Fotios M. Andreopoulos

Light-induced tailoring of PEG-hydrogel properties

We have previously reported (Andreopoulos et al. J Am Chem Soc 118 (1996) 6235-6240) the synthesis of hydrogels via the photopolymerization of water-soluble PEG molecules. In this paper, PEG-hydrogel membranes were prepared by the irradiation (> 300 nm) of aqueous solutions of photosensitive 4-armed PEG (nominal molecular weight of 20000), in the absence of photo-initiators. The hydroxyl termini of the PEGs were functionalized with cinnamylidene acetate groups to form photosensitive PEG macromers (PEG-CA), which upon irradiation (>300 nm) formed crosslinks between adjacent cinnamylidene groups resulting in highly crosslinked networks (hydrogels) (Andreopoulos et al. J Am Chem Soc 118 (1996) 6235-6240). The hydrogel membranes were highly swellable with equilibrium volume fractions ranging from 0.02 to 0.05. Their swellability was a function of irradiation light (>300 nm) and degree of modification of the PEG molecules. The effect of light on the permeation fluxes of myoglobin (Mb), hemoglobin (Hb), and lactate dehydrogenase-L (LDH) through PEG membranes was also assessed and the diffusion coefficients of the proteins were determined accordingly. The PEG-CA membranes exhibited photoscissive behavior upon exposure to UV irradiation (254 nm). Therefore, UV light was used as a trigger to control the mesh size of the membranes, and thereby the permeation fluxes of Mb, Hb, and LDH. Equilibrium swelling experiments with membranes prepared under different irradiation conditions were performed, and the Flory-Huggins model was utilized to determine the mesh size and the average molecular weight between crosslinks of the synthesized hydrogels.

bFGF-containing electrospun gelatin scaffolds with controlled nano-architectural features for directed angiogenesis

Current therapeutic angiogenesis strategies are focused on the development of biologically responsive scaffolds that can deliver multiple angiogenic cytokines and/or cells in ischemic regions. Herein, we report on a novel electrospinning approach to fabricate cytokine-containing nanofibrous scaffolds with tunable architecture to promote angiogenesis. Fiber diameter and uniformity were controlled by varying the concentration of the polymeric (i.e. gelatin) solution, the feed rate, needle to collector distance, and electric field potential between the collector plate and injection needle. Scaffold fiber orientation (random vs. aligned) was achieved by alternating the polarity of two parallel electrodes placed on the collector plate thus dictating fiber deposition patterns. Basic fibroblast growth factor (bFGF) was physically immobilized within the gelatin scaffolds at variable concentrations and human umbilical vein endothelial cells (HUVEC) were seeded on the top of the scaffolds. Cell proliferation and migration was assessed as a function of growth factor loading and scaffold architecture. HUVECs successfully adhered onto gelatin B scaffolds and cell proliferation was directly proportional to the loading concentrations of the growth factor (0-100 bFGF ng/mL). Fiber orientation had a pronounced effect on cell morphology and orientation. Cells were spread along the fibers of the electrospun scaffolds with the aligned orientation and developed a spindle-like morphology parallel to the scaffolds fibers. In contrast, cells seeded onto the scaffolds with random fiber orientation, did not demonstrate any directionality and appeared to have a rounder shape. Capillary formation (i.e. sprouts length and number of sprouts per bead), assessed in a 3-D in vitro angiogenesis assay, was a function of bFGF loading concentration (0 ng, 50 ng and 100 ng per scaffold) for both types of electrospun scaffolds (i.e. with aligned or random fiber orientation).

Co-delivery of FGF-2 and G-CSF from gelatin-based hydrogels as angiogenic therapy in a murine critical limb ischemic model

Peripheral artery disease and critical limb ischemia have become prevalent health risks in the United States due to an increasing elderly population and the prevalence of obesity and diabetes mellitus. Although highly invasive endarterectomy is the most popular method for treatment, angiogenic therapies based on growth factor administration are quickly becoming a popular alternative. Enzymatic degradation of these factors in vivo may be avoided by their incorporation in a delivery vehicle where the growth factors release rate can be controlled by altering the vehicles properties (i.e. cross-linking density, material selection, biodegradation, etc.). Herein, we report on the immobilization and controlled release of human recombinant basic fibroblast growth factor (FGF-2) and human recombinant granulocyte colony-stimulating factor (G-CSF) from ionic, gelatin-based hydrogel scaffolds to re-establish perfusion and induce capillary outgrowth in a murine hindlimb ischemic model. In vitro studies showed that endothelial cell proliferation was highly depended on FGF-2, whereas G-CSF stimulated migration and formation of a tubular network. When FGF-2 and G-CSF were used in combination there was an 82% increase in endothelial branch point formation compared to control groups. Leg reperfusion was assessed with laser Doppler perfusion imaging, while capillary outgrowth in the ischemic leg was evaluated using CD31(+) and alpha-SMA immunostaining. The co-delivery of G-CSF (1000 ngml(-1)) and FGF-2 (1000 ng ml(-1)) from the gelatin hydrogels resulted in a 3-fold increase in the perfusion levels and a 2-fold increase in capillary density and positive alpha-SMA vessels compared to the empty vehicle group. In conclusion, the co-delivery of FGF-2 and G-CSF was superior to bolus administration or the delivery of either factor alone in promoting reperfusion and mature vessel formation.

