Microscopic matrix remodeling precedes endothelial morphological changes during capillary morphogenesis.

The formation of microvascular networks (MVNs) is influenced by many aspects of the microenvironment, including soluble and insoluble biochemical factors and the biophysical properties of the surrounding matrix. It has also become clear that a dynamic and reciprocal interaction between the matrix and cells influences cell behavior. In particular, local matrix remodeling may play a role in driving cellular behaviors, such as MVN formation. In order to explore the role of matrix remodeling, an in vitro model of MVN formation involving suspending human umbilical vein endothelial cells within collagen hydrogels was used. The resulting cell and matrix morphology were microscopically observed and quantitative metrics of MVN formation and collagen gathering were applied to the resulting images. The macroscopic compaction of collagen gels correlates with the extent of MVN formation in gels of different stiffness values, with compaction preceding elongation leading to MVN formation. Furthermore, the microscopic analysis of collagen between cells at early timepoints demonstrates the alignment and gathering of collagen between individual adjacent cells. The results presented are consistent with the hypothesis that endothelial cells need to gather and align collagen between them as an early step in MVN formation.

Fibers in the extracellular matrix enable long-range stress transmission between cells.

Cells can sense, signal, and organize via mechanical forces. The ability of cells to mechanically sense and respond to the presence of other cells over relatively long distances (e.g., ∼100 µm, or ∼10 cell-diameters) across extracellular matrix (ECM) has been attributed to the strain-hardening behavior of the ECM. In this study, we explore an alternative hypothesis: the fibrous nature of the ECM makes long-range stress transmission possible and provides an important mechanism for long-range cell-cell mechanical signaling. To test this hypothesis, confocal reflectance microscopy was used to develop image-based finite-element models of stress transmission within fibroblast-seeded collagen gels. Models that account for the gel's fibrous nature were compared with homogenous linear-elastic and strain-hardening models to investigate the mechanisms of stress propagation. Experimentally, cells were observed to compact the collagen gel and align collagen fibers between neighboring cells within 24 h. Finite-element analysis revealed that stresses generated by a centripetally contracting cell boundary are concentrated in the relatively stiff ECM fibers and are propagated farther in a fibrous matrix as compared to homogeneous linear elastic or strain-hardening materials. These results support the hypothesis that ECM fibers, especially aligned ones, play an important role in long-range stress transmission.

Stability of a microvessel subject to structural adaptation of diameter and wall thickness.

Vascular adaptation--or structural changes of microvessels in response to physical and metabolic stresses--can influence physiological processes like angiogenesis and hypertension. To better understand the influence of these stresses on adaptation, Pries et al. (1998, 2001a,b, 2005) have developed a computational model for microvascular adaptation. Here, we reformulate this model in a way that is conducive to a dynamical systems analysis. Using th ese analytic methods, we determine the equilibrium geometries of a single vessel under different conditions and classify its type of stability. We demonstrate that our closed-form solution for vessel geometry exhibits the same regions of stability as the numerical predictions of Pries et al. (2005, Remodeling of blood vessels: responses of diameter and wall thickness to hemodynamic and metabolic stimuli. Hypertension, 46, 725-731). Our analytic approach allows us to predict the existence of limit-cycle oscillations and to extend the model to consider a fixed pressure across the vessel in addition to a fixed flow. Under these fixed pressure conditions, we show that the vessel stability is affected and that the multiple equilibria can exist.

Pericellular conditions regulate extent of cell-mediated compaction of collagen gels.

Cell-mediated compaction of the extracellular matrix (ECM) plays a critical role in tissue engineering, wound healing, embryonic development, and many disease states. The ECM is compacted as a result of cellular traction forces. We hypothesize that a cell mechanically remodels the nearby ECM until some target conditions are obtained, and then the cell stops compacting. A key feature of this hypothesis is that ECM compaction primarily occurs in the pericellular region and the properties of the ECM in the pericellular region govern cellular force generation. We developed a mathematical model to describe the amount of macroscopic compaction of cell-populated collagen gels in terms of the initial cell and collagen densities, as well as the final conditions of the pericellular environment (defined as the pericellular volume where the collagen is compacted (V(*)) and the mass of collagen within this volume (m(*))). This model qualitatively predicts the effects of varying initial cell and collagen concentrations on the extent of gel compaction, and by fitting V(*) and m(*), provides reasonable quantitative agreement with the extent of gel compaction observed in experiments with endothelial cells and fibroblasts. Microscopic analysis of compacted gels supports the assumption that collagen compaction occurs primarily in the pericellular environment.

