Salicylate-based poly(anhydride-esters) (PAEs) chemically incorporate salicylic acid (SA) into the polymer backbone, which is then delivered in a controlled manner upon polymer hydrolysis. In this work, a salicylate-based PAE is a carrier to encapsulate and deliver insulin. Polymer microspheres were formulated using a water/oil/water double-emulsion solvent evaporation technique. The microspheres obtained had a smooth surface, high protein encapsulation efficiency, and relatively low emulsifier content. Insulin was released in vitro for 15 days, with no signs of aggregation or unfolding of the secondary structure. The released insulin also retained bioactivity in vitro. Concurrently, SA was released from the microspheres with polymer degradation and anti-inflammatory activity was observed. Based upon these results, the formulated microspheres enable simultaneous delivery of insulin and SA, both retaining bioactivity following processing.

Morphine, a potent narcotic analgesic used for the treatment of acute and chronic pain, was chemically incorporated into a poly(anhydride-ester) backbone. The polymer termed "PolyMorphine", was designed to degrade hydrolytically releasing morphine in a controlled manner to ultimately provide analgesia for an extended time period. PolyMorphine was synthesized via melt-condensation polymerization and its structure was characterized using proton and carbon nuclear magnetic resonance spectroscopies, and infrared spectroscopy. The weight-average molecular weight and the thermal properties were determined. The hydrolytic degradation pathway of the polymer was determined by in vitro studies, showing that free morphine is released. In vitro cytocompatibility studies demonstrated that PolyMorphine is non-cytotoxic towards fibroblasts. In vivo studies using mice showed that PolyMorphine provides analgesia for 3 days, 20 times the analgesic window of free morphine. The animals retained full responsiveness to morphine after being subjected to an acute morphine challenge.

In previously reported work [1], we demonstrated that astrocytes cultured on synthetic polyamide nanofibrillar surfaces that mimic the architecture of the capillary basement membrane assumed morphological forms that recapitulated their physiology within the developing central nervous system. In the present work, atomic force microscopy was used to investigate astrocyte cell-cell interactions at 24 h, for cells cultured on nanofibrillar versus planar surfaces. For the nanofibrillar surfaces, high pass spatial filtering was required to distinguish the nanofibrillar background from the nanoscale astrocyte features. Using this approach, details of the physical interactions between astrocytes on nanofibrillar surfaces via connective extensions across ∼50 μm distances were identified, which were not observable in epi-fluorescent microscopy, or in tapping or deflection mode atomic force microscopy. Astrocyte cell-cell interactions were shown to differ in connective extension type, cell body type, and number of interactions. The connective bridges took the form of a filopodia network for planar surfaces but a single extension lamellipodia bridge for the nanofibrillar surfaces. Structures suggestive of adherens versus gap junctions that were part of the connective extensions were also identified. Cell-cell interactions via connective bridges (filopodia bridges, or tunneling nanotubes) over distances much larger than adjacent cell wall-cell wall contact distances have been previously reported for planar substrates. The present research supports this work and adds the dimension that nanofibrillar versus planar surface architectures can also be directive for specific implementations of such long-distance interactions. [1] Delgado-Rivera, R, Harris, SL, Ahmed, I, Babu, AN, Patel, R, Kamal, J, Ayres, V, Flowers, D, Meiners, S, 2009. Increased FGF-2 secretion and ability to support neurite outgrowth by astrocytes cultured on polyamide nanofibrillar matrices. Matrix Bio. 28: 137-147.

Microscale plasma-initiated patterning (μPIP) is a novel micropatterning technique used to create biomolecular micropatterns on polymer surfaces. The patterning method uses a polydimethylsiloxane (PDMS) stamp to selectively protect regions of an underlying substrate from oxygen plasma treatment resulting in hydrophobic and hydrophilic regions. Preferential adsorption of the biomolecules onto either the plasma-exposed (hydrophilic) or plasma-protected (hydrophobic) regions leads to the biomolecular micropatterns. In the current work, laminin-1 was applied to an electrospun polyamide nanofibrillar matrix following plasma treatment. Radial glial clones (neural precursors) selectively adhered to these patterned matrices following the contours of proteins on the surface. This work demonstrates that textured surfaces, such as nanofibrillar scaffolds, can be micropatterned to provide external chemical cues for cellular organization.

