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In situ additive biomanufacturing of structures may boost regenerative medicine.

Over the last decades, the fabrication of 3D tissues has become commonplace in tissue engineering and regenerative medicine. However, conventional 3D biofabrication techniques such as scaffolding, microengineering, and fiber and cell sheet engineering are limited in their capacity to fabricate complex tissue constructs with the required precision and controllability that is needed to replicate biologically relevant tissues. To this end, 3D bioprinting offers great versatility to fabricate biomimetic, volumetric tissues that are structurally and functionally relevant. It enables precise control of the composition, spatial distribution, and architecture of resulting constructs facilitating the recapitulation of the delicate shapes and structures of targeted organs and tissues. This Review systematically covers the history of bioprinting and the most recent advances in instrumentation and methods. It then focuses on the requirements for bioinks and cells to achieve optimal fabrication of biomimetic constructs. Next, emerging evolutions and future directions of bioprinting are discussed, such as freeform, high-resolution, multimaterial, and 4D bioprinting. Finally, the translational potential of bioprinting and bioprinted tissues of various categories are presented and the Review is concluded by exemplifying commercially available bioprinting platforms., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The field of regenerative medicine has progressed tremendously over the past few decades in its ability to fabricate functional tissue substitutes. Conventional approaches based on scaffolding and microengineering are limited in their capacity of producing tissue constructs with precise biomimetic properties. Three-dimensional (3D) bioprinting technology, on the other hand, promises to bridge the divergence between artificially engineered tissue constructs and native tissues. In a sense, 3D bioprinting offers unprecedented versatility to co-deliver cells and biomaterials with precise control over their compositions, spatial distributions, and architectural accuracy, therefore achieving detailed or even personalized recapitulation of the fine shape, structure, and architecture of target tissues and organs. Here we briefly describe recent progresses of 3D bioprinting technology and associated bioinks suitable for the printing process. We then focus on the applications of this technology in fabrication of biomimetic constructs of several representative tissues and organs, including blood vessel, heart, liver, and cartilage. We finally conclude with future challenges in 3D bioprinting as well as potential solutions for further development.

Bioprinting is an emerging technology with various applications in making functional tissue constructs to replace injured or diseased tissues. It is a relatively new approach that provides high reproducibility and precise control over the fabricated constructs in an automated manner, potentially enabling high-throughput production. During the bioprinting process, a solution of a biomaterial or a mixture of several biomaterials in the hydrogel form, usually encapsulating the desired cell types, termed the bioink, is used for creating tissue constructs. This bioink can be cross-linked or stabilized during or immediately after bioprinting to generate the final shape, structure, and architecture of the designed construct. Bioinks may be made from natural or synthetic biomaterials alone, or a combination of the two as hybrid materials. In certain cases, cell aggregates without any additional biomaterials can also be adopted for use as a bioink for bioprinting processes. An ideal bioink should possess proper mechanical, rheological, and biological properties of the target tissues, which are essential to ensure correct functionality of the bioprinted tissues and organs. In this review, we provide an in-depth discussion of the different bioinks currently employed for bioprinting, and outline some future perspectives in their further development.

Keratins extracted from human hair have emerged as a promising biomaterial for various biomedical applications, partly due to their wide availability, low cost, minimal immune response, and the potential to engineer autologous tissue constructs. However, the fabrication of keratin-based scaffolds typically relies on limited crosslinking mechanisms, such as via physical interactions or disulfide bond formation, which are time-consuming and result in relatively poor mechanical strength and stability. Here, we report the preparation of photocrosslinkable keratin-polyethylene glycol (PEG) hydrogels via the thiol-norbornene "click" reaction, which can be formed within one minute upon irradiation of visible light. The resulting keratin-PEG hydrogels showed highly tunable mechanical properties of up to 45 kPa in compressive modulus, and long-term stability in buffer solutions and cell culture media. These keratin-based hydrogels were tested as cell culture substrates in both two-dimensional surface seeding and three-dimensional cell encapsulation, demonstrating excellent cytocompatibility to support the attachment, spreading, and proliferation of fibroblast cells. Moreover, the photocrosslinking mechanism makes keratin-based hydrogel suitable for various microfabrication techniques, such as micropatterning and wet spinning, to fabricate cell-laden tissue constructs with different architectures. We believe that the unique features of this photocrosslinkable human hair keratin hydrogel promise new opportunities for their future biomedical applications.

