Roberto Delgado-Rivera

Salicylic acid‐derived poly(anhydride‐ester) electrospun fibers designed for regenerating the peripheral nervous system

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.

Increased FGF-2 secretion and ability to support neurite outgrowth by astrocytes cultured on polyamide nanofibrillar matrices

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.

Design and evaluation of novel polyanhydride blends as nerve guidance conduits.

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.

Biodegradable salicylate-based poly(anhydride-ester) microspheres for controlled insulin delivery.

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.

Microscale plasma-initiated patterning of electrospun polymer scaffolds

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.

Scanning probe recognition microscopy investigation of neural cell prosthetic properties

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.

Quantitative Investigations of Nanoscale Elasticity of Nanofibrillar Matrices

Recent research indicates that nanophysical properties as well as biochemical cues can influence cellular re-colonization of a tissue scaffold. It has also been shown nanoscale elasticity can strongly influence cellular responses. In the present work, quantitative investigations of the elasticity of a nanofibrillar matrix scaffold that has demonstrated promise for spinal cord injury repair are compared with complementary transmission electron microscopy investigations, performed to assess nanofiber internal structures. Interpretive model improvements are identified and discussed.

Astrocyte Cell-Cell Interactions via Long-Range Connective Bridges on Directive Surfaces

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.