During angiogenesis, endothelial cells (ECs) degrade their surrounding extracellular matrix (ECM) to facilitate invasion. How interactions between ECs and other cells within their microenvironment facilitate this process is only partially understood. We have utilized a tractable 3D co-culture model to investigate the proteolytic mechanisms by which pre-committed or more highly committed mesenchymal cells stimulate capillary formation. On their own, ECs invade their surrounding matrix, but do not form capillaries. However, in the presence of either mesenchymal stem cells (MSCs) or fibroblasts, ECs form polarized, tubular structures that are intimately associated with mesenchymal cells. Further, ECs upregulate gene expression of several extracellular proteases upon co-culture with either mesenchymal cell type. The administration of both broad spectrum and specific protease inhibitors demonstrated that MSC-stimulated capillary formation relied solely on membrane-type matrix metalloproteinases (MT-MMPs) while fibroblast-mediated sprouting proceeded independent of MMP inhibition unless the plasminogen activator/plasmin axis was inhibited in concert. While other studies have established a role for the ECM itself in dictating proteolysis and matrix degradation during capillary morphogenesis, the present study illustrates that heterotypic cellular interactions within the microenvironment can direct the proteolytic mechanisms required for capillary formation.

Identifying the mechanisms regulating angiogenesis in pathological conditions such as cancer and heart disease is crucial to develop successful therapies. The dependence of angiogenesis on characteristic properties of these conditions, such as alterations in tissue stiffness due to changes in the composition of the extracellular matrix (ECM), may shed light on potential therapeutic strategies. Prior studies have suggested that ECM compliance regulates capillary morphogenesis, but the mechanisms remain unclear. In this study, we hypothesized that ECM density, which influences substrate mechanics, may regulate angiogenesis via a mechanism involving actin-mediated cell-generated forces. To investigate this hypothesis, we utilized an in vitro model of angiogenesis in which endothelial cells coated on microcarrier beads are distributed within a three-dimensional (3-D) fibrin ECM. A monolayer of fibroblasts, which provides pro-angiogenic factors, is cultured on top of the gel. Variations in fibrin gel density, along with a library of pharmacological agents that inhibit forces generated by the actin cytoskeleton, were used to prove the necessity of cell-generated tractional forces in blood vessel formation. Our data demonstrate that cell-generated forces not only play a crucial role in the early sprouting stages of capillary morphogenesis but are also required in the later maintenance stages, and thereby suggest a broader interdependence among tissue stiffness, cell contractile forces, and angiogenesis.

Stem cell niches are composed of numerous microenvironmental features, including soluble and insoluble factors, cues from other cells, and the extracellular matrix (ECM), which collectively serve to maintain stem cell quiescence and promote their ability to support tissue homeostasis. A hallmark of many adult stem cell niches is their proximity to the vasculature in vivo, a feature common to neural stem cells, mesenchymal stem cells (MSCs) from bone marrow and adipose tissue, hematopoietic stem cells, and many tumor stem cells. In this study, we describe a novel 3D microfluidic device (MFD) as a model system in which to study the molecular regulation of perivascular stem cell niches. Endothelial cells (ECs) suspended within 3D fibrin gels patterned in the device adjacent to stromal cells (either fibroblasts or bone marrow-derived mesenchymal stem cells) executed a morphogenetic process akin to vasculogenesis, forming a primitive vascular plexus and maturing into a robust capillary network with hollow well-defined lumens. Both MSCs and fibroblasts formed pericytic associations with the ECs, but promoted capillary morphogenesis with distinct kinetics. Biochemical assays within the niche revealed that the perivascular association of MSCs required interaction between their α6β1 integrin receptor and EC-deposited laminin. These studies demonstrate the potential of this physiologically relevant ex vivo model system to study how proximity to blood vessels may influence stem cell multipotency.

The gonadotoxic effects of chemotherapy and radiation may result in premature ovarian failure in premenopausal oncology patients. Although autotransplantation of ovarian tissue has led to successful live births, reintroduction of latent malignant cells inducing relapse is a significant concern. In this report, we investigated the design of biomaterial grafts for transplantation of isolated ovarian follicles as a means to preserve fertility. Primordial and primary ovarian follicles from young female mice were extracted and encapsulated into biomaterials for subsequent transplantation into adult mice. Among the formulations tested, aggregated follicles encapsulated within fibrin had enhanced survival and integration with the host tissue following transplantation relative to the fibrin-alginate and fibrin-collagen composites. All mice transplanted with fibrin-encapsulated follicles resumed cycling, and live births were achieved only for follicles transplanted within VEGF-loaded fibrin beads. The extent to which these procedures reduce the presence of metastatic breast cancer cells among the isolated follicles was evaluated, with significantly reduced numbers of cancer cells present relative to intact ovaries. This ability to obtain live births by transplanting isolated primordial and primary follicles, while also reducing the risk of re-seeding disease relative to ovarian tissue transplantation, may ultimately provide a means to preserve fertility in premenopausal oncology patients.

Methods for tuning extracellular matrix (ECM) mechanics in 3D cell culture that rely on increasing the concentration of either protein or cross-linking molecules fail to control important parameters such as pore size, ligand density, and molecular diffusivity. Alternatively, ECM stiffness can be modulated independently from protein concentration by mechanically loading the ECM. We have developed a novel device for generating stiffness gradients in naturally derived ECMs, where stiffness is tuned by inducing strain, while local mechanical properties are directly determined by laser tweezers based active microrheology (AMR). Hydrogel substrates polymerized within 35 mm diameter Petri dishes are strained non-uniformly by the precise rotation of an embedded cylindrical post, and exhibit a position-dependent stiffness with little to no modulation of local mesh geometry. Here we present the device in the context of fibrin hydrogels. First AMR is used to directly measure local micromechanics in unstrained hydrogels of increasing fibrin concentration. Changes in stiffness are then mapped within our device, where fibrin concentration is held constant. Fluorescence confocal imaging and orbital particle tracking are used to quantify structural changes in fibrin on the micro and nano levels respectively. The micromechanical strain stiffening measured by microrheology is not accompanied by ECM microstructural changes under our applied loads, as measured by confocal microscopy. However, super-resolution orbital tracking reveals nanostructural straightening, lengthening, and reduced movement of fibrin fibers. Furthermore, we show that aortic smooth muscle cells cultured within our device are morphologically sensitive to the induced mechanical gradient. Our results demonstrate a powerful cell culture tool that can be used in the study of mechanical effects on cellular physiology in naturally derived 3D ECM tissues.