Painting blood vessels and atherosclerotic plaques with an adhesive drug depot.

The treatment of diseased vasculature remains challenging, in part because of the difficulty in implanting drug-eluting devices without subjecting vessels to damaging mechanical forces. Implanting materials using adhesive forces could overcome this challenge, but materials have previously not been shown to durably adhere to intact endothelium under blood flow. Marine mussels secrete strong underwater adhesives that have been mimicked in synthetic systems. Here we develop a drug-eluting bioadhesive gel that can be locally and durably glued onto the inside surface of blood vessels. In a mouse model of atherosclerosis, inflamed plaques treated with steroid-eluting adhesive gels had reduced macrophage content and developed protective fibrous caps covering the plaque core. Treatment also lowered plasma cytokine levels and biomarkers of inflammation in the plaque. The drug-eluting devices developed here provide a general strategy for implanting therapeutics in the vasculature using adhesive forces and could potentially be used to stabilize rupture-prone plaques.

A new model for in vitro testing of vitreous substitute candidates.

PURPOSE: To describe a new model for in vitro assessment of novel vitreous substitute candidates. METHODS: The biological impact of three vitreous substitute candidates was explored in a retinal explant culture model; a polyalkylimide hydrogel (Bio-Alcamid®), a two component hydrogel of 20 wt.% poly (ethylene glycol) in phosphate buffered saline (PEG) and a cross-linked sodium hyaluronic acid hydrogel (Healaflow®). The gels where applied to explanted adult rat retinas and then kept in culture for 2, 5 and 10 days. Gel-exposed explants were compared with explants incubated under standard tissue culture conditions. Cryosections of the specimens were stained with hematoxylin and eosin, immunohistochemical markers (GFAP, Vimentin, Neurofilament 160, PKC, Rhodopsin) and TUNEL. RESULTS: Explants kept under standard conditions as well as PEG-exposed explants displayed disruption of retinal layers with moderate pyknosis of all neurons. They also displayed moderate labeling of apoptotic cells. Bio-Alcamid®-exposed explants displayed severe thinning and disruption of retinal layers with massive cell death. Healaflow®-treated explants displayed normal retinal lamination with significantly better preservation of retinal neurons compared with control specimens, and almost no signs of apoptosis. Retinas exposed to Healaflow® and retinas kept under standard conditions showed variable labeling of GFAP with generally low expression and some areas of upregulation. PEG-exposed retinas showed increased GFAP labeling and Bio-Alcamid®-exposed retinas showed sparse labeling of GFAP. CONCLUSIONS: Research into novel vitreous substitutes has important implications for both medical and surgical vitreoretinal disease. The in vitro model presented here provides a method of biocompatibility testing prior to more costly and cumbersome in vivo experiments. The explant culture system imposes reactions within the retina including disruption of layers, cell death and gliosis, and the progression of these reactions can be used for comparison of vitreous substitute candidates. Bio-Alcamid® had strong adverse effects on the retina which is consistent with results of prior in vivo trials. PEG gel elicits reactions similar to the control retinas whereas Healaflow® shows protection from culture-induced trauma indicating favorable biocompatibility.

Blockade of peroxynitrite-induced neural stem cell death in the acutely injured spinal cord by drug-releasing polymer.

Therapeutic impact of neural stem cells (NSCs) for acute spinal cord injury (SCI) has been limited by the rapid loss of donor cells. Neuroinflammation is likely the cause. As there are close temporal-spatial correlations between the inducible nitric oxide (NO) synthase expression and the donor NSC death after neurotrauma, we reasoned that NO-associated radical species might be the inflammatory effectors which eliminate NSC grafts and kill host neurons. To test this hypothesis, human NSCs (hNSCs: 5 x 10(4) to 2 x 10(6) per milliliter) were treated in vitro with "plain" medium, 20 microM glutamate, or donors of NO and peroxynitrite (ONOO(-); 100 and 400 microM of spermine or DETA NONOate, and SIN-1, respectively). hNSC apoptosis primarily resulted from SIN-1 treatment, showing ONOO(-)-triggered protein nitration and the activation of p38 MAPK, cytochrome c release, and caspases. Therefore, cell death following post-SCI (p.i.) NO surge may be mediated through conversion of NO into ONOO(-). We subsequently examined such causal relationship in a rat model of dual penetrating SCI using a retrievable design of poly-lactic-co-glycolic acid (PLGA) scaffold seeded with hNSCs that was shielded by drug-releasing polymer. Besides confirming the ONOO(-)-induced cell death signaling, we demonstrated that cotransplantation of PLGA film embedded with ONOO(-) scavenger, manganese (III) tetrakis (4-benzoic acid) porphyrin, or uric acid (1 micromol per film), markedly protected hNSCs 24 hours p.i. (total: n = 10). Our findings may provide a bioengineering approach for investigating mechanisms underlying the host microenvironment and donor NSC interaction and help formulate strategies for enhancing graft and host cell survival after SCI.