Continuous biomaterial advances and the regenerating potential of the adult human peripheral nervous system offer great promise for restoring full function to innervated tissue following traumatic injury via synthetic nerve guidance conduits (NGCs). To most effectively facilitate nerve regeneration, a tissue engineering scaffold within a conduit must be similar to the linear microenvironment of the healthy nerve. To mimic the native nerve structure, aligned poly(lactic-co-glycolic acid)/bioactive polyanhydride fibrous substrates were fabricated through optimized electrospinning parameters with diameters of 600 ± 200 nm. Scanning electron microscopy images show fibers with a high degree of alignment. Schwann cells and dissociated rat dorsal root ganglia demonstrated elongated and healthy proliferation in a direction parallel to orientated electrospun fibers with significantly longer Schwann cell process length and neurite outgrowth when compared to randomly orientated fibers. Results suggest that an aligned polyanhydride fiber mat holds tremendous promise as a supplement scaffold for the interior of a degradable polymer NGC. Bioactive salicylic acid-based polyanhydride fibers are not limited to nerve regeneration and offer exciting promise for a wide variety of biomedical applications.

Long distance intercellular communication between astrocytes on nanofibrillar and planar surfaces was investigated by epi-fluorescence microscopy and atomic force microscopy. We found that astrocytes on nanofibrillar surfaces and astrocytes on planar surfaces diverged in apparent cell-cell contact structures. Astrocytes on nanofibrillar surfaces exhibited a “single cellular process” response, while astrocytes on planar surfaces exhibited a filopodial network response. The possibility that astrocytes can sense their geometrical environment and form different cell-to-cell contacts on nanofibrillar versus planar surfaces, with activation of different signaling pathways, is discussed.

The introduction of scaffolding materials with appropriate biochemical cues and physical properties into damaged sites within the central nervous system can encourage endogenous or exogenous cellular re-colonization. The scaffolding material currently under investigation is a synthetic electrospun polyamide nanofibrillar matrix that has demonstrated promise in tissue engineering approaches for the repair of the injured spinal cord and is architecturally mimetic for the capillary basement membrane at the blood brain barrier. We will present quantitative investigations of the nanofibrillar matrix, which are achieved through use of a dynamic new mode of atomic force microscopy, Scanning Probe Recognition Microscopy (SPRM). SPRM uniquely allows auto-tracking along individual nanofibers, which are then compiled into a statistical representation of the nanofibrillar matrix as a whole. Complementary transmission electron microscopy (TEM) and nuclear magnetic resonance (NMR) investigations are performed to assess nanofiber internal structures that contribute to elasticity, and growth factor binding site information, respectively. Results of investigations of unmodified nanofibers and nanofibers covalently modified with fibroblast growth factor-2 (FGF-2), a prevailing cytokine involved in regulation of the growth of astrocytes, neurons, and other neural cells, will be presented. Astrocytes are neural cells that can be considered to be the cellular bridge between the capillary basement membrane and neurons. They are therefore directly responsive to the biochemical cues and physical properties of the native or prosthetic basement membrane. We will present results achieved through SPRM, immunocytochemistry, and Western blot techniques that indicate that there are significant differences in the astrocyte response to 2D planar substrates versus 3D nanofibrillar substrates versus 3D nanofibrillar substrates that are covalently modified with FGF-2, mimicking the sequestration of growth factors on the basement membrane. Differences in astrocyte physiology, substrate probing through lamellipodia and filopodia extension and FGF-2 up-regulation will be discussed.