Development of an efficient sensing platform capable of continual monitoring of biomarkers is needed to assess the functionality of the in vitro organoids and to evaluate their biological responses toward pharmaceutical compounds or chemical species over extended periods of time. Here, a novel label-free microfluidic electrochemical (EC) biosensor with a unique built-in on-chip regeneration capability for continual measurement of cell-secreted soluble biomarkers from an organoid culture in a fully automated manner without attenuating the sensor sensitivity is reported. The microfluidic EC biosensors are integrated with a human liver-on-a-chip platform for continual monitoring of the metabolic activity of the organoids by measuring the levels of secreted biomarkers for up to 7 d, where the metabolic activity of the organoids is altered by a systemically applied drug. The variations in the biomarker levels are successfully measured by the microfluidic regenerative EC biosensors and agree well with cellular viability and enzyme-linked immunosorbent assay analyses, validating the accuracy of the unique sensing platform. It is believed that this versatile and robust microfluidic EC biosensor that is capable of automated and continual detection of soluble biomarkers will find widespread use for long-term monitoring of human organoids during drug toxicity studies or efficacy assessments of in vitro platforms.

A biofabrication strategy for creating planar multiscale protein, hydrogel, and cellular patterns, and simultaneously generating microscale topographical features is developed that laterally confines the patterned cells and direct their growth in cell permissive hydrogels.

Three‐dimensional (3D) printing is an emerging technique that has shown promising success in engineering human tissues in recent years. Further development of vat‐photopolymerization printing modalities has significantly enhanced the complexity level for 3D printing of various functional structures and components. Similarly, the development of microfluidic chip systems is an emerging research sector with promising medical applications. This work demonstrates the coupling of a digital light processing (DLP) printing procedure with a microfluidic chip system to produce size‐tunable, 3D‐printable porosities with narrow pore size distributions within a gelatin methacryloyl (GelMA) hydrogel matrix. It is found that the generation of size‐tunable gas bubbles trapped within an aqueous GelMA hydrogel‐precursor can be controlled with high precision. Furthermore, the porosities are printed in two‐dimensional (2D) as well as in 3D using the DLP printer. In addition, the cytocompatibility of the printed porous scaffolds is investigated using fibroblasts, where high cell viabilities as well as cell proliferation, spreading, and migration are confirmed. It is anticipated that the strategy is widely applicable in a range of application areas such as tissue engineering and regenerative medicine, among others. [ABSTRACT FROM AUTHOR]

Exosomes are membrane‐bound nanovesicles containing complex cargoes including proteins, lipids, and nucleic acids (mRNAs and microRNAs), which can be derived from most cells. Increasing evidence has implicated exosomes as key players in intercellular and even interorganismal communications. Exosomes confer stability and can direct their cargoes to specific cell types for promoting cell growth and tissue regeneration. Exosome cargoes also appear to act in a combinatorial manner to communicate directives to other cells. This Review focuses on recent developments and findings of exosomes applied towards applications in tissue engineering and regenerative medicine, including healing of the skin, cardiovascular, skeletal, nervous, and visceral systems. The underlying mechanisms of action of exosomes in tissue regeneration are also discussed. In addition, we highlight examples whereby exosomes have been integrated with hydrogels for biofabrication and other related biomedical utilities such as drug delivery. [ABSTRACT FROM AUTHOR]

Modern biomedical imaging has revolutionized life science by providing anatomical, functional, and molecular information of biological species with high spatial resolution, deep penetration, enhanced temporal responsiveness, and improved chemical specificity. In recent years, these imaging techniques have been increasingly tailored for characterizing biomaterials and probing their interactions with biological tissues. This in turn has spurred substantial advances in engineering material properties to accommodate different imaging modalities that was previously unattainable. Here, we review advances in engineering both imaging modalities and material properties with improved contrast, providing a timely practical guide to better assess biomaterial–tissue interactions both in vitro and in vivo . [ABSTRACT FROM AUTHOR]

