Microfluidic-enhanced 3D bioprinting of aligned myoblast-laden hydrogels leads to functionally organized myofibers in vitro and in vivo

3D bioprinting is a rapidly evolving industry that has been utilized for a variety of biomedical applications. It differs from traditional 3D printing in that it utilizes bioinks comprised of cells and other biomaterials to allow for the generation of complex functional tissues. Bioprinting involves computational modeling, bioink preparation, bioink deposition, and subsequent maturation of printed products; it is an intricate process where bioink composition, bioprinting approach, and bioprinter type must be considered during construct development. This technology has already found success in human studies, where a variety of functional tissues have been generated for both in vitro and in vivo applications. Although the main driving force behind innovation in 3D bioprinting has been utility in human medicine, recent efforts investigating its veterinary application have begun to emerge. To date, 3D bioprinting has been utilized to create bone, cardiovascular, cartilage, corneal and neural constructs in animal species. Furthermore, the use of animal-derived cells and various animal models in human research have provided additional information regarding its capacity for veterinary translation. While these studies have produced some promising results, technological limitations as well as ethical and regulatory challenges have impeded clinical acceptance. This article reviews the current understanding of 3D bioprinting technology and its recent advancements with a focus on recent successes and future translation in veterinary medicine.

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Liver microsystems in vitro for drug response

Journal of Biomedical Science ◽

10.1186/s12929-019-0575-0 ◽

2019 ◽

Vol 26 (1) ◽

Cited By ~ 6

Author(s):

Jyong-Huei Lee ◽

Kuan-Lun Ho ◽

Shih-Kang Fan

Keyword(s):

Soft Lithography ◽

Drug Response ◽

Study Drug ◽

Metabolic Function ◽

3D Bioprinting ◽

Cell Micropatterning ◽

Microfluidic Perfusion ◽

Parenchymal Cells

Abstract Engineering approaches were adopted for liver microsystems to recapitulate cell arrangements and culture microenvironments in vivo for sensitive, high-throughput and biomimetic drug screening. This review introduces liver microsystems in vitro for drug hepatotoxicity, drug-drug interactions, metabolic function and enzyme induction, based on cell micropatterning, hydrogel biofabrication and microfluidic perfusion. The engineered microsystems provide varied microenvironments for cell culture that feature cell coculture with non-parenchymal cells, in a heterogeneous extracellular matrix and under controllable perfusion. The engineering methods described include cell micropatterning with soft lithography and dielectrophoresis, hydrogel biofabrication with photolithography, micromolding and 3D bioprinting, and microfluidic perfusion with endothelial-like structures and gradient generators. We discuss the major challenges and trends of liver microsystems to study drug response in vitro.

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Three-dimensional bioprinted hepatorganoids prolong survival of mice with liver failure

Gut ◽

10.1136/gutjnl-2019-319960 ◽

2020 ◽

pp. gutjnl-2019-319960 ◽

Cited By ~ 2

Author(s):

Huayu Yang ◽

Lejia Sun ◽

Yuan Pang ◽

Dandan Hu ◽

Haifeng Xu ◽

...

Keyword(s):

Drug Metabolism ◽

Liver Failure ◽

Human Liver ◽

Three Dimensional ◽

3D Bioprinting ◽

Heparg Cells ◽

Liver Functions ◽

Human Specific

ObjectiveShortage of organ donors, a critical challenge for treatment of end-stage organ failure, has motivated the development of alternative strategies to generate organs in vitro. Here, we aim to describe the hepatorganoids, which is a liver tissue model generated by three-dimensional (3D) bioprinting of HepaRG cells and investigate its liver functions in vitro and in vivo.Design3D bioprinted hepatorganoids (3DP-HOs) were constructed using HepaRG cells and bioink, according to specific 3D printing procedures. Liver functions of 3DP-HOs were detected after 7 days of differentiation in vitro, which were later transplanted into Fah-deficient mice. The in vivo liver functions of 3DP-HOs were evaluated by survival time and liver damage of mice, human liver function markers and human-specific debrisoquine metabolite production.Results3DP-HOs broadly acquired liver functions, such as ALBUMIN secretion, drug metabolism and glycogen storage after 7 days of differentiation. After transplantation into abdominal cavity of Fah-/-Rag2-/- mouse model of liver injury, 3DP-HOs further matured and displayed increased synthesis of liver-specific proteins. Particularly, the mice acquired human-specific drug metabolism activities. Functional vascular systems were also formed in transplanted 3DP-HOs, further enhancing the material transport and liver functions of 3DP-HOs. Most importantly, transplantation of 3DP-HOs significantly improved the survival of mice.ConclusionsOur results demonstrated a comprehensive proof of principle, which indicated that 3DP-HO model of liver tissues possessed in vivo hepatic functions and alleviated liver failure after transplantation, suggesting that 3D bioprinting could be used to generate human liver tissues as the alternative transplantation donors for treatment of liver diseases.

