Multiphoton Lithography as a Promising Tool for Biomedical Applications

Multiphoton lithography (MPL) is a powerful and useful structuring tool capable of generating 2D and 3D arbitrary micro- and nanometer features of various materials with high spatial resolution down to nm-scale. This technology has received tremendous interest in tissue engineering and medical device manufacturing, due to its ability to print sophisticated structures, which is difficult to achieve through traditional printing methods. Thorough consideration of two-photon photoinitiators (PIs) and photoreactive biomaterials is key to the fabrication of such complex 3D micro- and nanostructures. In the current review, different types of two-photon PIs are discussed for their use in biomedical applications. Next, an overview of biomaterials (both natural and synthetic polymers) along with their crosslinking mechanisms is provided. Finally, biomedical applications exploiting MPL are presented, including photocleaving and photopatterning strategies, biomedical devices, tissue engineering, organoids, organ-on-chip, and photodynamic therapy. This review offers a helicopter view on the use of MPL technology in the biomedical field and defines the necessary considerations toward selection or design of PIs and photoreactive biomaterials to serve a multitude of biomedical applications.     

Biohybrid device with antagonistic skeletal muscle tissue for measurement of contractile force

ABSTRACT

This paper describes a fabrication method and driving property of a biohybrid device with an antagonistic pair of skeletal muscle tissues and a flexible substrate. Since two skeletal muscle tissues are symmetrically arranged with the flexible substrate as the central axis, the flexible substrate deforms according to differences in their tension. In the formation of the antagonistic pair of skeletal muscle tissues, we assembled myoblast-laden hydrogel sheets and cultured them to construct a single skeletal muscle tissue on each side of the flexible substrate. We confirmed that the skeletal muscle tissue on the flexible substrate had the fundamental morphology and function of skeletal muscle. Furthermore, we made the biohybrid device actuate with deformation of the flexible substrate by selective contractions of the skeletal muscle tissues. From the deformation of the flexible substrate, we estimated the contractile force of each skeletal muscle tissue in the biohybrid device using finite element analysis. This biohybrid device, composed of an antagonistic pair of skeletal muscle tissues and a flexible substrate, can potentially be used in biological studies and pharmacokinetic assays involving the antagonistic pair of skeletal muscles.     

Development of Endothelial Cell Networks in 3D Tissues by Combination of Melt Electrospinning Writing with Cell‐Accumulation Technology

A remaining challenge in tissue engineering approaches is the in vitro vascularization of engineered constructs or tissues. Current approaches in engineered vascularized constructs are often limited in the control of initial vascular network geometry, which is crucial to ensure full functionality of these constructs with regard to cell survival, metabolic activity, and potential differentiation ability. Herein, the combination of 3D‐printed poly‐ε‐caprolactone scaffolds via melt electrospinning writing with the cell‐accumulation technique to enable the formation and control of capillary‐like network structures is reported. The cell‐accumulation technique is already proven itself to be a powerful tool in obtaining thick (50 µm) tissues and its main advantage is the rapid production of tissues and its ease of performance. However, the applied combination yields tissue thicknesses that are doubled, which is of outstanding importance for an improved handling of the scaffolds and the generation of clinically relevant sample volumes. Moreover, a correlation of increasing vascular endothelial growth factor secretion to hypoxic conditions with increasing pore sizes and an assessment of the formation of neovascular like structures are included.     

[学科发展]5D打印——生物功能组织的制造

生物制造是制造技术与生命科学技术交叉融合产生的新兴学科方向,这一学科方向的发展将为巨大的人体组织与器官市场提供新技术,同时也给制造技术变革带来新机遇。面向生物制造未来发展,提出5D打印制造概念,论述了5D打印的内涵,分析了其关键技术。结合生物制造技术的现有进展,介绍了研究团队在心肌组织支架的制造、类脑神经组织制造、爬行生命机械混合机器人方面取得的初步研究进展,为生物制造技术拓展新方向提供新思路。     

Thiol–Ene Clickable Gelatin: A Platform Bioink for Multiple 3D Biofabrication Technologies

Bioprinting can be defined as the art of combining materials and cells to fabricate designed, hierarchical 3D hybrid constructs. Suitable materials, so called bioinks, have to comply with challenging rheological processing demands and rapidly form a stable hydrogel postprinting in a cytocompatible manner. Gelatin is often adopted for this purpose, usually modified with (meth‐)acryloyl functionalities for postfabrication curing by free radical photopolymerization, resulting in a hydrogel that is cross‐linked via nondegradable polymer chains of uncontrolled length. The application of allylated gelatin (GelAGE) as a thiol–ene clickable bioink for distinct biofabrication applications is reported. Curing of this system occurs via dimerization and yields a network with flexible properties that offer a wider biofabrication window than (meth‐)acryloyl chemistry, and without additional nondegradable components. An in‐depth analysis of GelAGE synthesis is conducted, and standard UV‐initiation is further compared with a recently described visible‐light‐initiator system for GelAGE hydrogel formation. It is demonstrated that GelAGE may serve as a platform bioink for several biofabrication technologies by fabricating constructs with high shape fidelity via lithography‐based (digital light processing) 3D printing and extrusion‐based 3D bioprinting, the latter supporting long‐term viability postprinting of encapsulated chondrocytes.     

