Labeling of endothelial cells with magnetic microbeads by angiophagy

Objectives Attachment of magnetic particles to cells is needed for a variety of applications but is not always possible or efficient. Simpler and more convenient methods are thus desirable. In this study, we tested the hypothesis that endothelial cells (EC) can be loaded with micron-size magnetic beads by the phagocytosis-like mechanism ‘angiophagy’. To this end, human umbilical vein EC (HUVEC) were incubated with magnetic beads conjugated or not (control) with an anti-VEGF receptor 2 antibody, either in suspension, or in culture followed by re-suspension using trypsinization. Results In all conditions tested, HUVEC incubation with beads induced their uptake by angiophagy, which was confirmed by (i) increased cell granularity assessed by flow cytometry, and (ii) the presence of an F-actin rich layer around many of the intracellular beads, visualized by confocal microscopy. For confluent cultures, the average number of beads per cell was 4.4 and 4.2, with and without the presence of the anti-VEGFR2 antibody, respectively. However, while the actively dividing cells took up 2.9 unconjugated beads on average, this number increased to 5.2 if binding was mediated by the antibody. Magnetic pulldown increased the cell density of beads-loaded cells in porous electrospun poly-capro-lactone scaffolds by a factor of 4.5 after 5 min, as compared to gravitational settling (p < 0.0001). Conclusion We demonstrated that EC can be readily loaded by angiophagy with micron-sized beads while attached in monolayer culture, then dispersed in single-cell suspensions for pulldown in porous scaffolds and for other applications

Biomaterial-Mediated Reprogramming of the Wound Interface to Enhance Meniscal Repair

Endogenous repair of fibrous connective tissues is limited, and there exist few successful strategies to improve healing after injury. As such, new methods that advance repair by enhancing cell migration to the wound interface, extracellular matrix (ECM) production, and tissue integration would represent a marked clinical advance. Using the adult meniscus as a test platform, we hypothesized that ECM density and stiffness increase throughout tissue maturation, and that these age-related changes present biophysical barriers to interstitial cell migration during wound healing. We further posited that modulating the matrix could remove these impediments, enabling endogenous cells to reach the injury site. To test our hypotheses, we compared the microenvironment of fetal and adult meniscal ECM via atomic force microscopy (AFM) indentation and second harmonic generation (SHG) imaging of the collagenous matrix. We also explored interstitial cell mobility through fetal and adult native tissue environments using a three-dimensional ex vivo system. We further investigated strategies that might expedite cell migration, including enzymatic degradation of the ECM with collagenase to reduce matrix stiffness and increase porosity. To restrict these biological manipulations to the wound interface, we fabricated a delivery system in which selected biofactors were stored inside composite electrospun nanofibrous scaffolds and released upon hydration. The ability for bioactive scaffolds to enhance the cellularity and integration of meniscal injuries was evaluated in vivo using tissue explants in a subcutaneous implantation model, as well as an orthotopic meniscal injury model. Our findings suggest that matrix stiffness, density, and organization increase with meniscal development at the expense of cell mobility. Our results also indicate that partial digestion of the wound interface with collagenase improves repair by creating a more compliant and porous microenvironment that facilitates cell migration. Furthermore, when scaffolds containing collagenase-releasing fibers were placed inside meniscal defects, enzymatic digestion was localized and resulted in improved cellular colonization and closure of the wound site, similar to treatment with aqueous collagenase. This innovative approach of targeted delivery may aid the many patients that exhibit meniscal tears by promoting integrative repair, thereby circumventing the pathologic consequences of partial meniscus removal, and may find widespread application in the treatment of injuries to a variety of dense connective tissues

Topologically defined composites of collagen type I and V as in vitro cell culture scaffolds

