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.

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.

Sonic Hedgehog-activated engineered blood vessels enhance bone tissue formation.

Large bone defects naturally regenerate via a highly vascularized tissue which progressively remodels into cartilage and bone. Current approaches in bone tissue engineering are restricted by delayed vascularization and fail to recapitulate this stepwise differentiation toward bone tissue. Here, we use the morphogen Sonic Hedgehog (Shh) to induce the in vitro organization of an endothelial capillary network in an artificial tissue. We show that endogenous Hedgehog activity regulates angiogenic genes and the formation of vascular lumens. Exogenous Shh further induces the in vitro development of the vasculature (vascular lumen formation, size, distribution). Upon implantation, the in vitro development of the vasculature improves the in vivo perfusion of the artificial tissue and is necessary to contribute to, and enhance, the formation of de novo mature bone tissue. Similar to the regenerating callus, the artificial tissue undergoes intramembranous and endochondral ossification and forms a trabecular-like bone organ including bone-marrow-like cavities. These findings open the door for new strategies to treat large bone defects by closely mimicking natural endochondral bone repair.

Tissue deformation spatially modulates VEGF signaling and angiogenesis.

Physical forces play a major role in the organization of developing tissues. During vascular development, physical forces originating from a fluid phase or from cells pulling on their environment can alter cellular signaling and the behavior of cells. Here, we observe how tissue deformation spatially modulates angiogenic signals and angiogenesis. Using soft lithographic templates, we assemble three-dimensional, geometric tissues. The tissues contract autonomously, change shape stereotypically and form patterns of vascular structures in regions of high deformations. We show that this emergence correlates with the formation of a long-range gradient of Vascular Endothelial Growth Factor (VEGF) in interstitial cells, the local overexpression of the corresponding receptor VEGF receptor 2 (VEGFR-2) and local differences in endothelial cells proliferation. We suggest that tissue contractility and deformation can induce the formation of gradients of angiogenic microenvironments which could contribute to the long-range patterning of the vascular system.

Embedded Printing of Hydrogels and Watery Suspensions of Cells in Patterned Granular Baths.

Bioprinting within support media has emerged as the superior alternative to conventional extrusion printing. Not only because it allows for more freedom over the shapes that can be printed but also because it allows for the printing of inks that would not retain shape fidelity in freeform deposition such as watery liquids. Apart from functioning as mechanical support during embedded printing, hydrogel microparticle support media can provide the unique advantage of offering distinct chemotactic cues to cells printed in the baths by varying the composition of the hydrogel microparticles. There is great potential in compartmentalized granular baths consisting of different hydrogel particle materials in the field of tissue engineering, as these allow for the local inclusion of properties or cues to guide tissue development. In this work, we present a method to create compartmentalized embedding baths by printing multiple granular hydrogel materials that are widely used in tissue engineering. After adapting the volume fraction (φp) of the particles in the bath, we print within them using both inks composed of hydrogel or of cells and other particles suspended in watery liquid. Our process consists of the following three steps: First, the hydrogel microparticles are packed at a φp that allows them to be extruded while being reversibly jammed, facilitating the localized deposition of the granular media to form a compartmentalized bath. Second, each granular media is deposited in succession to create a packed suspension compartment, and by adding liquid post deposition, φp is reduced to allow for embedded printing. Finally, we demonstrate the printing of multiple inks within the compartmentalized embedding bath and highlight the distinct differences between using inks composed of hydrogels or inks composed of particles suspended in watery liquid. This approach combines the advantages of embedded printing through the use of granular media with the added ability to pattern multiple bioactive granular materials to locally affect the behavior of cells printed within the bath. We expect that this workflow will allow researchers to create spatially compartmentalized, customized bioactive embedding baths that allow for the embedded printing of inks composed of hydrogels, cells, and other particles adapted to their need.

Switching to external flows: perturbations of developing vasculature within chicken chorioallantoic membrane.

