Injectable Self-Oxygenating Cardio-Protective and Tissue Adhesive Silk-Based Hydrogel for Alleviating Ischemia After Mi Injury.

Myocardial infarction (MI) is a significant cardiovascular disease that restricts blood flow, resulting in massive cell death and leading to stiff and noncontractile fibrotic scar tissue formation. Recently, sustained oxygen release in the MI area has shown regeneration ability; however, improving its therapeutic efficiency for regenerative medicine remains challenging. Here, a combinatorial strategy for cardiac repair by developing cardioprotective and oxygenating hybrid hydrogels that locally sustain the release of stromal cell-derived factor-1 alpha (SDF) and oxygen for simultaneous activation of neovascularization at the infarct area is presented. A sustained release of oxygen and SDF from injectable, mechanically robust, and tissue-adhesive silk-based hybrid hydrogels is achieved. Enhanced endothelialization under normoxia and anoxia is observed. Furthermore, there is a marked improvement in vascularization that leads to an increment in cardiomyocyte survival by ≈30% and a reduction of the fibrotic scar formation in an MI animal rodent model. Improved left ventricular systolic and diastolic functions by ≈10% and 20%, respectively, with a ≈25% higher ejection fraction on day 7 are also observed. Therefore, local delivery of therapeutic oxygenating and cardioprotective hydrogels demonstrates beneficial effects on cardiac functional recovery for reparative therapy.

A Modular Platform for Cytocompatible Hydrogels with Tailored Mechanical Properties Based on Monolithic Matrices and Particulate Building Blocks.

We establish a versatile hydrogel platform based on modular building blocks that allows the design of hydrogels with tailored physical architecture and mechanical properties. We demonstrate its versatility by assembling (i) a fully monolithic gelatin methacryloyl (Gel-MA) hydrogel, (ii) a hybrid hydrogel composed of 1:1 Gel-MA and gelatin nanoparticles, and (iii) a fully particulate hydrogel based on methacryloyl-modified gelatin nanoparticles. The hydrogels were formulated to exhibit the same solid content and comparable storage modulus but different stiffness and viscoelastic stress relaxation. The incorporation of particles resulted in softer hydrogels with enhanced stress relaxation. Murine osteoblastic cells cultured in two-dimensional (2D) on hydrogels showed proliferation and metabolic activity comparable to established collagen hydrogels. Furthermore, the osteoblastic cells showed a trend of increased cell numbers, cell expansion, and more defined protrusions on stiffer hydrogels. Hence, modular assembly allows the design of hydrogels with tailored mechanical properties and the potential to alter cell behavior.

Self-Oxygenation of Tissues Orchestrates Full-Thickness Vascularization of Living Implants.

Bioengineering of tissues and organs has the potential to generate functional replacement organs. However, achieving the full-thickness vascularization that is required for long-term survival of living implants has remained a grand challenge, especially for clinically sized implants. During the pre-vascular phase, implanted engineered tissues are forced to metabolically rely on the diffusion of nutrients from adjacent host-tissue, which for larger living implants results in anoxia, cell death, and ultimately implant failure. Here it is reported that this challenge can be addressed by engineering self-oxygenating tissues, which is achieved via the incorporation of hydrophobic oxygen-generating micromaterials into engineered tissues. Self-oxygenation of tissues transforms anoxic stresses into hypoxic stimulation in a homogenous and tissue size-independent manner. The in situ elevation of oxygen tension enables the sustained production of high quantities of angiogenic factors by implanted cells, which are offered a metabolically protected pro-angiogenic microenvironment. Numerical simulations predict that self-oxygenation of living tissues will effectively orchestrate rapid full-thickness vascularization of implanted tissues, which is empirically confirmed via in vivo experimentation. Self-oxygenation of tissues thus represents a novel, effective, and widely applicable strategy to enable the vascularization living implants, which is expected to advance organ transplantation and regenerative medicine applications.

3D Printed Cartilage-Like Tissue Constructs with Spatially Controlled Mechanical Properties.