Scanning probe recognition microscopy (SPRM) with auto-tracking of individual nanofibres is used for investigation of the key nanoscale properties of polyamide a nanofibrillar matrix that promotes more in vivo-like forms and functions for cultured cells. Both unmodified and fibroblast growth factor-2-modified nanofibres are considered. The contributions of nanofibrillar matrix elasticity and surface roughness to cellular behaviour are examined.

Implantable biodegradable nerve guidance conduits (NGCs) have the potential to align and support regenerating cells, as well as prevent scar formation. In this study in vitro bioassays and in vivo material evaluations were performed using a nerve guidance conduit material made from a novel polyanhydride blend. In vitro cytotoxicity studies with both fibroblasts and primary chick neurons demonstrated that the proposed polyanhydride blend was non-cytotoxic. Subcutaneous implantation for 7days in rats resulted in an initial fibrin matrix, minimal macrophage presence and angiogenesis in the surrounding tissues. Nerve guidance conduits fabricated from the proposed polyanhydride blend material may serve as favorable biocompatible tissue engineering devices.

An electrospun nonwoven matrix of polyamide nanofibers was employed as a new model for the capillary basement membrane at the blood-brain barrier (BBB). The basement membrane separates astrocytes from endothelial cells and is associated with growth factors, such as fibroblast growth factor-2 (FGF-2). FGF-2 is produced by astrocytes and induces specialized functions in endothelial cells, but also has actions on astrocytes. To investigate potential autocrine actions of FGF-2 at the BBB, astrocytes were cultured on unmodified nanofibers or nanofibers covalently modified with FGF-2. The former assumed an in vivo-like stellate morphology that was enhanced in the presence of cross-linked FGF-2. Furthermore, astrocyte monolayers established on unmodified nanofibers were more permissive for neurite outgrowth when cultured with an overlay of neurons than similar monolayers established on standard tissue culture surfaces, while astrocytes cultured on FGF-2-modifed nanofibers were yet more permissive. The observed differences were due in part to progressively increasing amounts of FGF-2 secreted by the astrocytes into the medium; hence FGF-2 increases its own expression in astrocytes to modulate astrocyte-neuron interactions. Soluble FGF-2 was unable to replicate the effects of cross-linked FGF-2. Nanofibers alone up-regulated FGF-2, albeit to a lesser extent than nanofibers covalently modified with FGF-2. These results underscore the importance of both surface topography and growth factor presentation on cellular function. Moreover, these results indicate that FGF-2-modified nanofibrillar scaffolds may demonstrate utility in tissue engineering applications for replacement and regeneration of lost tissue following central nervous system (CNS) injury or disease.

Preliminary data from in-vivo investigations (rat model) suggest that a nanofiber prosthetic device of FGF-2-modified nanofibers can correctly guide regenerating axons across an injury gap with aligned functional recovery. Scanning Probe Recognition Microscopy with auto-tracking of individual nanofibers is used for investigation of the key nanoscale properties of the nanofiber prosthetic device for spinal cord tissue engineering and repair.

BACKGROUND: The design of implants comprised of biodegradable electrospun nanofibers for the purpose of bridging injuries in damaged spinal cord is discussed. Electrospun nanofibers structurally mimic the extracellular matrix on which neurons and other cell types grow in vivo. This property has created great interest for their use in tissue engineering applications. However, their employment as biornimetic surfaces for such in vivo applications is still in its infancy.RESULTS: A nonwoven fabric comprised of electrospun polyamide nanofibers supported modest axonal regeneration in injured adult rat spinal cord. Covalent modification of the nanofibers with a bioactive peptide derived from the neuroregulatory extracellular matrix molecule tenascin-C enhanced the ability of the nanofibers to facilitate axonal regrowth. However, the random orientation of the nanofibrillar fabric folds was an impediment to the forward movement of axons.CONCLUSIONS: Polyamide nanofibers covalently modified with neuroactive molecules provide a promising material for grafts to promote spinal cord regeneration. However, for the proper guidance of regrowing axons, attention must be paid to the engineering of ordered nanofibrillar structures. (c) 2007 Society of Chemical Industry.