A new bioprinting method is reported for fabricating 3D tissue constructs replete with vasculature, multiple types of cells, and extracellular matrix. These intricate, heterogeneous structures are created by precisely co-printing multiple materials, known as bioinks, in three dimensions. These 3D micro-engineered environments open new -avenues for drug screening and fundamental studies of wound healing, angiogenesis, and stem-cell niches.

3D Bioprinting of Vascularized, Heterogeneous Cell‐Laden Tissue Constructs

3D bioprinting for engineering complex tissues.

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

Vascularization remains a critical challenge in tissue engineering. The development of vascular networks within densely populated and metabolically functional tissues facilitate transport of nutrients and removal of waste products, thus preserving cellular viability over a long period of time. Despite tremendous progress in fabricating complex tissue constructs in the past few years, approaches for controlled vascularization within hydrogel based engineered tissue constructs have remained limited. Here, we report a three dimensional (3D) micromolding technique utilizing bioprinted agarose template fibers to fabricate microchannel networks with various architectural features within photocrosslinkable hydrogel constructs. Using the proposed approach, we were able to successfully embed functional and perfusable microchannels inside methacrylated gelatin (GelMA), star poly(ethylene glycol-co-lactide) acrylate (SPELA), poly(ethylene glycol) dimethacrylate (PEGDMA) and poly(ethylene glycol) diacrylate (PEGDA) hydrogels at different concentrations. In particular, GelMA hydrogels were used as a model to demonstrate the functionality of the fabricated vascular networks in improving mass transport, cellular viability and differentiation within the cell-laden tissue constructs. In addition, successful formation of endothelial monolayers within the fabricated channels was confirmed. Overall, our proposed strategy represents an effective technique for vascularization of hydrogel constructs with useful applications in tissue engineering and organs on a chip.

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Hydrogel bioprinted microchannel networks for vascularization of tissue engineering constructs

Generating perfusable 3D microvessels in vitro is an important goal for tissue engineering, as well as for reliable modelling of blood vessel function. To date, in vitro blood vessel models have not been able to accurately reproduce the dynamics and responses of endothelial cells to grow perfusable and functional 3D vascular networks. Here we describe a microfluidic-based platform whereby we model natural cellular programs found during normal development and angiogenesis to form perfusable networks of intact 3D microvessels as well as tumor vasculatures based on the spatially controlled co-culture of endothelial cells with stromal fibroblasts, pericytes or cancer cells. The microvessels possess the characteristic morphological and biochemical markers of in vivo blood vessels, and exhibit strong barrier function and long-term stability. An open, unobstructed microvasculature allows the delivery of nutrients, chemical compounds, biomolecules and cell suspensions, as well as flow-induced mechanical stimuli into the luminal space of the endothelium, and exhibits faithful responses to physiological shear stress as demonstrated by cytoskeleton rearrangement and increased nitric oxide synthesis. This simple and versatile platform provides a wide range of applications in vascular physiology studies as well as in developing vascularized organ-on-a-chip and human disease models for pharmaceutical screening.

Engineering of functional, perfusable 3D microvascular networks on a chip

Microvascular networks support metabolic activity and define microenvironmental conditions within tissues in health and pathology. Recapitulation of functional microvascular structures in vitro could provide a platform for the study of complex vascular phenomena, including angiogenesis and thrombosis. We have engineered living microvascular networks in three-dimensional tissue scaffolds and demonstrated their biofunctionality in vitro. We describe the lithographic technique used to form endothelialized microfluidic vessels within a native collagen matrix; we characterize the morphology, mass transfer processes, and long-term stability of the endothelium; we elucidate the angiogenic activities of the endothelia and differential interactions with perivascular cells seeded in the collagen bulk; and we demonstrate the nonthrombotic nature of the vascular endothelium and its transition to a prothrombotic state during an inflammatory response. The success of these microvascular networks in recapitulating these phenomena points to the broad potential of this platform for the study of cardiovascular biology and pathophysiology.

https://www.pnas.org/content/pnas/109/24/9342.full.pdf

In vitro microvessels for the study of angiogenesis and thrombosis.