Developing biomimetic cartilaginous tissues that support locomotion while maintaining chondrogenic behavior is a major challenge in the tissue engineering field. Specifically, while locomotive forces demand tissues with strong mechanical properties, chondrogenesis requires a soft microenvironment. To address this challenge, 3D cartilage-like tissue is bioprinted using two biomaterials with different mechanical properties: a hard biomaterial to reflect the macromechanical properties of native cartilage, and a soft biomaterial to create a chondrogenic microenvironment. To this end, a hard biomaterial (MPa order compressive modulus) composed of an interpenetrating polymer network (IPN) of polyethylene glycol (PEG) and alginate hydrogel is developed as an extracellular matrix (ECM) with self-healing properties, but low diffusive capacity. Within this bath supplemented with thrombin, fibrinogen containing human mesenchymal stem cell (hMSC) spheroids is bioprinted forming fibrin, as the soft biomaterial (kPa order compressive modulus) to simulate cartilage's pericellular matrix and allow a fast diffusion of nutrients. The bioprinted hMSC spheroids improve viability and chondrogenic-like behavior without adversely affecting the macromechanical properties of the tissue. Therefore, the ability to print locally soft and cell stimulating microenvironments inside of a mechanically robust hydrogel is demonstrated, thereby uncoupling the micro- and macromechanical properties of the 3D printed tissues such as cartilage.

Concise Review: Organ Engineering: Design, Technology, and Integration.

Engineering complex tissues and whole organs has the potential to dramatically impact translational medicine in several avenues. Organ engineering is a discipline that integrates biological knowledge of embryological development, anatomy, physiology, and cellular interactions with enabling technologies including biocompatible biomaterials and biofabrication platforms such as three-dimensional bioprinting. When engineering complex tissues and organs, core design principles must be taken into account, such as the structure-function relationship, biochemical signaling, mechanics, gradients, and spatial constraints. Technological advances in biomaterials, biofabrication, and biomedical imaging allow for in vitro control of these factors to recreate in vivo phenomena. Finally, organ engineering emerges as an integration of biological design and technical rigor. An overall workflow for organ engineering and guiding technology to advance biology as well as a perspective on necessary future iterations in the field is discussed. Stem Cells 2017;35:51-60.

Nanostructured Fibrous Membranes with Rose Spike-Like Architecture.

Nanoparticles have been used for engineering composite materials to improve the intrinsic properties and/or add functionalities to pristine polymers. The majority of the studies have focused on the incorporation of spherical nanoparticles within the composite fibers. Herein, we incorporate anisotropic branched-shaped zinc oxide (ZnO) nanoparticles into fibrous scaffolds fabricated by electrospinning. The addition of the branched particles resulted in their protrusion from fibers, mimicking the architecture of a rose stem. We demonstrated that the encapsulation of different-shape particles significantly influences the physicochemical and biological activities of the resultant composite scaffolds. In particular, the branched nanoparticles induced heterogeneous crystallization of the polymeric matrix and enhance the ultimate mechanical strain and strength. Moreover, the three-dimensional (3D) nature of the branched ZnO nanoparticles enhanced adhesion properties of the composite scaffolds to the tissues. In addition, the rose stem-like constructs offered excellent antibacterial activity, while supporting the growth of eukaryote cells.

Gold Nanocomposite Bioink for Printing 3D Cardiac Constructs.

Bioprinting is the most convenient microfabrication method to create biomimetic three-dimensional (3D) cardiac tissue constructs, which can be used to regenerate damaged tissue and provide platforms for drug screening. However, existing bioinks, which are usually composed of polymeric biomaterials, are poorly conductive and delay efficient electrical coupling between adjacent cardiac cells. To solve this problem, we developed a gold nanorod (GNR) incorporated gelatin methacryloyl (GelMA)-based bioink for printing 3D functional cardiac tissue constructs. The GNR concentration was adjusted to create a proper microenvironment for the spreading and organization of cardiac cells. At optimized concentration of GNR, the nanocomposite bioink had a low viscosity, similar to pristine inks, which allowed for the easy integration of cells at high densities. As a result, rapid deposition of cell-laden fibers at a high resolution was possible, while reducing shear stress on the encapsulated cells. In the printed GNR constructs, cardiac cells showed improved cell adhesion and organization when compared to the constructs without GNRs. Furthermore, the incorporated GNRs bridged the electrically resistant pore walls of polymers, improved the cell-to-cell coupling, and promoted synchronized contraction of the bioprinted constructs. Given its advantageous properties, this gold nanocomposite bioink may find wide application in cardiac tissue engineering.