Despite tremendous advances in the field of regenerative medicine, it still remains challenging to repair the osteochondral interface and full-thickness articular cartilage defects. This inefficiency largely originates from the lack of appropriate tissue-engineered artificial matrices that can replace the damaged regions and promote tissue regeneration. Hydrogels are emerging as a promising class of biomaterials for both soft and hard tissue regeneration. Many critical properties of hydrogels, such as mechanical stiffness, elasticity, water content, bioactivity, and degradation, can be rationally designed and conveniently tuned by proper selection of the material and chemistry. Particularly, advances in the development of cell-laden hydrogels have opened up new possibilities for cell therapy. In this article, we describe the problems encountered in this field and review recent progress in designing cell-hydrogel hybrid constructs for promoting the reestablishment of osteochondral/cartilage tissues. Our focus centers on the effects of hydrogel type, cell type, and growth factor delivery on achieving efficient chondrogenesis and osteogenesis. We give our perspective on developing next-generation matrices with improved physical and biological properties for osteochondral/cartilage tissue engineering. We also highlight recent advances in biomanufacturing technologies ( e.g . molding, bioprinting, and assembly) for fabrication of hydrogel-based osteochondral and cartilage constructs with complex compositions and microarchitectures to mimic their native counterparts. Statement of significance Despite tremendous advances in the field of regenerative medicine, it still remains challenging to repair the osteochondral interface and full-thickness articular cartilage defects. This inefficiency largely originates from the lack of appropriate tissue-engineered biomaterials that replace the damaged regions and promote tissue regeneration. Cell-laden hydrogel systems have emerged as a promising tissue-engineering platform to address this issue. In this article, we describe the fundamental problems encountered in this field and review recent progress in designing cell-hydrogel constructs for promoting the reestablishment of osteochondral/cartilage tissues. Our focus centers on the effects of hydrogel composition, cell type, and growth factor delivery on achieving efficient chondrogenesis and osteogenesis. We give our perspective on developing next-generation hydrogel/inorganic particle/stem cell hybrid composites with improved physical and biological properties for osteochondral/cartilage tissue engineering. We also highlight recent advances in biomanufacturing and bioengineering technologies ( e.g. 3D bioprinting) for fabrication of hydrogel-based osteochondral and cartilage constructs. [ABSTRACT FROM AUTHOR]

Engineering cardiac tissues and organ models remains a great challenge due to the hierarchical structure of the native myocardium. The need of integrating blood vessels brings additional complexity, limiting the available approaches that are suitable to produce integrated cardiovascular organoids. In this work we propose a novel hybrid strategy based on 3D bioprinting, to fabricate endothelialized myocardium. Enabled by the use of our composite bioink, endothelial cells directly bioprinted within microfibrous hydrogel scaffolds gradually migrated towards the peripheries of the microfibers to form a layer of confluent endothelium. Together with controlled anisotropy, this 3D endothelial bed was then seeded with cardiomyocytes to generate aligned myocardium capable of spontaneous and synchronous contraction. We further embedded the organoids into a specially designed microfluidic perfusion bioreactor to complete the endothelialized-myocardium-on-a-chip platform for cardiovascular toxicity evaluation. Finally, we demonstrated that such a technique could be translated to human cardiomyocytes derived from induced pluripotent stem cells to construct endothelialized human myocardium. We believe that our method for generation of endothelialized organoids fabricated through an innovative 3D bioprinting technology may find widespread applications in regenerative medicine, drug screening, and potentially disease modeling. [ABSTRACT FROM AUTHOR]

Abstract: Cell patches are widely used for healing injuries on the surfaces or interfaces of tissues such as those of epidermis and myocardium. Here we report a novel type of porous scaffolds made of poly(d,l-lactic-co-glycolic acid) for fabricating cell patches. The scaffolds have a single layer of spherical pores arranged in a unique hexagonal pattern and are therefore referred to as “scaffolds with a hexagonal array of interconnected pores (SHAIPs)”. SHAIPs contain both uniform pores and interconnecting windows that can facilitate the exchange of biomacromolecules, ensure homogeneous cell seeding, and promote cell migration. As a proof-of-concept demonstration, we have created skeletal muscle patches with a thickness of approximately 150 μm using SHAIPs. The myoblasts seeded in the scaffolds maintained high viability and were able to differentiate into multi-nucleated myotubes. Moreover, neovasculature could efficiently develop into the patches upon subcutaneous implantation in vivo. [Copyright &y& Elsevier]

