PlGFMMP9-engineered iPS cells supported on a PEGfibrinogen hydrogel scaffold possess an enhanced capacity to repair damaged myocardium

Cell-based regenerative therapies are significantly improved by engineering allografts to express factors that increase vascularization and engraftment, such as placental growth factor (PlGF) and matrix metalloproteinase 9 (MMP9). Moreover, the seeding of therapeutic cells onto a suitable scaffold is of utmost importance for tissue regeneration. On these premises, we sought to assess the reparative potential of induced pluripotent stem (iPS) cells bioengineered to secrete PlGF or MMP9 and delivered to infarcted myocardium upon a poly(ethylene glycol)-fibrinogen scaffold. When assessing optimal stiffness of the PEG-fibrinogen (PF) scaffold, we found that the appearance of contracting cells after cardiogenic induction was accelerated on the support designed with an intermediate stiffness. Revascularization and hemodynamic parameters of infarcted mouse heart were significantly improved by injection into the infarct of this optimized PF scaffold seeded with both MiPS (iPS cells engineered to secrete MMP9) and PiPS (iPS cells engineered to secrete PlGF) cells as compared with nonengineered cells or PF alone. Importantly, allograft-derived cells and host myocardium were functionally integrated. Therefore, survival and integration of allografts in the ischemic heart can be significantly improved with the use of therapeutic cells bioengineered to secrete MMP9 and PlGF and encapsulated within an injectable PF hydrogel having an optimized stiffness

Visualizing cell-laden fibrin-based hydrogels using cryogenic scanning electron microscopy and confocal microscopy

The present investigation explores the microscopic aspects of cell-laden hydrogels at high resolutions, using three-dimensional cell cultures in semi-synthetic constructs that are of very high water content (>98% water). The study aims to provide an imaging strategy for these constructs, while minimizing artefacts. Constructs of poly(ethylene glycol)-fibrinogen and fibrin hydrogels containing embedded mesenchymal cells (human dermal fibroblasts) were first imaged by confocal microscopy. Next, high-resolution scanning electron microscopy (HR-SEM) was used to provide images of the cells within the hydrogels, at submicron resolutions. Because it was not possible to obtain artefact-free images of the hydrogels using room-temperature HR-SEM, a cryogenic HR-SEM imaging methodology was employed to visualize the sample while preserving the natural hydrated state of the hydrogel. The ultrastructural details of the constructs were observed at subcellular resolutions, revealing numerous cellular components, the biomaterial in its native configuration, and the uninterrupted cell membrane as it relates with the biomaterial in the hydrated state of the construct. Constructs containing microscopic albumin microbubbles were also imaged using these methodologies to reveal fine details of the interaction between the cells, the microbubbles, and the hydrogel. Taken together with the confocal microscopy, this imaging strategy provides a more complete picture of the hydrated state of the hydrogel network with cells inside. As such, this methodology addresses some of the challenges of obtaining this information in amorphous hydrogel systems containing a very high water content (>98%) with embedded cells. Such insight may lead to better hydrogel-based strategies for tissue engineering and regeneration

PlGFMMP9-engineered iPS cells supported on a PEGfibrinogen hydrogel scaffold possess an enhanced capacity to repair damaged myocardium

Cell-based regenerative therapies are significantly improved by engineering allografts to express factors that increase vascularization and engraftment, such as placental growth factor (PlGF) and matrix metalloproteinase 9 (MMP9). Moreover, the seeding of therapeutic cells onto a suitable scaffold is of utmost importance for tissue regeneration. On these premises, we sought to assess the reparative potential of induced pluripotent stem (iPS) cells bioengineered to secrete PlGF or MMP9 and delivered to infarcted myocardium upon a poly(ethylene glycol)fibrinogen scaffold. When assessing optimal stiffness of the PEGfibrinogen (PF) scaffold, we found that the appearance of contracting cells after cardiogenic induction was accelerated on the support designed with an intermediate stiffness. Revascularization and hemodynamic parameters of infarcted mouse heart were significantly improved by injection into the infarct of this optimized PF scaffold seeded with both MiPS (iPS cells engineered to secrete MMP9) and PiPS (iPS cells engineered to secrete PlGF) cells as compared with nonengineered cells or PF alone. Importantly, allograft-derived cells and host myocardium were functionally integrated. Therefore, survival and integration of allografts in the ischemic heart can be significantly improved with the use of therapeutic cells bioengineered to secrete MMP9 and PlGF and encapsulated within an injectable PF hydrogel having an optimized stiffness. © 2014 Macmillan Publishers Limited

Fibrinogen-Based Hydrogel Modulus and Ligand Density Effects on Cell Morphogenesis in Two-Dimensional and Three-Dimensional Cell Cultures

There is a need to further explore the convergence of mechanobiology and dimensionality with systematic investigations of cellular response to matrix mechanics in 2D and 3D cultures. Here, a semisynthetic hydrogel capable of supporting both 2D and 3D cell culture is applied to investigate cell response to matrix modulus and ligand density. The culture materials are fabricated from adducts of polyethylene glycol (PEG) or PluronicF127 and fibrinogen fragments, formed into hydrogels by free-radical polymerization, and characterized by shear rheology. Control over the modulus of the materials is accomplished by changing the concentration of synthetic PEG-diacrylate crosslinker (0.5% w/v), and by altering the molecular length of the PEG (10 and 20 kDa). Control over ligand density is accomplished by changing fibrinogen concentrations from 3 to 12 mg mL-1 . In 2D culture, cell motility parameters, including cell speed and persistence time are significantly increased with increasing modulus. In both 2D and 3D culture, cells express vinculin and there is evidence of focal adhesion formation in the high stiffness materials. The modulus- and ligand-dependent morphogenesis response from the cells in 2D culture is contradictory to the same measured response in 3D culture. In 2D culture, anchorage-dependent cells become more elongated and significantly increase their size with increasing ligand density and matrix modulus. In 3D culture, the same anchorage-dependent cells become less spindled and significantly reduce their size in response to increasing ligand density and matrix modulus. These differences arise from dimensionality constraints, most notably the encapsulation of cells in a non-porous hydrogel matrix. These insights underscore the importance of mechanical properties in regulating cell morphogenesis in a 3D culture milieu. The versatility of the hydrogel culture environment further highlights the significance of a modular approach when developing materials that aim to optimize the cell culture environment