Three-dimensional quantification and visualization of vascular networks in engineered tissues

Three-dimensional textural and volumetric image analysis holds great potential in understanding the image data produced by multi-photon microscopy. In this thesis, a tool that provides quantitative textural and morphometric analyzes of vasculature in engineered tissues, alongside with a fast three-dimensional volume rendering is proposed. The investigated 3D artificial tissues consist of Human Umbilical Vein Endothelial Cells (HUVEC) embedded in collagen exposed to two regimes of ultrasound standing wave fields under different pressure conditions. Textural features were evaluated over the extracted connected region in our samples using the normalized Gray Level Co-occurrence Matrix (GLCM) combined with Gray-Level Run Length Matrix (GLRLM) analysis. To minimize the error resulting from any possible volumetric rotation and to provide a comprehensive textural analysis, an averaged version of nine GLCM and GLRLM orientations is used. To evaluate volumetric features, parameters such as volume run length and percentage volume were utilized. The z-projection versions of the samples were used to estimate the tortuosity of the vessels, as well as, to measure the length and the angle of the branches. We utilized a three-dimensional volume rendering technique named MATVTK (derived from MATLAB and VTK) and runs under MATLAB that shows a great improvement on the processing time to reconstruct our volumes compared to MATLAB built-in functions. Results show that our analysis is able to differentiate among the exposed samples, due to morphological changes induced by the ultrasound standing wave fields. Furthermore, we demonstrate that providing more textural parameters than what is currently being reported in the literature, enhances the quantitative understanding of the heterogeneity of artificial tissues

Mathematical modelling of tissue-engineering angiogenesis

We present a mathematical model for the vascularisation of a porous scaffold following implantation in vivo. The model is given as a set of coupled non-linear ordinary differential equations (ODEs) which describe the evolution in time of the amounts of the different tissue constituents inside the scaffold. Bifurcation analyses reveal how the extent of scaffold vascularisation changes as a function of the parameter values. For example, it is shown how the loss of seeded cells arising from slow infiltration of vascular tissue can be overcome using a prevascularisation strategy consisting of seeding the scaffold with vascular cells. Using certain assumptions it is shown how the system can be simplified to one which is partially tractable and for which some analysis is given. Limited comparison is also given of the model solutions with experimental data from the chick chorioallantoic membrane (CAM) assay

Angiogenesis in tissue engineering : Breathing life into constructed tissue substitutes

Long-term function of three-dimensional (3D) tissue constructs depends on adequate vascularization after implantation. Accordingly, research in tissue engineering has focused on the analysis of angiogenesis. For this purpose, 2 sophisticated in vivo models (the chorioallantoic membrane and the dorsal skinfold chamber) have recently been introduced in tissue engineering research, allowing a more detailed analysis of angiogenic dysfunction and engraftment failure. To achieve vascularization of tissue constructs, several approaches are currently under investigation. These include the modification of biomaterial properties of scaffolds and the stimulation of blood vessel development and maturation by different growth factors using slow-release devices through pre-encapsulated microspheres. Moreover, new microvascular networks in tissue substitutes can be engineered by using endothelial cells and stem cells or by creating arteriovenous shunt loops. Nonetheless, the currently used techniques are not sufficient to induce the rapid vascularization necessary for an adequate cellular oxygen supply. Thus, future directions of research should focus on the creation of microvascular networks within 3D tissue constructs in vitro before implantation or by co-stimulation of angiogenesis and parenchymal cell proliferation to engineer the vascularized tissue substitute in situ

Computational fluid dynamic analysis of bioprinted self-supporting perfused tissue models

Natural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient‐rich oxygenated blood through the vasculature to support cell metabolism within most cell‐dense tissues. Since scaffold‐free biofabricated tissues being developed into clinical implants, research models, and pharmaceutical testing platforms should similarly exhibit perfused tissue‐like structures, we generated a generalizable biofabrication method resulting in self‐supporting perfused (SSuPer) tissue constructs incorporated with perfusible microchannels and integrated with the modular FABRICA perfusion bioreactor. As proof of concept, we perfused an MLO‐A5 osteoblast‐based SSuPer tissue in the FABRICA. Although our resulting SSuPer tissue replicated vascularization and perfusion observed in situ, supported its own weight, and stained positively for mineral using Von Kossa staining, our in vitro results indicated that computational fluid dynamics (CFD) should be used to drive future construct design and flow application before further tissue biofabrication and perfusion. We built a CFD model of the SSuPer tissue integrated in the FABRICA and analyzed flow characteristics (net force, pressure distribution, shear stress, and oxygen distribution) through five SSuPer tissue microchannel patterns in two flow directions and at increasing flow rates. Important flow parameters include flow direction, fully developed flow, and tissue microchannel diameters matched and aligned with bioreactor flow channels. We observed that the SSuPer tissue platform is capable of providing direct perfusion to tissue constructs and proper culture conditions (oxygenation, with controllable shear and flow rates), indicating that our approach can be used to biofabricate tissue representing primary tissues and that we can model the system in silico

Directional control of angiogenesis to produce a designed multiscale micro-vascular network with bioprinting