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Abstract MP207: A Precision Medicine Approach For Non-invasive, Longitudinal, And Quantitative Monitoring Of Cardiac Tissue-engineered Scaffolds

Circulation Research ◽

10.1161/res.129.suppl_1.mp207 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Carmen Gil ◽

Connor Evans ◽

Lan Li ◽

Merlyn Vargas ◽

Gabriella Kabboul ◽

...

Keyword(s):

Contrast Agents ◽

Precision Medicine ◽

Cardiac Tissue ◽

Ct Imaging ◽

3D Bioprinting ◽

Au Nps ◽

Non Invasive ◽

Functional Features

3D bioprinting has revolutionized personalized and precision medicine by enabling the manufacturing of tissue constructs that precisely recapitulate the cellular and functional features of native tissues. In cardiac regenerative medicine, printed scaffolds have shown tremendous potential in repairing damaged heart, however, their clinical applications have been limited by the lack of precise noninvasive tools to monitor the patch function following implantation. By integrating state-of-the-art 3D bioprinting and photon-counting computed tomography (PCCT), this study introduces a new approach for bioengineering defect-specific scaffolds and monitoring their function. We prepared distinct CT-visible bioinks containing a variety of molecular or nanoparticle (NP) contrast agents, including iodine and gadolinium molecules, Au NPs, Gd 2 O 3 NPs, and iodine-loaded liposomes ( Fig 1A-B ). In vitro release experiments showed relatively rapid diffusion-controlled depletion of molecular contrast agents from scaffolds. In contrast, NP agents showed more stable encapsulation and only a partial, degradation-mediated release for up to 3 weeks of incubation ( Fig 1C-D ). Next, PCCT imaging was performed on various scaffold geometries printed using bioinks laden with Gd 2 O 3 or Au NPs. Results demonstrated CT visibility with differential contrast between different patch regions that corresponded to the designed geometries ( Fig 1E ). Finally, we evaluated the in vivo CT imaging of bioprinted patches after their subcutaneous implantation in a mouse model. CT images demonstrated adequate signal from implanted grafts ( Fig 1F ). Together, these results establish a novel precision medicine platform for non-invasive monitoring of medical devices which can open new prospects for a broad range of tissue engineering applications. Figure 1. 3D Bioprinting of CT-visible cardiac patches. A-B: Design of bioinks functionalized with molecular (left) and nanoparticle (right) CT contrast agents ( A ) and their bioprinting ( B ). C-D: In vitro release of contrast agents from printed patches. E: CAD design (left), CT image (middle), and PCCT material decomposition (right) for multi-contrast bioprinted scaffolds. F: In vivo CT imaging of printed patch, laden with Au NPs, implanted subcutaneously into a mouse torso.

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3D Bioprinting for Vascularized Tissue-Engineered Bone Fabrication

Materials ◽

10.3390/ma13102278 ◽

2020 ◽

Vol 13 (10) ◽

pp. 2278 ◽

Cited By ~ 1

Author(s):

Fei Xing ◽

Zhou Xiang ◽

Pol Maria Rommens ◽

Ulrike Ritz

Keyword(s):

Three Dimensional ◽

Current Status ◽

Layer By Layer ◽

3D Bioprinting ◽

Vascular Networks ◽

Waste Products ◽

Tissue Microenvironment ◽

Spatial Architecture

Vascularization in bone tissues is essential for the distribution of nutrients and oxygen, as well as the removal of waste products. Fabrication of tissue-engineered bone constructs with functional vascular networks has great potential for biomimicking nature bone tissue in vitro and enhancing bone regeneration in vivo. Over the past decades, many approaches have been applied to fabricate biomimetic vascularized tissue-engineered bone constructs. However, traditional tissue-engineered methods based on seeding cells into scaffolds are unable to control the spatial architecture and the encapsulated cell distribution precisely, which posed a significant challenge in constructing complex vascularized bone tissues with precise biomimetic properties. In recent years, as a pioneering technology, three-dimensional (3D) bioprinting technology has been applied to fabricate multiscale, biomimetic, multi-cellular tissues with a highly complex tissue microenvironment through layer-by-layer printing. This review discussed the application of 3D bioprinting technology in the vascularized tissue-engineered bone fabrication, where the current status and unique challenges were critically reviewed. Furthermore, the mechanisms of vascular formation, the process of 3D bioprinting, and the current development of bioink properties were also discussed.