Degradation is among the most important properties of biomaterial scaffolds, which are indispensable for regenerative medicine. The currently used method relies on the measurement of mass loss across different samples and cannot track the degradation of an individual scaffold in situ. Here we report, for the first time, the use of multiscale photoacoustic microscopy to non-invasively monitor the degradation of an individual scaffold. We could observe alterations to the morphology and structure of a scaffold at high spatial resolution and deep penetration, and more significantly, quantify the degradation of an individual scaffold as a function of time, both in vitro and in vivo. In addition, the remodeling of vasculature inside a scaffold can be visualized simultaneously using a dual-wavelength scanning mode in a label-free manner. This optoacoustic method can be used to monitor the degradation of individual scaffolds, offering a new approach to non-invasively analyze and quantify biomaterial-tissue interactions in conjunction with the assessment of in vivo vascular parameters. [ABSTRACT FROM AUTHOR]

Inverse opal scaffolds are finding widespread use in tissue engineering and regenerative medicine. Herein, the way in which the pore sizes and related physical properties of poly( D, L-lactide- co-glycolide) inverse opal scaffolds are affected by the fabrication conditions is systematically investigated. It is found that the window size of an inverse opal scaffold is mainly determined by the annealing temperature rather than the duration of time, and the surface pore size is largely determined by the concentration of the infiltration solution. Although scaffolds with larger pore or window sizes facilitate faster migration of cells, they show slightly lower compressive moduli than scaffolds with smaller pore or window sizes. [ABSTRACT FROM AUTHOR]

Biochemically modified proteins have attracted significant attention due to their widespread applications as biomaterials. For instance, chemically modified gelatin derivatives have been widely explored to develop hydrogels for tissue engineering and regenerative medicine applications. Among the reported methods, modification of gelatin with methacrylic anhydride (MA) stands out as a convenient and efficient strategy to introduce functional groups and form hydrogels via photopolymerization. Combining light-activation of modified gelatin with soft lithography has enabled the materialization of microfabricated hydrogels. So far, this gelatin derivative has been referred to in the literature as gelatin methacrylate, gelatin methacrylamide, or gelatin methacryloyl, with the same abbreviation of GelMA. Considering the complex composition of gelatin and the presence of different functional groups on the amino acid residues, both hydroxyl groups and amine groups can possibly react with methacrylic anhydride during functionalization of the protein. This can also apply to the modification of other proteins, such as recombinant human tropoelastin to form MA-modified tropoelastin (MeTro). Here, we employed analytical methods to quantitatively determine the amounts of methacrylate and methacrylamide groups in MA-modified gelatin and tropoelastin to better understand the reaction mechanism. By combining two chemical assays with instrumental techniques, such as proton nuclear magnetic resonance ( 1 H NMR) and liquid chromatography tandem-mass spectrometry (LC-MS/MS), our results indicated that while amine groups had higher reactivity than hydroxyl groups and resulted in a majority of methacrylamide groups, modification of proteins by MA could lead to the formation of both methacrylamide and methacrylate groups. It is therefore suggested that the standard terms for GelMA and MeTro should be defined as gelatin methacryloyl and methacryloyl-substituted tropoelastin, respectively, to remain consistent with the widespread abbreviations used in literature. [ABSTRACT FROM AUTHOR]

Graphene and its chemical derivatives have been a pivotal new class of nanomaterials and a model system for quantum behavior. The material's excellent electrical conductivity, biocompatibility, surface area and thermal properties are of much interest to the scientific community. Two-dimensional graphene materials have been widely used in various biomedical research areas such as bioelectronics, imaging, drug delivery, and tissue engineering. In this review, we will highlight the recent applications of graphene-based materials in tissue engineering and regenerative medicine. In particular, we will discuss the application of graphene-based materials in cardiac, neural, bone, cartilage, skeletal muscle, and skin/adipose tissue engineering. We will also discuss the potential risk factors of graphene-based materials in tissue engineering. In conclusion, we will outline the opportunities in the usage of graphene-based materials for clinical applications. [ABSTRACT FROM AUTHOR]