Department of Biomedical EngineeringThe biomimetic vascular network is a key element in regeneration of viable, functional and scalable artificial tissues. In this study, we developed a multiscale vascular network that can be patterned freely by using bioprinting technology. An endothelialized channel of several hundred micrometer scale was directly printed. The micro-vascular network consisting of tubular structures of several tens of micrometers was generated through the direction control of angiogenic sprouting using the chemotaxis effect. For this purpose, human umbilical vein endothelial cells (HUVEC) and angiogenic factor secreting cells, normal human dermal fibroblasts (NHDF), were co-patterned at 1 to 2 mm intervals using water soluble bio-ink and alginate based bio-ink, respectively. Then, a bridge pattern connecting the two patterned gels was made with fibrin gel. After printing, an endothelialized channel of about 800 ??m was formed by selective removal of water soluble bio-ink. The angiogenic sprouting was induced at about 200 ??m/day along the bridge pattern from the channel. It was also possible to fabricate a multiscale micro-vascular network with diagonal, wave and branch shapes using bridge patterns of various designs. In this study, we investigated the functionality of hepatocytes by co-culturing mouse primary hepatocytes after fabricating a vascular construct with hepatic lobule-shaped pattern to confirm the utility of the constructed process. As a result, we could confirm largely improved albumin and urea secretion. Based on these results, we confirmed that the tissue specific multiscale vascular network could be constructed. This technique should provide a useful tool for the development of functional and scalable vascularized tissues.clos

Multi-scale cellular engineering: From molecules to organ-on-a-chip.

Recent technological advances in cellular and molecular engineering have provided new insights into biology and enabled the design, manufacturing, and manipulation of complex living systems. Here, we summarize the state of advances at the molecular, cellular, and multi-cellular levels using experimental and computational tools. The areas of focus include intrinsically disordered proteins, synthetic proteins, spatiotemporally dynamic extracellular matrices, organ-on-a-chip approaches, and computational modeling, which all have tremendous potential for advancing fundamental and translational science. Perspectives on the current limitations and future directions are also described, with the goal of stimulating interest to overcome these hurdles using multi-disciplinary approaches

Big bottlenecks in cardiovascular tissue engineering.

Although tissue engineering using human-induced pluripotent stem cells is a promising approach for treatment of cardiovascular diseases, some limiting factors include the survival, electrical integration, maturity, scalability, and immune response of three-dimensional (3D) engineered tissues. Here we discuss these important roadblocks facing the tissue engineering field and suggest potential approaches to overcome these challenges

Decellularized diaphragmatic muscle drives a constructive angiogenic response in vivo

Skeletal muscle tissue engineering (TE) aims to efficiently repair large congenital and acquired defects. Biological acellular scaffolds are considered a good tool for TE, as decellularization allows structural preservation of tissue extracellular matrix (ECM) and conservation of its unique cytokine reservoir and the ability to support angiogenesis, cell viability, and proliferation. This represents a major advantage compared to synthetic scaffolds, which can acquire these features only after modification and show limited biocompatibility. In this work, we describe the ability of a skeletal muscle acellular scaffold to promote vascularization both ex vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array confirmed the presence of pro-angiogenic molecules in the decellularized tissue such as HGF, VEGF, and SDF-1\u3b1. The acellular muscle was implanted in BL6/J mice both subcutaneously and ortotopically. In the first condition, the ECM-derived scaffold appeared vascularized 7 days post-implantation. When the decellularized diaphragm was ortotopically applied, newly formed blood vessels containing CD31+, \u3b1SMA+, and vWF+ cells were visible inside the scaffold. Systemic injection of Evans Blue proved function and perfusion of the new vessels, underlying a tissue-regenerative activation. On the contrary, the implantation of a synthetic matrix made of polytetrafluoroethylene used as control was only surrounded by vWF+ cells, with no cell migration inside the scaffold and clear foreign body reaction (giant cells were visible). The molecular profile and the analysis of macrophages confirmed the tendency of the synthetic scaffold to enhance inflammation instead of regeneration. In conclusion, we identified the angiogenic potential of a skeletal muscle-derived acellular scaffold and the pro-regenerative environment activated in vivo, showing clear evidence that the decellularized diaphragm is a suitable candidate for skeletal muscle tissue engineering and regeneration

Layered Fabrication of Branched Networks Using Lindenmayer Systems

A current challenge impeding the growth of bone tissue engineering is the lack of functional scaffolds of geometric sizes greater than 10mm due to the inability of cells to survive deep within the scaffold. It is hypothesized that these scaffolds must have an inbuilt nutrient distribution network to sustain the uniform growth of cells. In this paper, we seek to enhance the design and layered fabrication of scaffold internal architecture through the development of Lindenmayer systems, a graphical language based theory to create nutrient delivery networks. The scaffolds are fabricated using the Texas Instruments DLP™ system through UV‐photopolymerization to produce polyethylene glycol hydrogels with internal branch structures. The paper will discuss the Lindenmayer system, process planning algorithms, layered fabrication of samples, challenges and future tasks.Mechanical Engineerin