A Novel Photoscissile Poly(ethylene glycol)‐Based Hydrogel

drawbacks, such as slow gelation rate, need for potentially toxic initiators, and thermal or storage instability. In addition, a limited number of these photoinduced systems demonstrate photoreversibility. [2,3,9,10] Andreopoulos et al. [3] have synthesized a partially reversible hydrogel via photopolymerization of cinnamylidene-terminated PEG. The physical properties of the hydrogel membrane such as pore size and swellability were controlled in a predictive way by alternating the wavelength (>300/254 nm) and sequence of irradiation. The photoreversibility efficiency of the PEG-cinnamylidene hydrogel, however, was compromised by photoscission light inefficiency, cinnamylidene photodegradation, and side polymerization reactions. [10] In the work reported here, we designed a new photocrosslinked and photoscissile hydrogel based on an eight-branched PEG with nitrocinnamate as pendant groups. Cinnamate is known to undergo trans‐cis isomerization and [2+2] cycloaddition upon UV irradiation at wavelengths longer than 290 nm, and the formed cyclobutane ring can be cleaved to regenerate the starting cinnamate groups at wavelengths below 260 nm. [11] The photocrosslinking property of cinnamates has been broadly utilized in the field of photolithography and the semiconductor industry. [12] However, the application of nitrocinnamate photoreactivity in PEG chemistry, especially in PEG-based hydrogel formation, has rarely been reported. [6,13] On the other hand, cinnamate derivatives possess excellent thermal and storage stability superior to cinnamylidene systems. [14] PEG-cinnamylidene polymers have been shown to be unstable and undergo gelation when they are kept at room temperature for a period of a few weeks. Nitrocinnamate demonstrates the stability characteristics of the unsubstituted cinnamate and at the same time is 350 times more photoreactive. [11,15]

Photoimmobilization of organophosphorus hydrolase within a PEG-based hydrogel

Organophosphorous hydrolase (OPH) was physically and covalently immobilized within photosensitive polyethylene glycol (PEG)-based hydrogels. The hydroxyl ends of branched polyethylene glycol (b-PEG, four arms, MW = 20,000) were modified with cinnamylidene acetate groups to give water-soluble, photosensitive PEG macromers (b-PEG-CA). The b-PEG-CA macromers underwent photocrosslinking reaction and formed gels upon UV irradiation (>300 nm) in the presence of erythrosin B. Native OPH was pegylated with cinnamylidene-terminated PEG chains (MW = 3400) to be covalently linked with the b-PEG-CA macromers during photogelation. The effect of pegylation on the stability of the enzyme was determined. Furthermore, the effect of enzyme concentration, wavelength of irradiation, and photosensitizer on the stability of the entrapped enzyme was also investigated. The pegylated OPH was more stable than the native enzyme, and the OPH-containing gels exhibited superior stability than the soluble enzyme preparations.

Tissue engineering the retinal ganglion cell nerve fiber layer.

Retinal degenerative diseases, such as glaucoma and macular degeneration, affect millions of people worldwide and ultimately lead to retinal cell death and blindness. Cell transplantation therapies for photoreceptors demonstrate integration and restoration of function, but transplantation into the ganglion cell layer is more complex, requiring guidance of axons from transplanted cells to the optic nerve head in order to reach targets in the brain. Here we create a biodegradable electrospun (ES) scaffold designed to direct the growth of retinal ganglion cell (RGC) axons radially, mimicking axon orientation in the retina. Using this scaffold we observed an increase in RGC survival and no significant change in their electrophysiological properties. When analyzed for alignment, 81% of RGCs were observed to project axons radially along the scaffold fibers, with no difference in alignment compared to the nerve fiber layer of retinal explants. When transplanted onto retinal explants, RGCs on ES scaffolds followed the radial pattern of the host retinal nerve fibers, whereas RGCs transplanted directly grew axons in a random pattern. Thus, the use of this scaffold as a cell delivery device represents a significant step towards the use of cell transplant therapies for the treatment of glaucoma and other retinal degenerative diseases.