Cell fate is known to be triggered by cues from the extracellular matrix including its chemical, biological and physical characteristics. Specifically, mechanical and topological properties are increasingly recognized as important signals. The aim of this work was to provide an easy-accessible biomimetic in vitro platform of topologically defined collagen I matrices to dissect cell behaviour under various conditions in vitro. We reconstituted covalently bound layers of three-dimensional (3D) networks of collagen type I and collagen type V with a defined network topology. A new erosion algorithm enabled us to analyse the mean pore diameter and fibril content, while the mean fibril diameter was examined by an autocorrelation method. Different concentrations and ratios of collagen I and V resulted in pore diameters from 2.4 μm to 4.5 μm and fibril diameters from 0.6 to 0.8 μm. A comparison of telopeptide intact collagen I to telopeptide deficient collagen I revealed obvious differences in network structure. The good correlation of the topological data to measurements of network stiffness as well as invasion of human dermal fibroblasts proofed the topological analysis to provide meaningful measures of the functional characteristics of the reconstituted 3D collagen matrices

Doctor of Philosophy

dissertationSpinal cord injury (SCI) is extremely debilitating to patients and costly to our healthcare system. Since it is an important contributor to mortality and morbidity, various therapeutic strategies have been investigated, either experimentally or clinically, to improve patients' quality of life. Studies utilizing pharmacological methods to mitigate the inhibitory components of the glial scar and facilitate axonal regeneration have been the primary experimental approaches in the field. However, the results are still not satisfactory. In this research, we aimed to tackle the issue from a novel perspective by developing cell derived, tissue engineered biomaterials that can be used in combination with other therapeutic approaches to improve the efficacy of current treatments. In this dissertation, a simple method to create either cellularized or acellular ECM biomaterial constructs is described. In particular, by utilizing patterned surface ligands, organized orientation can be introduced to the entire astrocyte derived construct morphologically and with regard to its associated matrix proteins, which mimics the native astrocyte framework within the spinal cord fiber tracts and provides these constructs the ability to guide axonal regeneration in vitro. In addition, meningeal fibroblast based biomaterial constructs are also developed taking advantage of the same engineering approach. It has been demonstrated that repairing damaged dura mater with allografts also benefits the regeneration process of the damaged spinal cord. In particular, acellular meningeal ECM constructs preserve a similar matrix protein profile as the native rat dura mater and support allogeneic meningeal cell adhesion and promote proliferation. The results suggest these engineered biomaterial constructs derived particularly from cells residing within tissue targeted for repair may carry appropriate tissue specific biological cues and hold therapeutic potentials for spinal cord injury repair as well as dual defect reconstruction

Proteolytic and Mechanical Matrix Remodeling During Capillary Morphogenesis

Engineering large viable tissues requires techniques for encouraging rapid capillary bed formation to prevent necrosis. A convenient means of creating this micro-vascular network is through spontaneous neovascularization, which occurs when endothelial cells (ECs) and supportive stromal cells are co-encapsulated within a variety of hydrogel-based extracellular matrices (ECM) and self-assemble into an interconnected network of endothelial tubules. Although this is a robust phenomenon, the environmental and cell-specific determinants that affect the rate and quality of micro-vascular network formation still require additional characterization to improve clinical translatability. This thesis investigates how the proteolytic susceptibility of engineered matrices effects neovascular self-assembly in poly(ethylene glycol) (PEG) hydrogels and provides characterization of changes to matrix mechanics that accompany neovascular morphogenesis in fibrin and PEG hydrogels. Proteolytic ECM remodeling is essential for the process of capillary morphogenesis. Pharmacological inhibitor studies suggested a role for both matrix metalloproteinases (MMP)- and plasmin-mediated mechanisms of ECM remodeling in an EC-fibroblast co-culture model of vasculogenesis in fibrin. To further investigate the potential contribution of plasmin mediated matrix degradation in facilitating capillary morphogenesis we employed PEG hydrogels engineered with proteolytic specificity to either MMPs, plasmin, or both. Although fibroblasts spread in plasmin-selective hydrogels, we only observed robust capillary morphogenesis in MMP-sensitive matrices, with no added benefit in dual susceptible hydrogels. Enhanced capillary morphogenesis was observed, however, in PEG hydrogels engineered with increased susceptibility to MMPs without altering proteolytic selectivity or hydrogel mechanical properties. These findings highlight the critical importance of MMP-mediated ECM degradation during vasculogenesis and justify the preferential selection of MMP-degradable peptide crosslinkers in the design of synthetic hydrogels used to promote vascularization. Matrix stiffness is a well-established cue in cellular morphogenesis, however, the converse effect of cellular remodeling on environmental mechanics is comparatively under characterized. In fibrin hydrogels, we applied traditional bulk rheology and laser tweezers-based active microrheology to demonstrate that both ECs and fibroblasts progressively stiffen the ECM across length scales, with the changes in bulk properties dominated by fibroblasts. Despite a lack of fibrillar architecture, a similar stiffening effect was observed in MMP-degradable PEG hydrogels. This stiffening tightly correlated with degree of vessel formation and critically depended on active cellular contractility. To a lesser degree, deposition of ECM proteins also appeared to contribute to progressive hydrogel stiffening. Blocking cell-mediated hydrogel degradation abolished stiffening, demonstrating that matrix metalloproteinase (MMP)-mediated remodeling is required for stiffening to occur. EC co-culture with mesenchymal stem cells (MSCs) in PEG resulted in reduced vessel formation compared to fibroblast co-cultures and no change in hydrogel mechanics over time. The correlation between matrix stiffening and enhanced vessel formation, and dependence on cellular contractility, suggests differences in vessel formation between fibroblasts and MSCs may be partially mediated by differences in cellular contractility. Collectively, these findings provide a deeper understanding of mechanobiological effects during capillary morphogenesis and highlight the dynamic reciprocity between cells and their mechanical environmentPHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/162935/2/bjuliar_1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162935/1/bjuliar_2.pd