The impact of fluid flow shear stresses, generated by the movement of blood through vasculature, on the organization and maturation of vessels is widely recognized. Nevertheless, it remains uncertain whether external fluid flows outside of the vasculature in the surrounding tissue can similarly play a role in governing these processes. In this research, we introduce an innovative technique called superfusion-induced vascular steering (SIVS). SIVS involves the controlled imposition of external fluid flow patterns onto the vascularized chick chorioallantoic membrane (CAM), allowing us to observe how this impacts the organization of vascular networks. To investigate the concept of SIVS, we conducted superfusion experiments on the intact chick CAM cultured within an engineered eggshell system, using phosphate buffered saline (PBS). To capture and analyze the effects of superfusion, we employed a custom-built microscopy setup, enabling us to image both superfused and non-superfused regions within the developing CAM. This study provides valuable insights into the practical application of fluid superfusion within an in vivo context, shedding light on its significance for understanding tissue development and manipulation in an engineering setting.

Toward a disruptive, minimally invasive small finger joint implant concept: Cellular and molecular interactions with materials in vivo.

Osteoarthritis (OA) poses significant therapeutic challenges, particularly OA that affects the hand. Currently available treatment strategies are often limited in terms of their efficacy in managing pain, regulating invasiveness, and restoring joint function. The APRICOTⓇ implant system developed by Aurora Medical Ltd (Chichester, UK) introduces a minimally invasive, bone-conserving approach for treating hand OA (https://apricot-project.eu/). By utilizing polycarbonate urethane (PCU), this implant incorporates a caterpillar track-inspired design to promote the restoration of natural movement to the joint. Surface modifications of PCU have been proposed for the biological fixation of the implant. This study investigated the biocompatibility of PCU alone or in combination with two surface modifications, namely dopamine-carboxymethylcellulose (dCMC) and calcium-phosphate (CaP) coatings. In a rat soft tissue model, native and CaP-coated PCU foils did not increase cellular migration or cytotoxicity at the implant-soft tissue interface after 3 d, showing gene expression of proinflammatory cytokines similar to that in non-implanted sham sites. However, dCMC induced an amplified initial inflammatory response that was characterized by increased chemotaxis and cytotoxicity, as well as pronounced gene activation of proinflammatory macrophages and neoangiogenesis. By 21 d, inflammation subsided in all the groups, allowing for implant encapsulation. In a rat bone model, 6 d and 28 d after release of the periosteum, all implant types were adapted to the bone surface with a surrounding fibrous capsule and no protracted inflammatory response was observed. These findings demonstrated the biocompatibility of native and CaP-coated PCU foils as components of APRICOTⓇ implants. STATEMENT OF SIGNIFICANCE: Hand osteoarthritis treatments require materials that minimize irritation of the delicate finger joints. Differing from existing treatments, the APRICOTⓇ implant leverages polycarbonate urethane (PCU) for minimally invasive joint replacement. This interdisciplinary, preclinical study investigated the biocompatibility of thin polycarbonate urethane (PCU) foils and their surface modifications with calcium-phosphate (CaP) or dopamine-carboxymethylcellulose (dCMC). Cellular and morphological analyses revealed that both native and Ca-P coated PCU elicit transient inflammation, similar to sham sites, and a thin fibrous encapsulation in soft tissues and on bone surfaces. However, dCMC surface modification amplified initial chemotaxis and cytotoxicity, with pronounced activation of proinflammatory and neoangiogenesis genes. Therefore, native and CaP-coated PCU possess sought-for biocompatible properties, crucial for patient safety and performance of APRICOTⓇ implant.

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.

Visualization of micro-agents and surroundings by real-time multicolor fluorescence microscopy.