Biodegradable scaffolds promote tissue remodeling and functional improvement in non-human primates with acute spinal cord injury.

Tissue loss significantly reduces the potential for functional recovery after spinal cord injury. We previously showed that implantation of porous scaffolds composed of a biodegradable and biocompatible block copolymer of Poly-lactic-co-glycolic acid and Poly-l-lysine improves functional recovery and reduces spinal cord tissue injury after spinal cord hemisection injury in rats. Here, we evaluated the safety and efficacy of porous scaffolds in non-human Old-World primates (Chlorocebus sabaeus) after a partial and complete lateral hemisection of the thoracic spinal cord. Detailed analyses of kinematics and muscle activity revealed that by twelve weeks after injury fully hemisected monkeys implanted with scaffolds exhibited significantly improved recovery of locomotion compared to non-implanted control animals. Twelve weeks after injury, histological analysis demonstrated that the spinal cords of monkeys with a hemisection injury implanted with scaffolds underwent appositional healing characterized by a significant increase in remodeled tissue in the region of the hemisection compared to non-implanted controls. The number of glial fibrillary acidic protein immunopositive astrocytes was diminished within the inner regions of the remodeled tissue layer in treated animals. Activated macrophage and microglia were present diffusely throughout the remodeled tissue and concentrated at the interface between the preserved spinal cord tissue and the remodeled tissue layer. Numerous unphosphorylated neurofilament H and neuronal growth associated protein positive fibers and myelin basic protein positive cells may indicate neural sprouting inside the remodeled tissue layer of treated monkeys. These results support the safety and efficacy of polymer scaffolds in a primate model of acute spinal cord injury. A device substantially similar to the device described here is the subject of an ongoing human clinical trial.

Evaluation of viscoelastic poly(ethylene glycol) sols as vitreous substitutes in an experimental vitrectomy model in rabbits.

The aim of this study was to employ an experimental protocol for in vivo evaluation of sols of 5 wt.% poly(ethylene glycol) (PEG) in phosphate-buffered saline as artificial vitreous substitutes. A 20 gauge pars plana vitrectomy and posterior vitreous detachment were performed in the right eye of eight pigmented rabbits. Approximately 1 ml of the viscoelastic PEG sols was then injected into the vitreous space of six eyes. PEG with an average molecular weight of 300,000 and 400,000 g mol(-1) was used in two and four eyes, respectively. Two eyes received balanced salt solution and served as controls. Full-field electroretinography was carried out and intra-ocular pressure (IOP, palpation) measured pre- and post-operatively at regular intervals up to 41 days. The rabbits were killed and the eyes examined by retinal photography, gross macroscopic examination and histology. The viscoelastic sols were successfully injected and remained translucent throughout the post-operative period, with some inferior formation of precipitates. None of the eyes displayed IOP elevation post-operatively, but in three of the PEG sol injected eyes transient hypotony was noted. One eye sustained retinal detachment during surgery and another two in the post-operative period. ERG recordings confirmed preservation of retinal function in three out of four eyes injected with 400,000 g mol(-1) PEG. Histological examination revealed up-regulation of glial acidic fibrillary protein in Müller cells in PEG sol injected eyes, but normal overall morphology in eyes with attached retinas. The viscosity of the sol was not retained throughout the post-operative period, indicating the demand for polymer cross-linking to increase residence time. The results provide promising preliminary results on the use of PEG hydrogels as a vitreous substitute.

An injectable thiol-acrylate poly(ethylene glycol) hydrogel for sustained release of methylprednisolone sodium succinate.

Clinically available injectable hydrogels face technical challenges associated with swelling after injection and toxicity from unreacted constituents that impede their performance as surgical biomaterials. To overcome these challenges, we developed a system where chemical gelation was controlled by a conjugate Michael addition between thiol and acrylate in aqueous media, with 97% monomer conversion and 6 wt.% sol fraction. The hydrogel exhibited syneresis on equilibration, reducing to 59.7% of its initial volume. It had mechanical properties similar to soft human tissue with an elastic modulus of 189.8 kPa. Furthermore, a mesh size of 6.9 nm resulted in sustained release of methylprednisolone sodium succinate with a loading efficiency of 2 mg/mL. Functionalization with 50 µg/mL of an oligolysine peptide resulted in attachment of freshly isolated murine mesenchymal stem cells. The rational design of the physical, chemical and biological properties of the hydrogel makes it a potentially promising candidate for injectable applications.

Retinal transplantation using surface modified poly(glycerol-co-sebacic acid) membranes.