Centering Single Cells in Microgels via Delayed Crosslinking Supports Long-Term 3D Culture by Preventing Cell Escape.

Single-cell-laden microgels support physiological 3D culture conditions while enabling straightforward handling and high-resolution readouts of individual cells. However, their widespread adoption for long-term cultures is limited by cell escape. In this work, it is demonstrated that cell escape is predisposed to off-center encapsulated cells. High-speed microscopy reveals that cells are positioned at the microgel precursor droplets' oil/water interface within milliseconds after droplet formation. In conventional microencapsulation strategies, the droplets are typically gelled immediately after emulsification, which traps cells in this off-center position. By delaying crosslinking, driving cells toward the centers of microgels is succeeded. The centering of cells in enzymatically crosslinked microgels prevents their escape during at least 28 d. It thereby uniquely enables the long-term culture of individual cells within <5-µm-thick 3D uniform hydrogel coatings. Single cell analysis of mesenchymal stem cells in enzymatically crosslinked microgels reveals unprecedented high cell viability (>90%), maintained metabolic activity (>70%), and multilineage differentiation capacity (>60%) over a period of 28 d. The facile nature of this microfluidic cell-centering method enables its straightforward integration into many microencapsulation strategies and significantly enhances control, reproducibility, and reliability of 3D single cell cultures.

Biomechanical Strain Exacerbates Inflammation on a Progeria-on-a-Chip Model.

Organ-on-a-chip platforms seek to recapitulate the complex microenvironment of human organs using miniaturized microfluidic devices. Besides modeling healthy organs, these devices have been used to model diseases, yielding new insights into pathophysiology. Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disease showing accelerated vascular aging, leading to the death of patients due to cardiovascular diseases. HGPS targets primarily vascular cells, which reside in mechanically active tissues. Here, a progeria-on-a-chip model is developed and the effects of biomechanical strain are examined in the context of vascular aging and disease. Physiological strain induces a contractile phenotype in primary smooth muscle cells (SMCs), while a pathological strain induces a hypertensive phenotype similar to that of angiotensin II treatment. Interestingly, SMCs derived from human induced pluripotent stem cells of HGPS donors (HGPS iPS-SMCs), but not from healthy donors, show an exacerbated inflammatory response to strain. In particular, increased levels of inflammation markers as well as DNA damage are observed. Pharmacological intervention reverses the strain-induced damage by shifting gene expression profile away from inflammation. The progeria-on-a-chip is a relevant platform to study biomechanics in vascular biology, particularly in the setting of vascular disease and aging, while simultaneously facilitating the discovery of new drugs and/or therapeutic targets.

Spatially and Temporally Controlled Hydrogels for Tissue Engineering.

Recent years have seen tremendous advances in the field of hydrogel-based biomaterials. One of the most prominent revolutions in this field has been the integration of elements or techniques that enable spatial and temporal control over hydrogels' properties and functions. Here, we critically review the emerging progress of spatiotemporal control over biomaterial properties towards the development of functional engineered tissue constructs. Specifically, we will highlight the main advances in the spatial control of biomaterials, such as surface modification, microfabrication, photo-patterning, and three-dimensional (3D) bioprinting, as well as advances in the temporal control of biomaterials, such as controlled release of molecules, photocleaving of proteins, and controlled hydrogel degradation. We believe that the development and integration of these techniques will drive the engineering of next-generation engineered tissues.

Metabolic programming of mesenchymal stromal cells by oxygen tension directs chondrogenic cell fate.

