Hydrogels are hydrophilic polymer-based materials with high water content and physical characteristics that resemble the native extracellular matrix. Because of their remarkable properties, hydrogel systems are used for a wide range of biomedical applications, such as three-dimensional (3D) matrices for tissue engineering, drug-delivery vehicles, composite biomaterials, and as injectable fillers in minimally invasive surgeries. In addition, the rational design of hydrogels with controlled physical and biological properties can be used to modulate cellular functionality and tissue morphogenesis. Here, the development of advanced hydrogels with tunable physiochemical properties is highlighted, with particular emphasis on elastomeric, light-sensitive, composite, and shape-memory hydrogels. Emerging technologies developed over the past decade to control hydrogel architecture are also discussed and a number of potential applications and challenges in the utilization of hydrogels in regenerative medicine are reviewed. It is anticipated that the continued development of sophisticated hydrogels will result in clinical applications that will improve patient care and quality of life.

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25th Anniversary Article: Rational Design and Applications of Hydrogels in Regenerative Medicine

Organ printing can be defined as layer-by-layer additive robotic biofabrication of three-dimensional functional living macrotissues and organ constructs using tissue spheroids as building blocks. The microtissues and tissue spheroids are living materials with certain measurable, evolving and potentially controllable composition, material and biological properties. Closely placed tissue spheroids undergo tissue fusion - a process that represents a fundamental biological and biophysical principle of developmental biology-inspired directed tissue self-assembly. It is possible to engineer small segments of an intraorgan branched vascular tree by using solid and lumenized vascular tissue spheroids. Organ printing could dramatically enhance and transform the field of tissue engineering by enabling large-scale industrial robotic biofabrication of living human organ constructs with "built-in" perfusable intraorgan branched vascular tree. Thus, organ printing is a new emerging enabling technology paradigm which represents a developmental biology-inspired alternative to classic biodegradable solid scaffold-based approaches in tissue engineering.

Organ Printing: Tissue Spheroids as Building Blocks

Direct 3D bioprinting of perfusable vascular constructs using a blend bioink

A novel bioink and a dispensing technique for 3D tissue-engineering applications are presented. The technique incorporates a coaxial extrusion needle using a low-viscosity cell-laden bioink to produce highly defined 3D biostructures. The extrusion system is then coupled to a microfluidic device to control the bioink arrangement deposition, demonstrating the versatility of the bioprinting technique. This low-viscosity cell-responsive bioink promotes cell migration and alignment within each fiber organizing the encapsulated cells.

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Microfluidic Bioprinting of Heterogeneous 3D Tissue Constructs Using Low-Viscosity Bioink.

The cell microenvironment has emerged as a key determinant of cell behavior and function in development, physiology, and pathophysiology. The extracellular matrix (ECM) within the cell microenvironment serves not only as a structural foundation for cells but also as a source of three-dimensional (3D) biochemical and biophysical cues that trigger and regulate cell behaviors. Increasing evidence suggests that the 3D character of the microenvironment is required for development of many critical cell responses observed in vivo, fueling a surge in the development of functional and biomimetic materials for engineering the 3D cell microenvironment. Progress in the design of such materials has improved control of cell behaviors in 3D and advanced the fields of tissue regeneration, in vitro tissue models, large-scale cell differentiation, immunotherapy, and gene therapy. However, the field is still in its infancy, and discoveries about the nature of cell–microenvironment interactions continue to overturn much earl...

Functional and Biomimetic Materials for Engineering of the Three-Dimensional Cell Microenvironment

Artificial reconstruction of fibre-shaped cellular constructs could greatly contribute to tissue assembly in vitro. Here we show that, by using a microfluidic device with double-coaxial laminar flow, metre-long core-shell hydrogel microfibres encapsulating ECM proteins and differentiated cells or somatic stem cells can be fabricated, and that the microfibres reconstitute intrinsic morphologies and functions of living tissues. We also show that these functional fibres can be assembled, by weaving and reeling, into macroscopic cellular structures with various spatial patterns. Moreover, fibres encapsulating primary pancreatic islet cells and transplanted through a microcatheter into the subrenal capsular space of diabetic mice normalized blood glucose concentrations for about two weeks. These microfibres may find use as templates for the reconstruction of fibre-shaped functional tissues that mimic muscle fibres, blood vessels or nerve networks in vivo.

