direct bioprinting of cell-laden tissue constructs 24 (biocompatible fabrication process). This paper, presenting a first-time comprehensive review of 25 the VP-based bioprinting process, provides an in-depth analysis and comparison of the various 26 biocompatible PIs and highlights the important considerations and bioprinting requirements. This 27 review paper reports a detailed analysis of its printing process and the influence of light-based 28 curing modality and PIs on living cells. Lastly, this review also highlights the significance of VP-29 based bioprinting, the regulatory challenges and presents future directions to transform the VP-30 Page 1 of 47 AUTHOR SUBMITTED MANUSCRIPT -BF-102156.R2based printing technology into imperative tools in the field of tissue engineering and regenerative 31 medicine. The readers will be informed on the current limitations and achievements of the VP-32 based bioprinting techniques. Notably, the readers will realize the importance and value of 33 highly-automated platforms for tissue engineering applications and be able to develop objective 34 viewpoints towards this field.

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Three-dimensional (3D) pigmented human skin constructs have been fabricated using a 3D bioprinting approach. The 3D pigmented human skin constructs are obtained from using three different types of skin cells (keratinocytes, melanocytes and fibroblasts from three different skin donors) and they exhibit similar constitutive pigmentation (pale pigmentation) as the skin donors. A two-step drop-on-demand bioprinting strategy facilitates the deposition of cell droplets to emulate the epidermal melanin units (pre-defined patterning of keratinocytes and melanocytes at the desired positions) and manipulation of the microenvironment to fabricate 3D biomimetic hierarchical porous structures found in native skin tissue. The 3D bioprinted pigmented skin constructs are compared to the pigmented skin constructs fabricated by conventional a manual-casting approach; in-depth characterization of both the 3D pigmented skin constructs has indicated that the 3D bioprinted skin constructs have a higher degree of resemblance to native skin tissue in term of the presence of well-developed stratified epidermal layers and the presence of a continuous layer of basement membrane proteins as compared to the manually-cast samples. The 3D bioprinting approach facilitates the development of 3D in vitro pigmented human skin constructs for potential toxicology testing and fundamental cell biology research.

Bioprinting is an emerging research field that has attracted tremendous attention for various applications; it offers a highly automated, advanced manufacturing platform for the fabrication of complex bioengineered constructs. Different bio-inks comprising multiple types of printable biomaterials and cells are utilized during the bioprinting process to improve the homology to native tissues and/or organs in a highly reproducible manner. This paper, presenting a first-time comprehensive yet succinct review of microvalve-based bioprinting, provides an in-depth analysis and comparison of different drop-on-demand bioprinting systems and highlights the important considerations for microvalve-based bioprinting systems. This review paper reports a detailed analysis of its printing process, bio-ink properties and cellular components on the printing outcomes. Lastly, this review highlights the significance of drop-on-demand bioprinting for various applications such as high-throughput screening, fundamental cell biology research, in situ bioprinting and fabrication of in vitro tissue constructs and also presents future directions to transform the microvalve-based bioprinting technology into imperative tools for tissue engineering and regenerative medicine.

Bioprinting is a promising automated platform that enables the simultaneous deposition of multiple types of cells and biomaterials to fabricate complex three-dimensional (3D) tissue constructs. Collagen-based biomaterial used in most of the previous works on skin bioprinting has poor printability and long crosslinking time. This posed an immense challenge to create 3D constructs with pre-determined shape and configuration at high throughput. Recently, the use of chitosan for wound healing applications has attracted huge attention due to its attractive traits such as its antimicrobial properties and ability to trigger hemostasis. In this paper, we optimized polyelectrolyte gelatin-chitosan hydrogel for 3D bioprinting. Modification to the chitosan was carried out via the oppositely charged functional groups from chitosan and gelatin at a specific pH of ~pH 6.5 to form polyelectrolyte complexes. The polyelectrolyte hydrogels were evaluated in terms of physical interactions within polymer blend, rheological properties (viscosities, storage and loss modulus), printing resolution at varying pressures and feed rates and biocompatibility. The polyelectrolyte gelatin-chitosan hydrogels formulated in this work was optimized for 3D bioprinting at room temperature to achieve high shape fidelity of the printed 3D constructs and good biocompatibility with fibroblast skin cells.

One of the major challenges in the field of soft tissue engineering using bioprinting is fabricating complex tissue constructs with desired structure integrity and mechanical property. To accomplish such requirements, most of the reported works incorporated reinforcement materials such as poly( ϵ -caprolactone) (PCL) polymer within the 3D bioprinted constructs. Although this approach has made some progress in constructing soft tissue-engineered scaffolds, the mechanical compliance mismatch and long degradation period are not ideal for soft tissue engineering. Herein, we present a facile bioprinting strategy that combines the rapid extrusion-based bioprinting technique with an in-built ultraviolet (UV) curing system to facilitate the layer-by-layer UV curing of bioprinted photo-curable GelMA-based hydrogels to achieve soft yet stable cell-laden constructs with high aspect ratio for soft tissue engineering. GelMA is supplemented with a viscosity enhancer (gellan gum) to improve the bio-ink printability and shape fidelity while maintaining the biocompatibility before crosslinking via a layer-by-layer UV curing process. This approach could eventually fabricate soft tissue constructs with high aspect ratio (length to diameter) of ≥ 5. The effects of UV source on printing resolution and cell viability were also studied. As a proof-of-concept, small building units (3D lattice and tubular constructs) with high aspect ratio are fabricated. Furthermore, we have also demonstrated the ability to perform multi-material printing of tissue constructs with high aspect ratio along both the longitudinal and transverse directions for potential applications in tissue engineering of soft tissues. This layer-by-layer ultraviolet assisted extrusion-based (UAE) Bioprinting may provide a novel strategy to develop soft tissue constructs with desirable structure integrity.

In 3D bioprinting, printing resolution represents the deposited material in the x-and y-axes, while dimensionality defines the structural resolution of printed constructs. Dimensionality in 3D bioprinting can be defined as the resolution in the z-axis. The printing resolution, together with dimensionality, contributes to the overall shape fidelity of the bioprinted constructs. The in-depth understanding of physical processes for different printing technologies is imperative in controlling the print resolution and definition. In this article, bioprinting technologies are classified according to the physical processes that deposit or form the bioprinted construct. Due to the different fabrication processes in forming fundamental printed units (voxels), the definition of printability differs for each bioprinting technique. Another aspect of resolution is the spatial positioning of cells within each fundamental building unit. The proximity of cells in the bioprinted construct affects the physiological outcomes. The second aspect of 3D bioprinting technologies is the ability to control shape fidelity. Different strategies have been used to improve the construction of a 3D engineered tissue or organ. Lastly, moving toward complex tissue printing involves adding functionalities to the bioprinted construct. Data processing, material formulations, and integration of different fabrication technologies are key areas in bioprinting that can recapture the different hierarchical aspects of native tissues. This article presents a comprehensive overview of enhancing the resolution of the bioprinting construct and identifying methods to improve functionalities of bioprinted tissues.