One strategy of adipose tissue engineering is to transplant adipocytes or adipocyte precursor cells in combination with polymeric materials. However, a satisfying formation of fat tissue and its long-term survival still remain major problems. There is increasing evidence that treatment of the cells prior to implantation plays a critical role in the success of adipose tissue growth. In a previous study, we established a model system based on 3T3-L1 cells that allows for reproducible engineering of mature, coherent adipose tissues in vitro. We utilized this model system in the current study and systematically investigated the long-term in vivo development of cellular constructs with varying stages of adipogenic development at the time point of implantation. Blank polyglycolic acid fiber meshes, scaffolds seeded with uninduced 3T3-L1 preadipocytes, and cell–polymer constructs precultivated under adipogenic conditions for 2, 9, or 35 days were implanted subcutaneously into nude mice. Histological analysis rev...

In vivo development and long-term survival of engineered adipose tissue depend on in vitro precultivation strategy.

Considering the limited physiological relevance of 2D cell culture experiments, significant effort was devoted to the development of materials that could more accurately recreate the in vivo cellular microenvironment, and support 3D cell cultures in vitro. (1) One such class of materials is conducting polymers, which are promising due to their compliant mechanical properties, compatibility with biological systems, mixed electrical and ionic conductivity, and ability to form porous structures. (2) In this work, we report the fabrication of a single component, macroporous scaffold made from poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) via an ice-templating method. (3) PEDOT:PSS scaffolds offer tunable pore size, morphology and shape through facile changes in preparation conditions, and are capable of supporting 3D cell cultures due to their biocompatibility and tissue-like elasticity. Moreover, these materials are functional: they exhibit excellent electrochemical switching behavior and significantly lower impedance compared to films. Their electrochemical activity enables their use in the active channel of a state of the art diagnostic tool in the field of bioelectronics, i.e., the organic electrochemical transistor (OECT). The inclusion of cells within the porous architecture affects the impedance of the electrically-conducting polymer network and, thus, may be used as a method to quantify cell growth. The adhesion and pro-angiogenic secretions of mouse fibroblasts cultured within the scaffolds can be controlled by switching the electrochemical state of the polymer prior to cell-seeding. In summary, these smart materials hold promise not only as extracellular matrix-mimicking structures for cell culture, but also as high-performance bioelectronic tools for diagnostic and signaling applications. 

References 
[1] M. Holzwarth, P. X. Ma, Journal of Materials Chemistry, 21, 10243‐10251 (2011). 
[2] L. H. Jimison, J. Rivnay, R. M. Owens, in Organic Electronics, Wiley‐VCH Verlag GmbH and Co. KGaA, 27‐6 (2013).
[3] A. M.-D. Wan, S. Inal, T. Williams et al. Journal of Materials Chemistry B, DOI: 10.1039/C5TB00390C (2015).

Conducting polymer scaffolds for electrical control of cellular functions (Conference Presentation)

There is a growing interest in patterning biomolecules and cells on modified surfaces, as they enable the direct study of biological entities with their environment We demonstrate the fabrication and use of a polymer (Parylene C) lift-off template, to pattern a variety of biological materials and cells, with feature sizes ranging from micrometers to a minimum width of 300 nm Our goal is to create highly reproducible arrays, for studying biomolecule-cell and cell-cell interactions

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Claudia Fischbach-Teschl

Papers

In vitro microvessels for the study of angiogenesis and thrombosis.

In vivo development and long-term survival of engineered adipose tissue depend on in vitro precultivation strategy.

Conducting polymer scaffolds for electrical control of cellular functions (Conference Presentation)

Surface modification and patterning of biomolecules and cells, using a polymer lift-off template