Writing 3D in vitro models of human tendon within a biomimetic fibrillar support platform

Dissertação de mestrado em Engenharia Biomédica (especialização em Biomateriais, Reabilitação e Biomecânica)As patologias do tendão são doenças altamente debilitantes, para as quais os tratamentos atuais permanecem desafiadores e têm resultados de recuperação pouco relevantes. Deste modo, modelos in vitro relevantes, que permitam o estudo da tendinopatia e a testagem de novas abordagens regenerativas para desenvolver melhores tratamentos são altamente necessários. Neste trabalho, propomos o fabrico automatizado de sistemas microfisiológicos bioimpressos em 3D (MPS), incorporados numa plataforma de suporte fibrilar biomimética baseada na automontagem de nanocristais de celulose (CNCs). A matriz extracelular descelularizada do tendão (dECM) foi usada para produzir biotinta que recapitula de perto as pistas biofísicas e bioquímicas do nicho de células do tendão e, assim, autoinduz a diferenciação tenogénica de células-estaminais derivadas do tecido adiposo humano (hASCs). Dois MPS foram desenvolvidos: um sistema de monocultura que recria os padrões celulares e o fenótipo do tendão; e um sistema multicelular, com a incorporação de células endoteliais para estudar a comunicação entre o tendão e o sistema vascular, que desempenha papéis críticos na tendinopatia e no desenvolvimento do tendão. Ambos os MPS mostraram alta viabilidade celular, proliferação e alinhamento durante a cultura até 21 dias, e o hidrogel de dECM induziu a diferenciação de células-estaminais em direção à linhagem tenogénica, mostrado pela expressão de marcadores relacionados com o tendão, como Scleraxis (SCX) e Tenomodulin (TNMD). Notavelmente, as células endoteliais migram em direção ao compartimento do tendão, mostrando a atração química existente entre os dois compartimentos, mas não o invadiram. A comunicação com células endoteliais parece aumentar a diferenciação tenogénica das hASCs. No geral, o sistema proposto pode ser promissor para o fabrico automatizado de modelos organotípicos de tendão num-chip que será uma nova ferramenta valiosa para estudar a fisiologia e as patologias do tendão, bem como o efeito de medicamentos para o tratamento de tendinopatias.Tendon pathologies are highly debilitating diseases, for which current treatments remains challenging, and has poor recovery outcomes. Therefore, relevant in vitro models allowing to study tendinopathies and test new regenerative approaches to develop better treatments are highly needed. Here we propose the automated fabrication of 3D bioprinted microphysiological systems (MPS) embedded into a biomimetic fibrillar support platform based on self-assembling of cellulose nanocrystals (CNCs). Tendon decellularized extracellular matrix (dECM) was used to produce bioink that closely recapitulate the biophysical and biochemical cues of tendon cell niche, and thus self-induce the tenogenic differentiation of human adipose derived stem cells (hASCs). Two MPS were developed: a monoculture system that recreates the cellular patterns and phenotype of tendon core; and a multicellular system, incorporating endothelial cells to study the crosstalk between the tendon and the vascular compartments, which plays critical roles in tendinopathy and tendon development. Both MPS showed high cell viability, proliferation, and alignment during culture up to 21 days, and the dECM hydrogel induced stem cell differentiation towards tenogenic lineage, as shown by the expression of tendon-related markers such as Scleraxis (SCX) and Tenomodulin (TNMD). Remarkably, endothelial cells migrate towards tendon compartment, showing the existing chemoattraction between the two compartments, but did not invade it. The crosstalk with endothelial cells seem to boost hASCs tenogenesis. Overall, the proposed system might be promising for the automated fabrication of organotypic tendon-on-chip models that will be a valuable new tool to study tendon physiology and pathologies, as well as the effect of drugs for the treatment of tendinopathy