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Tissue-Specific Decellularized Extracellular Matrix Bioinks for Musculoskeletal Tissue Regeneration and Modeling Using 3D Bioprinting Technology

International Journal of Molecular Sciences ◽

10.3390/ijms22157837 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7837

Author(s):

Wonbin Park ◽

Ge Gao ◽

Dong-Woo Cho

Keyword(s):

Extracellular Matrix ◽

Donor Site ◽

Leading Edge ◽

Donor Site Morbidity ◽

3D Bioprinting ◽

Musculoskeletal Tissue ◽

Decellularized Extracellular Matrix ◽

Homeostatic Function

The musculoskeletal system is a vital body system that protects internal organs, supports locomotion, and maintains homeostatic function. Unfortunately, musculoskeletal disorders are the leading cause of disability worldwide. Although implant surgeries using autografts, allografts, and xenografts have been conducted, several adverse effects, including donor site morbidity and immunoreaction, exist. To overcome these limitations, various biomedical engineering approaches have been proposed based on an understanding of the complexity of human musculoskeletal tissue. In this review, the leading edge of musculoskeletal tissue engineering using 3D bioprinting technology and musculoskeletal tissue-derived decellularized extracellular matrix bioink is described. In particular, studies on in vivo regeneration and in vitro modeling of musculoskeletal tissue have been focused on. Lastly, the current breakthroughs, limitations, and future perspectives are described.

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The Use of Collagen with High Concentration in Cartilage Tissue Engineering by Means of 3D-Bioprinting

Cell and Tissue Biology ◽

10.1134/s1990519x21050059 ◽

2021 ◽

Vol 15 (5) ◽

pp. 493-502

Author(s):

E. V. Isaeva ◽

E. E. Beketov ◽

V. V. Yuzhakov ◽

N. V. Arguchinskaya ◽

A. A. Kisel ◽

...

Keyword(s):

Granulomatous Inflammation ◽

Small Cells ◽

Cartilage Tissue ◽

3D Bioprinting ◽

High Concentration ◽

Tissue Scaffold ◽

Scaffold Fabrication ◽

Morphological Criteria

Abstract 3D-bioprinting is a promising technology for a tissue scaffold fabrication in the case of damaged tissue/organ replacement. Collagen is one of the most appropriate hydrogel for the purpose, due to its exceptional biocompatibility. However, the use of collagen with conventionally low concentration makes bioprinting process difficult and does not provide its high accuracy. The purpose of the study was evaluation of suitability of collagen with high concentration in case of chondrocyte-laden scaffold fabrication via 3D-bioprinting for cartilage regeneration in vitro and in vivo. The results of the study showed that inherent porosity of 4% collagen was not enough for cell survival in the case of long-term incubation in vitro. With the beginning of the scaffold incubation, cell migration to the surface and out of the scaffold was observed. The residual cells died mostly within 4 weeks. As for in vivo study, in 2 weeks after implantation of the scaffold, a weak granulomatous inflammation was observed. In 6 weeks, a connective tissue was formed in the area of implantation. In the tissue, macrophages and groups of small cells with round nuclei were found. In accordance with morphological criteria, these cells could be considered as young chondrocytes. However, its amount was not enough to initiate the formation of cartilage.

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3D Bioprinting of Vascularized Tissues for in vitro and in vivo Applications

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.664188 ◽

2021 ◽

Vol 9 ◽

Author(s):

Earnest P. Chen ◽

Zeren Toksoy ◽

Bruce A. Davis ◽

John P. Geibel

Keyword(s):

Tissue Engineering ◽

Drug Testing ◽

Rapid Development ◽

Three Dimensional ◽

3D Bioprinting ◽

Vascular Networks ◽

Main Challenge ◽

3D Printed

With a limited supply of organ donors and available organs for transplantation, the aim of tissue engineering with three-dimensional (3D) bioprinting technology is to construct fully functional and viable tissue and organ replacements for various clinical applications. 3D bioprinting allows for the customization of complex tissue architecture with numerous combinations of materials and printing methods to build different tissue types, and eventually fully functional replacement organs. The main challenge of maintaining 3D printed tissue viability is the inclusion of complex vascular networks for nutrient transport and waste disposal. Rapid development and discoveries in recent years have taken huge strides toward perfecting the incorporation of vascular networks in 3D printed tissue and organs. In this review, we will discuss the latest advancements in fabricating vascularized tissue and organs including novel strategies and materials, and their applications. Our discussion will begin with the exploration of printing vasculature, progress through the current statuses of bioprinting tissue/organoids from bone to muscles to organs, and conclude with relevant applications for in vitro models and drug testing. We will also explore and discuss the current limitations of vascularized tissue engineering and some of the promising future directions this technology may bring.