Directed 3D cell alignment and elongation in microengineered hydrogels.

Organized cellular alignment is critical to controlling tissue microarchitecture and biological function. Although a multitude of techniques have been described to control cellular alignment in 2D, recapitulating the cellular alignment of highly organized native tissues in 3D engineered tissues remains a challenge. While cellular alignment in engineered tissues can be induced through the use of external physical stimuli, there are few simple techniques for microscale control of cell behavior that are largely cell-driven. In this study we present a simple and direct method to control the alignment and elongation of fibroblasts, myoblasts, endothelial cells and cardiac stem cells encapsulated in microengineered 3D gelatin methacrylate (GelMA) hydrogels, demonstrating that cells with the intrinsic potential to form aligned tissues in vivo will self-organize into functional tissues in vitro if confined in the appropriate 3D microarchitecture. The presented system may be used as an in vitro model for investigating cell and tissue morphogenesis in 3D, as well as for creating tissue constructs with microscale control of 3D cellular alignment and elongation, that could have great potential for the engineering of functional tissues with aligned cells and anisotropic function.

VASP involvement in force-mediated adherens junction strengthening.

Strengthening of cell-matrix adhesions in response to applied force has been well documented. However, while implied by various lines of evidence, the force-mediated strengthening of cell-cell adhesions has not been directly demonstrated. In the current study, we present results consistent with force strengthening in adherens junctions, obtained by application of different force profiles to VE-cadherin-coated magnetic beads attached to endothelial cells. When force is ramped from a low to high value over time, fewer beads detach than with the immediate application of high force. Cells treated with cytochalasin D or lacking Ena/VASP activity show similar levels of detachment relative to controls, but force strengthening is lost. Further, cells overexpressing VASP show stronger adhesion in response to low and high force, but adhesion weakening in response to ramped forces. These results indicate that force-mediated adhesion strengthening occurs in endothelial adherens junctions and that dynamic VASP activity is necessary for this process.

Ena/VASP is required for endothelial barrier function in vivo.

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are key actin regulators that localize at regions of dynamic actin remodeling, including cellular protrusions and cell-cell and cell-matrix junctions. Several studies have suggested that Ena/VASP proteins are involved in the formation and function of cellular junctions. Here, we establish the importance of Ena/VASP in endothelial junctions in vivo by analysis of Ena/VASP-deficient animals. In the absence of Ena/VASP, the vasculature exhibits patterning defects and lacks structural integrity, leading to edema, hemorrhaging, and late stage embryonic lethality. In endothelial cells, we find that Ena/VASP activity is required for normal F-actin content, actomyosin contractility, and proper response to shear stress. These findings demonstrate that Ena/VASP is critical for actin cytoskeleton remodeling events involved in the maintenance of functional endothelia.

Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis.

Cell migration on 2D surfaces is governed by a balance between counteracting tractile and adhesion forces. Although biochemical factors such as adhesion receptor and ligand concentration and binding, signaling through cell adhesion complexes, and cytoskeletal structure assembly/disassembly have been studied in detail in a 2D context, the critical biochemical and biophysical parameters that affect cell migration in 3D matrices have not been quantitatively investigated. We demonstrate that, in addition to adhesion and tractile forces, matrix stiffness is a key factor that influences cell movement in 3D. Cell migration assays in which Matrigel density, fibronectin concentration, and beta1 integrin binding are systematically varied show that at a specific Matrigel density the migration speed of DU-145 human prostate carcinoma cells is a balance between tractile and adhesion forces. However, when biochemical parameters such as matrix ligand and cell integrin receptor levels are held constant, maximal cell movement shifts to matrices exhibiting lesser stiffness. This behavior contradicts current 2D models but is predicted by a recent force-based computational model of cell movement in a 3D matrix. As expected, this 3D motility through an extracellular environment of pore size much smaller than cellular dimensions does depend on proteolytic activity as broad-spectrum matrix metalloproteinase (MMP) inhibitors limit the migration of DU-145 cells and also HT-1080 fibrosarcoma cells. Our experimental findings here represent, to our knowledge, discovery of a previously undescribed set of balances of cell and matrix properties that govern the ability of tumor cells to migration in 3D environments.

Micropatterned polymer surfaces improve retention of endothelial cells exposed to flow-induced shear stress.