Enhanced angiogenic efficacy through controlled and sustained delivery of FGF-2 and G-CSF from fibrin hydrogels containing ionic-albumin microspheres.

Neo-vessel formation in ischemic tissues relies on numerous growth factors and cell fractions for the formation of mature, stable, functional vasculature. However, the efforts to regenerate tissues typically rely on the administration of a single growth factor or cells alone. Conversely, polymeric matrices have been investigated extensively to deliver multiple growth factors at pre-determined rates to form stable blood vessels in ischemic tissues. We report on a novel sequential delivery system of a fibrin hydrogel containing ionic-albumin microspheres that allows for the controlled release of two growth factors. The use of this system was investigated in the context of therapeutic angiogenesis. Material properties were determined based on degree of swelling measurements and degradation characteristics. Release kinetics of model angiogenic polypeptides FGF-2 and G-CSF were determined using ELISA and the bioactivity of released protein was evaluated in human endothelial cell cultures. The release of growth factors from ionic-albumin microspheres was significantly delayed compared to the growth factor released from fibrin matrices in the absence of spheres. The scaffolds were implanted in a murine critical limb ischemia model at two concentrations, 40 ng (low) and 400 ng (high), restoring 92% of the blood flow in a normally perfused limb using a fibrin hydrogel releasing FGF-2 containing albumin–PLL microspheres releasing G-CSF (measured by LDPI at the high concentration), a 3.2-fold increase compared to untreated limbs. The extent of neo-vessel formation was delineated by immunohistochemical staining for capillary density (CD-31+) and mature vessel formation (α-SMA+). In conclusion, our study demonstrated that the release kinetics from our scaffold have distinct kinetics previously unpublished and the delivery of these factors resulted in hindlimb reperfusion, and robust capillary and mature vessel formation after 8 weeks compared to either growth factor alone or bolus administration of growth factor.

Comparative studies of surface topography and mechanical properties of a new, photo-switchable PEG-based hydrogel

Abstract We have recently synthesized a novel nitrocinnamate-modified poly(ethylene glycol) hydrogel, further referred to as PEG–NC hydrogel, via photo-crosslinking of the nitrocinnamoyl groups. The practical advantage of photo-gelation is that it allows facile control of the gelation process and thereby properties of formed hydrogel in situ. In this paper, we present an investigation of the physico-chemical properties of the photo-sensitive PEG–NC hydrogel. Using environmental scanning electron microscopy (ESEM) and atomic force scans (AFM force scans) techniques, we explored the changes in surface topography and mechanical properties of this new photo-switchable hydrogel in its different stages, i.e. photo-crosslinked and photo-cleaved, and a hydrogel created by crosslinking of the previously photo-cleaved one. We have observed distinct differences in both the surface topography and the mechanical properties between the photo-crosslinked and photo-cleaved stages of the hydrogel, and have demonstrated this to be a reversible process. NMR experiments were also carried out to illustrate the photochemical process. We believe that this novel, potentially biocompatible hydrogel could have biomedical applications, especially in the areas of wound healing, surgical implants, tissue engineering and artificial muscles.

Retinal ganglion cell polarization using immobilized guidance cues on a tissue-engineered scaffold

Cell transplantation therapies to treat diseases related to dysfunction of retinal ganglion cells (RGCs) are limited in part by an inability to navigate to the optic nerve head within the retina. During development, RGCs are guided by a series of neurotrophic factors and guidance cues; however, these factors and their receptors on the RGCs are developmentally regulated and often not expressed during adulthood. Netrin-1 is a guidance factor capable of guiding RGCs in culture and relevant to guiding RGC axons toward the optic nerve head in vivo. Here we immobilized Netrin-1 using UV-initiated crosslinking to form a gradient capable of guiding the axonal growth of RGCs on a radial electrospun scaffold. Netrin-gradient scaffolds promoted both the percentage of RGCs polarized with a single axon, and also the percentage of cells polarized toward the scaffold center, from 31% to 52%. Thus, an immobilized protein gradient on a radial electrospun scaffold increases RGC axon growth in a direction consistent with developmental optic nerve head guidance, and may prove beneficial for use in cell transplant therapies for the treatment of glaucoma and other optic neuropathies.

Initial experience with the tandemheart circulatory support system in children

Options for mechanical ventricular assistance in pediatric patients are limited. Extracorporeal membrane oxygenation is used in most cases for short-term support. The TandemHeart circulatory support system is an established device that is used in adult patients to provide short-term ventricular support. In this article, we report three children in whom a TandemHeart ventricular assist device was used for right ventricular support, two after heart transplantation and another for failed Fontan physiology. Herein, we report the novel application of this technology to pediatric patients, and we discuss the lessons learned from its utilization.