Out‐of‐Plane 3D‐Printed Microfibers Improve the Shear Properties of Hydrogel Composites

One challenge in biofabrication is to fabricate a matrix that is soft enough to elicit optimal cell behavior while possessing the strength required to withstand the mechanical load that the matrix is subjected to once implanted in the body. Here, melt electrowriting (MEW) is used to direct‐write poly(ε‐caprolactone) fibers “out‐of‐plane” by design. These out‐of‐plane fibers are specifically intended to stabilize an existing structure and subsequently improve the shear modulus of hydrogel–fiber composites. The stabilizing fibers (diameter = 13.3 ± 0.3 µm) are sinusoidally direct‐written over an existing MEW wall‐like structure (330 µm height). The printed constructs are embedded in different hydrogels (5, 10, and 15 wt% polyacrylamide; 65% poly(2‐hydroxyethyl methacrylate) (pHEMA)) and a frequency sweep test (0.05–500 rad s^?1, 0.01% strain, n = 5) is performed to measure the complex shear modulus. For the rheological measurements, stabilizing fibers are deposited with a radial‐architecture prior to embedding to correspond to the direction of the stabilizing fibers with the loading of the rheometer. Stabilizing fibers increase the complex shear modulus irrespective of the percentage of gel or crosslinking density. The capacity of MEW to produce well‐defined out‐of‐plane fibers and the ability to increase the shear properties of fiber‐reinforced hydrogel composites are highlighted.     

Volumetric Bioprinting of Complex Living‐Tissue Constructs within Seconds

Biofabrication technologies, including stereolithography and extrusion‐based printing, are revolutionizing the creation of complex engineered tissues. The current paradigm in bioprinting relies on the additive layer‐by‐layer deposition and assembly of repetitive building blocks, typically cell‐laden hydrogel fibers or voxels, single cells, or cellular aggregates. The scalability of these additive manufacturing technologies is limited by their printing velocity, as lengthy biofabrication processes impair cell functionality. Overcoming such limitations, the volumetric bioprinting of clinically relevant sized, anatomically shaped constructs, in a time frame ranging from seconds to tens of seconds is described. An optical‐tomography‐inspired printing approach, based on visible light projection, is developed to generate cell‐laden tissue constructs with high viability (>85%) from gelatin‐based photoresponsive hydrogels. Free‐form architectures, difficult to reproduce with conventional printing, are obtained, including anatomically correct trabecular bone models with embedded angiogenic sprouts and meniscal grafts. The latter undergoes maturation in vitro as the bioprinted chondroprogenitor cells synthesize neo‐fibrocartilage matrix. Moreover, free‐floating structures are generated, as demonstrated by printing functional hydrogel‐based ball‐and‐cage fluidic valves. Volumetric bioprinting permits the creation of geometrically complex, centimeter‐scale constructs at an unprecedented printing velocity, opening new avenues for upscaling the production of hydrogel‐based constructs and for their application in tissue engineering, regenerative medicine, and soft robotics.     

Emerging Biofabrication Strategies for Engineering Complex Tissue Constructs

The demand for organ transplantation and repair, coupled with a shortage of available donors, poses an urgent clinical need for the development of innovative treatment strategies for long‐term repair and regeneration of injured or diseased tissues and organs. Bioengineering organs, by growing patient‐derived cells in biomaterial scaffolds in the presence of pertinent physicochemical signals, provides a promising solution to meet this demand. However, recapitulating the structural and cytoarchitectural complexities of native tissues in vitro remains a significant challenge to be addressed. Through tremendous efforts over the past decade, several innovative biofabrication strategies have been developed to overcome these challenges. This review highlights recent work on emerging three‐dimensional bioprinting and textile techniques, compares the advantages and shortcomings of these approaches, outlines the use of common biomaterials and advanced hybrid scaffolds, and describes several design considerations including the structural, physical, biological, and economical parameters that are crucial for the fabrication of functional, complex, engineered tissues. Finally, the applications of these biofabrication strategies in neural, skin, connective, and muscle tissue engineering are explored.