Actively steering the chondrogenic differentiation of mesenchymal stromal cells (MSCs) into either permanent cartilage or hypertrophic cartilage destined to be replaced by bone has not yet been possible. During limb development, the developing long bone is exposed to a concentration gradient of oxygen, with lower oxygen tension in the region destined to become articular cartilage and higher oxygen tension in transient hypertrophic cartilage. Here, we prove that metabolic programming of MSCs by oxygen tension directs chondrogenesis into either permanent or transient hyaline cartilage. Human MSCs chondrogenically differentiated in vitro under hypoxia (2.5% O2) produced more hyaline cartilage, which expressed typical articular cartilage biomarkers, including established inhibitors of hypertrophic differentiation. In contrast, normoxia (21% O2) prevented the expression of these inhibitors and was associated with increased hypertrophic differentiation. Interestingly, gene network analysis revealed that oxygen tension resulted in metabolic programming of the MSCs directing chondrogenesis into articular- or epiphyseal cartilage-like tissue. This differentiation program resembled the embryological development of these distinct types of hyaline cartilage. Remarkably, the distinct cartilage phenotypes were preserved upon implantation in mice. Hypoxia-preconditioned implants remained cartilaginous, whereas normoxia-preconditioned implants readily underwent calcification, vascular invasion, and subsequent endochondral ossification. In conclusion, metabolic programming of MSCs by oxygen tension provides a simple yet effective mechanism by which to direct the chondrogenic differentiation program into either permanent articular-like cartilage or hypertrophic cartilage that is destined to become endochondral bone.

Nitric Oxide Mediates Crosstalk between Interleukin 1ß and WNT Signaling in Primary Human Chondrocytes by Reducing DKK1 and FRZB Expression.

Interleukin 1 beta (IL1ß) and Wingless-Type MMTV Integration Site Family (WNT) signaling are major players in Osteoarthritis (OA) pathogenesis. Despite having a large functional overlap in OA onset and development, the mechanism of IL1ß and WNT crosstalk has remained largely unknown. In this study, we have used a combination of computational modeling and molecular biology to reveal direct or indirect crosstalk between these pathways. Specifically, we revealed a mechanism by which IL1ß upregulates WNT signaling via downregulating WNT antagonists, DKK1 and FRZB. In human chondrocytes, IL1ß decreased the expression of Dickkopf-1 (DKK1) and Frizzled related protein (FRZB) through upregulation of nitric oxide synthase (iNOS), thereby activating the transcription of WNT target genes. This effect could be reversed by iNOS inhibitor 1400W, which restored DKK1 and FRZB expression and their inhibitory effect on WNT signaling. In addition, 1400W also inhibited both the matrix metalloproteinase (MMP) expression and cytokine-induced apoptosis. We concluded that iNOS/NO play a pivotal role in the inflammatory response of human OA through indirect upregulation of WNT signaling. Blocking NO production may inhibit the loss of the articular phenotype in OA by preventing downregulation of the expression of DKK1 and FRZB.

Advancing Tissue Engineering: A Tale of Nano-, Micro-, and Macroscale Integration.

Tissue engineering has the potential to revolutionize the health care industry. Delivering on this promise requires the generation of efficient, controllable and predictable implants. The integration of nano- and microtechnologies into macroscale regenerative biomaterials plays an essential role in the generation of such implants, by enabling spatiotemporal control of the cellular microenvironment. Here we review the role, function and progress of a wide range of nano- and microtechnologies that are driving the advancements in the field of tissue engineering.

Single-Step Biofabrication of In Situ Spheroid-Forming Compartmentalized Hydrogel for Clinical-Sized Cartilage Tissue Formation.

3D cellular spheroids offer more biomimetic microenvironments than conventional 2D cell culture technologies, which has proven value for many tissue engineering applications. Despite beneficiary effects of 3D cell culture, clinical translation of spheroid tissue engineering is challenged by limited scalability of current spheroid formation methods. Although recent adoption of droplet microfluidics can provide a continuous production process, use of oils and surfactants, generally low throughput, and requirement of additional biofabrication steps hinder clinical translation of spheroid culture. Here, the use of clean (e.g., oil-free and surfactant-free), ultra-high throughput (e.g., 8.5 mL min-1 , 10 000 spheroids s-1 ), single-step, in-air microfluidic biofabrication of spheroid forming compartmentalized hydrogels is reported. This novel technique can reliably produce 1D fibers, 2D planes, and 3D volumes compartmentalized hydrogel constructs, which each allows for distinct (an)isotropic orientation of hollow spheroid-forming compartments. Spheroids produced within ink-jet bioprinted compartmentalized hydrogels outperform 2D cell cultures in terms of chondrogenic behavior. Moreover, the cellular spheroids can be harvested from compartmentalized hydrogels and used to build shape-stable centimeter-sized biomaterial-free living tissues in a bottom-up manner. Consequently, it is anticipated that in-air microfluidic production of spheroid-forming compartmentalized hydrogels can advance production and use of cellular spheroids for various biomedical applications.