Optical microscopy techniques are a popular choice for visualizing micro-agents. They generate images with relatively high spatiotemporal resolution but do not reveal encoded information for distinguishing micro-agents and surroundings. This study presents multicolor fluorescence microscopy for rendering color-coded identification of mobile micro-agents and dynamic surroundings by spectral unmixing. We report multicolor microscopy performance by visualizing the attachment of single and cluster micro-agents to cancer spheroids formed with HeLa cells as a proof-of-concept for targeted drug delivery demonstration. A microfluidic chip is developed to immobilize a single spheroid for the attachment, provide a stable environment for multicolor microscopy, and create a 3D tumor model. In order to confirm that multicolor microscopy is able to visualize micro-agents in vascularized environments, in vitro vasculature network formed with endothelial cells and ex ovo chicken chorioallantoic membrane are employed as experimental models. Full visualization of our models is achieved by sequential excitation of the fluorophores in a round-robin manner and synchronous individual image acquisition from three-different spectrum bands. We experimentally demonstrate that multicolor microscopy spectrally decomposes micro-agents, organic bodies (cancer spheroids and vasculatures), and surrounding media utilizing fluorophores with well-separated spectrum characteristics and allows image acquisition with 1280 [Formula: see text] 1024 pixels up to 15 frames per second. Our results display that real-time multicolor microscopy provides increased understanding by color-coded visualization regarding the tracking of micro-agents, morphology of organic bodies, and clear distinction of surrounding media.

Spatiotemporally controlled, aptamers-mediated growth factor release locally manipulates microvasculature formation within engineered tissues.

Spatiotemporally controlled growth factor (GF) delivery is crucial for achieving functional vasculature within engineered tissues. However, conventional GF delivery systems show inability to recapitulate the dynamic and heterogeneous nature of developing tissue's biochemical microenvironment. Herein, an aptamer-based programmable GF delivery platform is described that harnesses dynamic affinity interactions for facilitating spatiotemporal control over vascular endothelial GF (VEGF165) bioavailability within gelatin methacryloyl matrices. The platform showcases localized VEGF165 sequestration from the culture medium (offering spatial-control) and leverages aptamer-complementary sequence (CS) hybridization for triggering VEGF165 release (offering temporal-control), without non-specific leakage. Furthermore, extensive 3D co-culture studies (human umbilical vein-derived endothelial cells & mesenchymal stromal cells), in bi-phasic hydrogel systems revealed its fundamentally novel capability to selectively guide cell responses and manipulate lumen-like microvascular networks via spatiotemporally controlling VEGF165 bioavailability within 3D microenvironment. This platform utilizes CS as an external biochemical trigger for guiding vascular morphogenesis which is suitable for creating dynamically controlled engineered tissues.

Embedded 3D printing of dilute particle suspensions into dense complex tissue fibers using shear thinning xanthan baths.

In order to fabricate functional organoids and microtissues, a high cell density is generally required. As such, the placement of cell suspensions in molds or microwells to allow for cell concentration by sedimentation is the current standard for the production of organoids and microtissues. Even though molds offer some level of control over the shape of the resulting microtissue, this control is limited as microtissues tend to compact towards a sphere after sedimentation of the cells. 3D bioprinting on the other hand offers complete control over the shape of the resulting structure. Even though the printing of dense cell suspensions in the ink has been reported, extruding dense cellular suspensions is challenging and generally results in high shear stresses on the cells and a poor shape fidelity of the print. As such, additional materials such as hydrogels are added in the bioink to limit shear stresses, and to improve shape fidelity and resolution. The maximum cell concentration that can be incorporated in a hydrogel-based ink before the ink's rheological properties are compromised, is significantly lower than the concentration in a tissue equivalent. Additionally, the hydrogel components often interfere with cellular self-assembly processes. To circumvent these limitations, we report a simple and inexpensive xanthan bath based embedded printing method to 3D print dense functional linear tissues using dilute particle suspensions consisting of cells, spheroids, hydrogel beads, or combinations thereof. Using this method, we demonstrated the self-organization of functional cardiac tissue fibers with a layer of epicardial cells surrounding a body of cardiomyocytes.

Serial imaging of micro-agents and cancer cell spheroids in a microfluidic channel using multicolor fluorescence microscopy.