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3D Bioprinting of Cardiovascular Tissues for In Vivo and In Vitro Applications Using Hybrid Hydrogels Containing Silk Fibroin: State of the Art and Challenges

Current Tissue Microenvironment Reports ◽

10.1007/s43152-020-00026-5 ◽

2020 ◽

Vol 1 (4) ◽

pp. 261-276

Author(s):

Laura Vettori ◽

Poonam Sharma ◽

Jelena Rnjak-Kovacina ◽

Carmine Gentile

Keyword(s):

Silk Fibroin ◽

Mechanical Stability ◽

Biological Properties ◽

Cardiac Cells ◽

3D Bioprinting ◽

Natural Polymers ◽

Cardiovascular Tissues ◽

Hybrid Hydrogels

Abstract Purpose of Review 3D bioprinting of cardiovascular tissues for in vitro and in vivo applications is currently investigated as a potential solution to better mimic the microenvironment typical of the human heart. However, optimal cell viability and tissue vascularization remain two of the main challenges in this regard. Silk fibroin (SF) as a natural biomaterial with unique features supports cell survival and tissue vascularization. This review aims to evaluate the potential of hydrogels containing SF in 3D bioprinting of cardiac tissue that better recapitulate the native cardiac microenvironment. Recent Findings SF hydrogels spontaneously develop nanocrystals, which limit their use for 3D bioprinting applications. Nevertheless, the printability of SF is improved in hybrid hydrogels by mixing it with other natural polymers (such as alginate and gelatin). This is achieved by adding SF with other polymers or by crosslinking it by peroxidase catalysis (i.e., with alginate). Compared to only SF-based hydrogels, hybrid hydrogels provide a durable bioprinted construct with improved mechanical stability and biological properties. To date, studies using cardiac cells in bioprinted SF constructs are yet to be performed. Summary Mixing SF with other polymers in bioprinted hybrid hydrogels improves the printability and durability of 3D bioprinted tissues. Studies using these hydrogels with cardiac cells will be required to evaluate the biocompatibility of SF hybrid hydrogels and to establish their potential use for cardiovascular applications.

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Development and Application of an Additively Manufactured Calcium Chloride Nebulizer for Alginate 3D-Bioprinting Purposes

Journal of Functional Biomaterials ◽

10.3390/jfb9040063 ◽

2018 ◽

Vol 9 (4) ◽

pp. 63 ◽

Cited By ~ 9

Author(s):

Lukas Raddatz ◽

Antonina Lavrentieva ◽

Iliyana Pepelanova ◽

Janina Bahnemann ◽

Dominik Geier ◽

...

Keyword(s):

Cell Culture ◽

Culture Media ◽

Single Cells ◽

Matrix Material ◽

Three Dimensional ◽

Culture Cell ◽

3D Bioprinting ◽

Cacl2 Solution

Three-dimensional (3D)-bioprinting enables scientists to mimic in vivo micro-environments and to perform in vitro cell experiments under more physiological conditions than is possible with conventional two-dimensional (2D) cell culture. Cell-laden biomaterials (bioinks) are precisely processed to bioengineer tissue three-dimensionally. One primarily used matrix material is sodium alginate. This natural biopolymer provides both fine mechanical properties when gelated and high biocompatibility. Commonly, alginate is 3D bioprinted using extrusion based devices. The gelation reaction is hereby induced by a CaCl2 solution in the building chamber after material extrusion. This established technique has two main disadvantages: (1) CaCl2 can have toxic effects on the cell-laden hydrogels by oxygen diffusion limitation and (2) good printing resolution in the CaCl2 solution is hard to achieve, since the solution needs to be removed afterwards and substituted by cell culture media. Here, we show an innovative approach of alginate bioprinting based on a CaCl2 nebulizer. The device provides CaCl2 mist to the building platform inducing the gelation. The necessary amount of CaCl2 could be decreased as compared to previous gelation strategies and limitation of oxygen transfer during bioprinting can be reduced. The device was manufactured using the MJP-3D printing technique. Subsequently, its digital blueprint (CAD file) can be modified and additive manufactured easily and mounted in various extrusion bioprinters. With our approach, a concept for a more gentle 3D Bioprinting method could be shown. We demonstrated that the concept of an ultrasound-based nebulizer for CaCl2 mist generation can be used for 3D bioprinting and that the mist-induced polymerization of alginate hydrogels of different concentrations is feasible. Furthermore, different cell-laden alginate concentrations could be used: Cell spheroids (mesenchymal stem cells) and single cells (mouse fibroblasts) were successfully 3D printed yielding viable cells and stable hydrogels after 24 h cultivation. We suggest our work to show a different and novel approach on alginate bioprinting, which could be useful in generating cell-laden hydrogel constructs for e.g., drug screening or (soft) tissue engineering applications.

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