Towards single-cell bioprinting: micropatterning tools for organ-on-chip development.

Organs-on-chips (OoCs) hold promise to engineer progressively more human-relevant in vitro models for pharmaceutical purposes. Recent developments have delivered increasingly sophisticated designs, yet OoCs still lack in reproducing the inner tissue physiology required to fully resemble the native human body. This review emphasizes the need to include microarchitectural and microstructural features, and discusses promising avenues to incorporate well-defined microarchitectures down to the single-cell level. We highlight how their integration will significantly contribute to the advancement of the field towards highly organized structural and hierarchical tissues-on-chip. We discuss the combination of state-of-the-art micropatterning technologies to achieve OoCs resembling human-intrinsic complexity. It is anticipated that these innovations will yield significant advances in realization of the next generation of OoC models.

Osmolarity-Induced Altered Intracellular Molecular Crowding Drives Osteoarthritis Pathology.

Osteoarthritis (OA) is a multifactorial degenerative joint disease of which the underlying mechanisms are yet to be fully understood. At the molecular level, multiple factors including altered signaling pathways, epigenetics, metabolic imbalance, extracellular matrix degradation, production of matrix metalloproteinases, and inflammatory cytokines, are known to play a detrimental role in OA. However, these factors do not initiate OA, but are mediators or consequences of the disease, while many other factors causing the etiology of OA are still unknown. Here, it is revealed that microenvironmental osmolarity can induce and reverse osteoarthritis-related behavior of chondrocytes via altered intracellular molecular crowding, which represents a previously unknown mechanism underlying OA pathophysiology. Decreased intracellular crowding is associated with increased sensitivity to proinflammatory triggers and decreased responsiveness to anabolic stimuli. OA-induced lowered intracellular molecular crowding could be renormalized via exposure to higher extracellular osmolarity such as those found in healthy joints, which reverse OA chondrocyte's sensitivity to catabolic stimuli as well as its glycolytic metabolism.

Aqueous Two-Phase Enabled Low Viscosity 3D (LoV3D) Bioprinting of Living Matter.

Embedded 3D bioprinting has great value for the freeform fabrication of living matter. However, embedded 3D bioprinting is currently limited to highly viscous liquid baths or liquid-like solid baths. In contrast, prior to crosslinking, most hydrogels are formulated as low-viscosity solutions and are therefore not directly compatible with bioprinting due to low shape fidelity and poor print stability. The authors here present a method to enable low-viscosity ink 3D (LoV3D) bioprinting, based on aqueous two-phase stabilization of the ink-bath interface. LoV3D allows for the printing of living constructs at high extrusion speeds (up to 1.8 m s-1 ) with high viability due to its exceedingly low-viscosity. Moreover, LoV3D liquid/liquid interfaces offer unique advantages for fusing printed structures, creating intricate vasculature, and modifying surfaces at higher efficiencies than traditional systems. Furthermore, the low interfacial tension of LoV3D bioprinting offers unprecedented nozzle-independent control over filament diameter via large-dimension strand-thinning, which allows for the printing of an exceptionally wide range of diameters down to the width of a single cell. Overall, LoV3D bioprinting is a unique all-aqueous approach with broad material compatibility without the need for rheological ink adaption, which opens new avenues of application in cell patterning, drug screening, engineered meat, and organ fabrication.

Scalable fabrication, compartmentalization and applications of living microtissues.