Metre-long cell-laden microfibres exhibit tissue morphologies and functions

In this paper, a tubular 3D microenvironment created in a calcium alginate hydrogel microtube with respect to the effect of scaffold dimensions on the differentiation of mouse neuronal stem cells (mNSCs) is evaluated. Five types of hydrogel microtubes with different core diameters (≈65-200 μm) and shell thicknesses (≈30-110 μm) are fabricated by using a double coaxial microfluidic device, and differentiation of encapsulated mNSCs is induced by changing the growth medium to the differentiation medium. The influence of the microtube geometries is examined by using quantitative real-time polymerase chain reaction and fluorescent immunocytochemistry. The analyses reveal that differences in microtube thickness within 30-110 μm affected the relative Tuj1 expression but do not affect the morphology of encapsulated mNSCs. The diameters of cores influence both the relative Tuj1 expression and morphology of the differentiated neurons. It is found that the tubular microenvironment with a core diameter of less than ≈100 μm contributes to forming highly viable and aligned neural tissue. The tubular microenvironment can provide an effective method for constructing microfiber-shaped neural tissues with geometrically controlled differentiation induction.

Differentiation Induction of Mouse Neural Stem Cells in Hydrogel Tubular Microenvironments with Controlled Tube Dimensions.

This paper describes a co-culture method using cell fiber technology. Cell fibers are cell-laden hydrogel microfibers, in which cells are cultured three-dimensionally and allowed to reach more mature state than the conventional two-dimensional cell culture. Cells in the cell fibers are encapsulated by alginate shell. Only cellular secretome is released into the surrounding environment through the shell while the cells were retained by the fiber. With their high handleability and retrievability, we propose to use the cell fibers for co-culture to ensure steady supply of cellular secretome. We cultured mouse C2C12 myoblasts with mouse 3T3 fibroblasts encapsulated in the cell fibers for two days. The number of C2C12 cells increased proportionally to the number of co-cultured 3T3 fibers, suggesting that the secretome of 3T3 fibers promoted survival and proliferation of C2C12 cells. We believe that cell fiber technology is a useful tool for co-culturing cells, and it will contribute to both basic cell biology and tissue engineering with its unique features.

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Cell fibers promote proliferation of co-cultured cells on a dish.

Current microfluidic methods for cell-laden microfiber fabrication generally require larger than 100 μl of cell-suspensions. Since some 'rare' cells can be only acquired in small amounts, the preparation of >100 μl cell-suspensions with high-cell density can be both expensive and time consuming. Here, we present a facile method capable of fabricating cell-laden microfibers using small-volume cell-suspensions. The method utilizes a 3D-printed coaxial microfluidic device featured with a 'luer-lock inlet' to effectively load cell-suspensions in a deterministic volume (down to 5 μl) with a low sample-loss. In experiments, we demonstrate the formation of fibrous tissues consisting of various kinds of cells. Investigations on the morphology and function of the encapsulated cells show the viability of the cells is not significantly affected by the fabrication process, and also indicate the potential of using our method to perform quantitative assays on fiber-shaped tissues, while reducing the overall material and time consumption.

Cell-laden microfibers fabricated using μL cell-suspension.

This paper describes long-term implantation of primary pancreatic islet cell fibers into diabetic mice. Primary rat pancreatic islet cells were encapsulated in core-shell hydrogel microfibers and implanted into renal capsular space of diabetic mice by using a microcatheter. The blood glucose concentration of the recipient mice were normalized for more than 36 days. This result shows that our cell fiber approach can be applied to long-term implantation and can provide primary islet cells with sufficient microenvironment to promote cellular functions in vivo.

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Akane Itou

Papers

Metre-long cell-laden microfibres exhibit tissue morphologies and functions

Differentiation Induction of Mouse Neural Stem Cells in Hydrogel Tubular Microenvironments with Controlled Tube Dimensions.

Cell fibers promote proliferation of co-cultured cells on a dish.

Cell-laden microfibers fabricated using μL cell-suspension.

Long-term implantation of primary islet cell-encapsulating hydrogel microfibers in diabetic mice