Multicolor fluorescence microscopy is a powerful technique to fully visualize many biological phenomena by acquiring images from different spectrum channels. This study expands the scope of multicolor fluorescence microscopy by serial imaging of polystyrene micro-beads as surrogates for drug carriers, cancer spheroids formed using HeLa cells, and microfluidic channels. Three fluorophores with different spectral characteristics are utilized to perform multicolor microscopy. According to the spectrum analysis of the fluorophores, a multicolor widefield fluorescence microscope is developed. Spectral crosstalk is corrected by exciting the fluorophores in a round-robin manner and synchronous emitted light collection. To report the performance of the multicolor microscopy, a simplified 3D tumor model is created by placing beads and spheroids inside a channel filled with the cell culture medium is imaged at varying exposure times. As a representative case and a method for bio-hybrid drug carrier fabrication, a spheroid surface is coated with beads in a channel utilizing electrostatic forces under the guidance of multicolor microscopy. Our experiments show that multicolor fluorescence microscopy enables crosstalk-free and spectrally-different individual image acquisition of beads, spheroids, and channels with the minimum exposure time of 5.5 ms. The imaging technique has the potential to monitor drug carrier transportation to cancer cells in real-time.

Assessment of flow within developing chicken vasculature and biofabricated vascularized tissues using multimodal imaging techniques.

Fluid flow shear stresses are strong regulators for directing the organization of vascular networks. Knowledge of structural and flow dynamics information within complex vasculature is essential for tuning the vascular organization within engineered tissues, by manipulating flows. However, reported investigations of vascular organization and their associated flow dynamics within complex vasculature over time are limited, due to limitations in the available physiological pre-clinical models, and the optical inaccessibility and aseptic nature of these models. Here, we developed laser speckle contrast imaging (LSCI) and side-stream dark field microscopy (SDF) systems to map the vascular organization, spatio-temporal blood flow fluctuations as well as erythrocytes movements within individual blood vessels of developing chick embryo, cultured within an artificial eggshell system. By combining imaging data and computational simulations, we estimated fluid flow shear stresses within multiscale vasculature of varying complexity. Furthermore, we demonstrated the LSCI compatibility with bioengineered perfusable muscle tissue constructs, fabricated via molding techniques. The presented application of LSCI and SDF on perfusable tissues enables us to study the flow perfusion effects in a non-invasive fashion. The gained knowledge can help to use fluid perfusion in order to tune and control multiscale vascular organization within engineered tissues.

Hyaluronic Acid Oligomer Immobilization as an Angiogenic Trigger for the Neovascularization of TE Constructs.

Tissue engineered (TE) substitutes of clinically relevant sizes need an adequate vascular system to ensure function and proper tissue integration after implantation. However, the predictable vascularization of TE substitutes is yet to be achieved. Molecular weight variations in hyaluronic acid (HA) have been pointed to trigger angiogenesis. Thus, this study investigates HA oligomer immobilization as a promoter for TE construct vascularization. As a proof-of-concept, the surface of methacrylated gelatin (GelMA) hydrogels were functionalized with high molecular weight (HMW; 1.5 to 1.8 MDa) and low molecular weight (LMW; < 10 kDa) HA, previously modified with aldehyde groups to enable the immobilization through Schiff's base formation. The ability of A-HA to bind amine-presenting surfaces was confirmed by Surface Plasmon Resonance (SPR). Human Umbilical Vein Endothelial Cells (HUVECs) seeded over hydrogels functionalized with LMW HA showed higher proliferation and expression of angiogenic markers (KDR and CD31), than those grown in HMW HA conjugated- or plain surfaces, in line with the activation of HA ERK1/2 mediated downstream signaling. Moreover, when cocultured with human dental pulp cells (hDPCs) encapsulated into the GelMA, an increase in endothelial cell migration was observed for the LMW HA functionalized formulations. Overall LMW HA functionalization enhanced endothelial cell response showing potential as an angiogenesis inducer for TE applications.

Supply of nutrients to cells in engineered tissues.

A proper supply of nutrients to cells in engineered tissues is paramount for an optimal development and survival of these tissues. However, especially in tissues with clinically relevant sizes, the mass transport of nutrients into the tissue is often insufficient to sustain all the cells within the tissue. This is not only the case during in vitro culture. After implantation of an engineered tissue, a vascular network is not directly established. Therefore, the mass transport of nutrients is also critical during the initial period after implantation. This review introduces the basics of mass transport, leading to the conclusion that three main concepts can be used to increase nutrient supply in tissue engineering. These are; increasing the overall diffusion coefficient, decreasing the diffusion distance, or increasing convective transport. Based on these concepts, the main strategies that have been developed to enhance the supply of nutrients to cells in engineered tissues will be discussed.