Living microtissues are used in a multitude of applications as they more closely resemble native tissue physiology, as compared to 2D cultures. Microtissues are typically composed of a combination of cells and materials in varying combinations, which are dictated by the applications' design requirements. Their applications range wide, from fundamental biological research such as differentiation studies to industrial applications such as cruelty-free meat production. However, their translation to industrial and clinical settings has been hindered due to the lack of scalability of microtissue production techniques. Continuous microfluidic processes provide an opportunity to overcome this limitation as they offer higher throughput production rates as compared to traditional batch techniques, while maintaining reproducible control over microtissue composition and size. In this review, we provide a comprehensive overview of the current approaches to engineer microtissues with a focus on the advantages of, and need for, the use of continuous processes to produce microtissues in large quantities. Finally, an outlook is provided that outlines the required developments to enable large-scale microtissue fabrication using continuous processes.

Spatial control of self-organizing vascular networks with programmable aptamer-tethered growth factor photopatterning.

Given the dynamic nature of engineered vascular networks within biofabricated tissue analogues, it is instrumental to have control over the constantly evolving biochemical cues within synthetic matrices throughout tissue remodeling. Incorporation of pro-angiogenic vascular endothelial growth factor (VEGF165) specific aptamers into cell-instructive polymer networks is shown to be pivotal for spatiotemporally controlling the local bioactivity of VEGF that selectively elicit specific cell responses. To harness this effect and quantitatively unravel its spatial resolution, herein, bicomponent micropatterns consisting of VEGF165 specific aptamer-functionalized gelatin methacryloyl (GelMA) (aptamer regions) overlaid with pristine GelMA regions using visible-light photoinitiators (Ru/SPS) were fabricated via two-step photopatterning approach. For the 3D co-culture study, human umbilical vein-derived endothelial cells and mesenchymal stromal cells were used as model cell types. Bicomponent micropatterns with spatially defined spacings (300/500/800 â€‹µm) displayed high aptamer retention, aptamer-fluorescent complementary sequence (CSF) molecular recognition and VEGF sequestration localized within patterned aptamer regions. Stiffness gradient at the interface of aptamer and GelMA regions was observed with high modulus in aptamer region followed by low stiffness GelMA regions. Leveraging aptamer-tethered VEGF's dynamic affinity interactions with CS that upon hybridization facilitates triggered VEGF release, co-culture studies revealed unique characteristics of aptamer-tethered VEGF to form spatially defined luminal vascular networks covered with filopodia-like structures in vitro (spatial control) and highlights their ability to control network properties including orientation over time using CS as an external trigger (temporal control). Moreover, the comparison of single and double exposed regions within micropatterns revealed differences in cell behavior among both regions. Specifically, the localized aptamer-tethered VEGF within single exposed aptamer regions exhibited higher cellular alignment within the micropatterns till d5 of culture. Taken together, this study highlights the potential of photopatterned aptamer-tethered VEGF to spatiotemporally regulate vascular morphogenesis as a tool for controlling vascular remodeling in situ.

Steering Stem Cell Fate within 3D Living Composite Tissues Using Stimuli-Responsive Cell-Adhesive Micromaterials.

Engineered living microtissues such as cellular spheroids and organoids have enormous potential for the study and regeneration of tissues and organs. Microtissues are typically engineered via self-assembly of adherent cells into cellular spheroids, which are characterized by little to no cell-material interactions. Consequently, 3D microtissue models currently lack structural biomechanical and biochemical control over their internal microenvironment resulting in suboptimal functional performance such as limited stem cell differentiation potential. Here, this work report on stimuli-responsive cell-adhesive micromaterials (SCMs) that can self-assemble with cells into 3D living composite microtissues through integrin binding, even under serum-free conditions. It is demonstrated that SCMs homogeneously distribute within engineered microtissues and act as biomechanically and biochemically tunable designer materials that can alter the composite tissue microenvironment on demand. Specifically, cell behavior is controlled based on the size, stiffness, number ratio, and biofunctionalization of SCMs in a temporal manner via orthogonal secondary crosslinking strategies. Photo-based mechanical tuning of SCMs reveals early onset stiffness-controlled lineage commitment of differentiating stem cell spheroids. In contrast to conventional encapsulation of stem cell spheroids within bulk hydrogel, incorporating cell-sized SCMs within stem cell spheroids uniquely provides biomechanical cues throughout the composite microtissues' volume, which is demonstrated to be essential for osteogenic differentiation.