In retinal transplantation experiments it is hypothesized that remaining diseased photoreceptor cells in the host retina and inner retinal cells in transplants physically obstruct the development of graft-host neuronal contacts which are required for vision. Recently, we developed methods for the isolation of donor photoreceptor layers in vitro, and the selective removal of host photoreceptors in vivo using biodegradable elastomeric membranes composed of poly(glycerol-co-sebacic acid) (PGS). We also coated PGS membranes with electrospun nanofibers, composed of laminin and poly(epsilon-caprolactone) (PCL), to promote attachment of embryonic retinal explants, allowing the resulting composites to be handled surgically as a single entity. Here, we report subretinal transplantation of these composites into adult porcine eyes. In hematoxylin and eosin stained sections of composite explants after 5-7 days in vitro, excellent fusion of retinas and biomaterial membranes was noted, with the immature retinal components showing laminated as well as folded and rosetted areas. The composite grafts could be transplanted in all cases and, 3 months after surgery, eyes displayed clear media, attached retinas and the grafts located subretinally. Histological examination revealed that the biomaterial membrane had degraded without any signs of inflammation. Transplanted retinas displayed areas of rosettes as well as normal lamination. In most cases inner retinal layers were present in the grafts. Laminated areas displayed well-developed photoreceptors adjacent to an intact host retinal pigment epithelium and degeneration of the host outer nuclear layer (ONL) was often observed together with occasional fusion of graft and host inner layers.

The use of surface modified poly(glycerol-co-sebacic acid) in retinal transplantation.

Retinal transplantation experiments have advanced considerably during recent years, but remaining diseased photoreceptor cells in the host retina and inner retinal cells in the transplant physically obstruct the development of graft-host neuronal contacts which are required for vision. Recently, we developed methods for the isolation of donor photoreceptor layers in vitro, and the selective removal of host photoreceptors in vivo using biodegradable elastomeric membranes composed of poly(glycerol-co-sebacic acid) (PGS). Here, we report the surface modification of PGS membranes to promote the attachment of photoreceptor layers, allowing the resulting composite to be handled surgically as a single entity. PGS membranes were chemically modified with peptides containing an arginine-glycine-aspartic acid (RGD) extracellular matrix ligand sequence. PGS membranes were also coated with electrospun nanofiber meshes, containing laminin and poly(epsilon-caprolactone) (PCL). Following in vitro co-culture of biomaterial membranes with isolated embryonic retinal tissue, composites were tested for surgical handling and examined with hematoxylin and eosin staining and immunohistochemical markers. Electrospun nanofibers composed of laminin and PCL promoted sufficient cell adhesion for simultaneous transplantation of isolated photoreceptor layers and PGS membranes. Composites developed large populations of recoverin and rhodopsin labeled photoreceptors. Furthermore, ganglion cells, rod bipolar cells and AII amacrine cells were absent in co-cultured retinas as observed by neurofilament, PKC and parvalbumin labeling respectively. These results facilitate retinal transplantation experiments in which a composite graft composed of a biodegradable membrane adhered to an immature retina dominated by photoreceptor cells may be delivered in a single surgery, with the possibility of improving graft-host neuronal connections.

Establishing a model spinal cord injury in the African green monkey for the preclinical evaluation of biodegradable polymer scaffolds seeded with human neural stem cells.

Given the involvement of post-mitotic neurons, long axonal tracts and incompletely elucidated injury and repair pathways, spinal cord injury (SCI) presents a particular challenge for the creation of preclinical models to robustly evaluate longitudinal changes in neuromotor function in the setting in the presence and absence of intervention. While rodent models exhibit high degrees of spontaneous recovery from SCI injury, animal care concerns preclude complete cord transections in non-human primates and other larger vertebrate models. To overcome such limitations a segmental thoracic (T9-T10) spinal cord hemisection was created and characterized in the African green monkey. Physiological tolerance of the model permitted behavioral analyses for a prolonged period post-injury, extending to predefined study termination points at which histological and immunohistochemical analyses were performed. Four monkeys were evaluated (one receiving no implant at the lesion site, one receiving a poly(lactide-co-glycolide) (PLGA) scaffold, and two receiving PLGA scaffolds seeded with human neural stem cells (hNSC)). All subjects exhibited Brown-Séquard syndrome 2 days post-injury consisting of ipsilateral hindlimb paralysis and contralateral hindlimb hypesthesia with preservation of bowel and bladder function. A 20-point observational behavioral scoring system allowed quantitative characterization of the levels of functional recovery. Histological endpoints including silver degenerative staining and Iba1 immunohistochemistry, for microglial and macrophage activation, were determined to reliably define lesion extent and correlate with neurobehavioral data, and justify invasive telemetered electromyographic and kinematic studies to more definitively address efficacy and mechanism.