Co-culture of Smooth Muscle Cells and Endothelial Cells on Porous 3D Polyurethane Scaffolds for Vascular Tissue Engineering

One of the challenges in the designing of clinically-relevant vascular substitutes is our lack of understanding on how vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs) interact in the graft. The aim of this study was to examine the factors that play a role in VSMC and VEC interaction in 3D co-culture. Highly porous 3D poly(carbonate urethane) scaffolds were fabricated using a solvent casting and particulate leaching method. VSMCs and VECs were co-cultured for 48 hours. Immunofluorescence staining showed that VSMCs readily attached to the scaffold and formed dense confluent layers which facilitated the organization of VECs of into a monolayer above the VSMC layer. Western blot analysis showed that co-culture induced an up-regulation of the contractile phenotype in VSMCs. A small interfereing RNA knockdown study of Jagged1 established a direct link between Jagged1 expression in VECs and contractile protein expression in VSMCs

Doctor of Philosophy

dissertationChronic heart failure (CHF) is a life-altering long-term condition that contributes a substantial burden to our healthcare system. It is caused by a maladaptive remodeling of the heart mediated through fibroblast synthesis, degradation, and modification of extracellular matrix (ECM). It is currently managed through pharmacologic intervention or medical device treatment, but can be reversed only through heart transplantation. Cell therapy is a new approach to treating CHF that promises to prevent and potentially reverse cardiac remodeling through interaction with cardiac fibroblasts. Adherent bone marrow derived stem cells (MSC) are one of the most promising candidates for use in cell therapies. The major challenge hindering standard clinical application of MSC therapy is limited understanding of how MSC interact with heart cells to reverse remodeling. Numerous techniques are available to harvest, isolate, and modify MSC, however these techniques are believed to influence the efficacy of the treatment. Current techniques for evaluating efficacy of MSC treatments are either prohibitively difficult or significantly limited in their ability to assess functional changes. Establishing an in vitro platform for evaluating the influence of MSC coculture on performance characteristics of cardiac fibroblasts is a logical and efficient step prior to successful clinical implementation of MSC therapy. The objective of this research was to develop a threedimensional (3D) tissue model that allows investigation of the underlying mechanism responsible for MSC mediated cardiac regeneration. The three phases of this work included: (1) development of a biomaterial substrate capable of sustaining fibroblast attachment, proliferation, and alignment, (2) development of a culture platform and seeding techniques capable of providing sufficient mass transport to sustain a relatively thick 3D scaffold populated with both fibroblasts and MSC (3) application of the substrate and culture platform to evaluate changes in mechanical properties and cell distribution resulting from MSC coculture with cardiac fibroblasts