Bioengineered 3D Models to Recapitulate Tissue Fibrosis.

Fibrosis, characterized by progressive tissue stiffening resulting in organ failure, is a growing health problem affecting millions of people worldwide. Currently, therapeutic options for tissue fibrosis are severely limited and organ transplantation is the only effective treatment for the end-stage fibrotic diseases with inherent limitations. Recent advancements in engineered 3D in vitro human disease mimic models, recapitulating the tissue pathophysiology, have provided unique state-of-the-art platforms for: (i) understanding the biological mechanisms involved in the disease pathogenesis; and (ii) high-throughput and reproducible drug screening. This review focuses on the recent multidisciplinary developments made towards advanced 3D biomimetic fibrotic tissue (liver, kidney, and lung) models that combine highly precision manufacturing techniques with high cellular functionality and biophysical (mechanical) properties.

Regulation of endothelial cell arrangements within hMSC - HUVEC co-cultured aggregates.

BACKGROUND: Micro-mass culturing or cellular aggregation is an effective method used to form mineralised bone tissue. Poor core cell viability, however, is often an impeding characteristic of large micro-mass cultures, and equally for large tissue-engineered bone grafts. Because of this, efforts are being made to enhance large graft perfusion, often through pre-vascularisation, which involves the co-culture of endothelial cells and bone cells or stem cells. METHODS: This study investigated the effects of different aggregation techniques and culture conditions on endothelial cell arrangements in mesenchymal stem cell and human umbilical vein endothelial cell co-cultured aggregates when endothelial cells constituted just 5%. Two different cellular aggregation techniques, i.e. suspension culture aggregation and pellet culture aggregation, were applied alongside two subsequent culturing techniques, i.e. hydrostatic loading and static culturing. Endothelial cell arrangements were assessed under such conditions to indicate potential pre-vascularisation. RESULTS: Our study found that the suspension culture aggregates cultured under hydrostatic loading offered the best environment for enhanced endothelial cell regional arrangements, closely followed by the pellet culture aggregates cultured under hydrostatic loading, the suspension culture aggregates cultured under static conditions, and the pellet culture aggregates cultured under static conditions. CONCLUSIONS: The combination of particular aggregation techniques with dynamic culturing conditions appeared to have a synergistic effect on the cellular arrangements within the co-cultured aggregates.

Vascularization in tissue engineering.

Tissue engineering has been an active field of research for several decades now. However, the amount of clinical applications in the field of tissue engineering is still limited. One of the current limitations of tissue engineering is its inability to provide sufficient blood supply in the initial phase after implantation. Insufficient vascularization can lead to improper cell integration or cell death in tissue-engineered constructs. This review will discuss the advantages and limitations of recent strategies aimed at enhancing the vascularization of tissue-engineered constructs. We will illustrate that combining the efforts of different research lines might be necessary to obtain optimal results in the field.

Engineering vascularized skeletal muscle tissue.

One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. Here we describe the induction of endothelial vessel networks in engineered skeletal muscle tissue constructs using a three-dimensional multiculture system consisting of myoblasts, embryonic fibroblasts and endothelial cells coseeded on highly porous, biodegradable polymer scaffolds. Analysis of the conditions for induction and stabilization of the vessels in vitro showed that addition of embryonic fibroblasts increased the levels of vascular endothelial growth factor expression in the construct and promoted formation and stabilization of the endothelial vessels. We studied the survival and vascularization of the engineered muscle implants in vivo in three different models. Prevascularization improved the vascularization, blood perfusion and survival of the muscle tissue constructs after transplantation.

Microphysiological systems: analysis of the current status, challenges and commercial future.

The field of microphysiological systems (or organs-on-a-chip) experienced, in the past decade, a surge in publications and efforts towards commercialization. Such systems hold the promise to advance drug discovery, diagnostics, and many other areas. In this review we summarize and analyze the current status of the field, describe the commercial advances and discuss standing challenges and the commercial outlook of the field.