The use of synthetic polymeric vascular grafts is limited by the thrombogenecity of most biomaterials. Efforts to reduce thrombogenicity by seeding grafts with endothelial cells, the natural non-thrombogenic lining of blood vessels, have been thwarted by flow-induced cell detachment. We hypothesized that by creating well-defined micro-textured patterns on a surface, fluid flow at the surface can be altered to create discrete regions of low shear stress. We further hypothesized that, due to reduced shear stress, these regions will serve as sanctuaries for endothelial cells and promote their retention. To test these hypotheses, well-defined micro-textured polyurethane (PU) surfaces consisting of arrays of parallel 95-micron wide and 32-micron deep channels were created using an etched silicon template and solvent casting techniques. Based on computational fluid dynamics, under identical bulk flow conditions, the average local shear stress in the channels (46 dyn/cm2) was 28% lower than unpatterned surfaces (60 dyn/cm2). When PU surfaces pre-seeded with endothelial cells (EC) were exposed to the same bulk flow rate, EC retention was significantly improved on the micropatterned surfaces relative to un-patterned surfaces (92% vs. 58% retention).

Self-assembling short oligopeptides and the promotion of angiogenesis.

Because an adequate blood supply to and within tissues is an essential factor for successful tissue regeneration, promoting a functional microvasculature is a crucial factor for biomaterials. In this study, we demonstrate that short self-assembling peptides form scaffolds that provide an angiogenic environment promoting long-term cell survival and capillary-like network formation in three-dimensional cultures of human microvascular endothelial cells. Our data show that, in contrast to collagen type I, the peptide scaffold inhibits endothelial cell apoptosis in the absence of added angiogenic factors, accompanied by enhanced gene expression of the angiogenic factor VEGF. In addition, our results suggest that the process of capillary-like network formation and the size and spatial organization of cell networks may be controlled through manipulation of the scaffold properties, with a more rigid scaffold promoting extended structures with a larger inter-structure distance, as compared with more dense structures of smaller size observed in a more compliant scaffold. These findings indicate that self-assembling peptide scaffolds have potential for engineering vascularized tissues with control over angiogenic processes. Since these peptides can be modified in many ways, they may be uniquely valuable in regeneration of vascularized tissues.

Salmon fibrin supports an increased number of sprouts and decreased degradation while maintaining sprout length relative to human fibrin in an in vitro angiogenesis model.

Salmon-derived fibrin has been proposed as a preferred alternative to human or bovine fibrin because of its reduced potential for disease transmission. Here we evaluate salmon fibrin as an alternative ECM support for therapeutic angiogenesis applications, such as vascularizing engineered tissues. Human umbilical vein endothelial cells (HUVEC) seeded on gelatin beads and suspended in either salmon or human fibrin sprouted and formed capillary-like structures. Sprout length was generally increased with the addition of bFGF and VEGF and further increased with the addition of phorbol myristate acetate (PMA). The number of sprouts per bead was increased 61-188% in salmon fibrin relative to human fibrin (alpha < 0.0005) in cultures receiving growth factors and PMA, while average sprout lengths were similar for HUVEC within human or salmon fibrin. Additionally, under these conditions in the absence of a protease inhibitor, HUVEC appeared to degrade human, but not salmon, fibrin. These results support the idea that salmon fibrin may be an attractive alternative ECM able to support microvascular network formation.

Differential effects of growth factors on tissue-engineered cartilage.

The effects of four regulatory factors on tissue-engineered cartilage were examined with specific focus on the ability to increase construct growth rate and concentrations of glycosaminoglycans (GAG) and collagen, the major extracellular matrix (ECM) components. Bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid (PGA) scaffolds and cultured in medium with or without supplemental insulin-like growth factor (IGF-I), interleukin-4 (IL-4), transforming growth factor-beta1 (TGF-beta1) or platelet-derived growth factor (PDGF). IGF-I, IL-4, and TGF-beta1 increased construct wet weights by 1.5-2.9-fold over 4 weeks of culture and increased amounts of cartilaginous ECM components. IGF-I (10-300 ng/mL) maintained wet weight fractions of GAG in constructs seeded at high cell density and increased by up to fivefold GAG fractions in constructs seeded at lower cell density. TGF-beta1 (30 ng/mL) increased wet weight fractions of total collagen by up to 1.4-fold while maintaining a high fraction of type II collagen (79 plus minus 11% of the total collagen). IL-4 (1-100 ng/mL) minimized the thickness of the GAG-depleted region at the construct surfaces. PDGF (1-100 ng/mL) decreased construct growth rate and ECM fractions. Different regulatory factors thus elicit significantly different chondrogenic responses and can be used to selectively control the growth rate and improve the composition of engineered cartilage.