Rutledge Ellis-Behnke

Temperature and pH effects on biophysical and morphological properties of self-assembling peptide RADA16-I.

It has been found that the self‐assembling peptide RADA 16‐I forms a β‐sheet structure and self‐assembles into nanofibers and scaffolds in favor of cell growth, hemostasis and tissue‐injury repair. But its biophysical and morphological properties, especially for its β‐sheet and self‐assembling properties in heat‐ and pH‐denatured conditions, remain largely unclear. In order to better understand and design nanobiomaterials, we studied the self‐assembly behaviors of RADA16‐I using CD and atomic force microscopy (AFM) measurements in various pH and heat‐denatured conditions. Here, we report that the peptide, when exposed to pH 1.0 and 4.0, was still able to assume a typical β‐sheet structure and self‐assemble into long nanofiber, although its β‐sheet content was dramatically decreased by 10% in a pH 1.0 solution. However, the peptide, when exposed to pH 13.0, drastically lost its β‐sheet structure and assembled into different small‐sized globular aggregates. Similarly, the peptide, when heat‐denatured from 25 to 70 °C, was still able to assume a typical β‐sheet structure with 46% content, but self‐assembled into small‐sized globular aggregates at much higher temperature. Titration experiments showed that the peptide RADA16‐I exists in three types of ionic species: acidic (fully protonated peptide), zwitterionic (electrically neutral peptide carrying partial positive and negative charges) and basic (fully deprotonated peptide) species, called ‘super ions’. The unordered structure and β‐turn of these ‘super ions’ via hydrogen or ionic bonds, and heat Brownian motion under the above denatured conditions would directly affect the stability of the β‐sheet and nanofibers. These results help us in the design of future nanobiomaterials, such as biosensors, based on β‐sheets and environmental changes. These results also help understand the pathogenesis of the β‐sheet‐mediated neuronal diseases such as Alzheimers disease and the mechanism of hemostasis. Copyright

Effect of self-assembled nanofibrous silk/polycaprolactone layer on the osteoconductivity and mechanical properties of biphasic calcium phosphate scaffolds

We here present the first successful report on combining nanostructured silk and poly(ε-caprolactone) (PCL) with a ceramic scaffold to produce a composite scaffold that is highly porous (porosity ∼85%, pore size ∼500 μm, ∼100% interconnectivity), strong and non-brittle with a surface that resembles extracellular matrix (ECM). The ECM-like surface was developed by self-assembly of nanofibrous structured silk (20-80 nm diameter, similar to native collagen found in ECM) over a thin PCL layer which is coated on biphasic calcium phosphate (BCP) scaffolds. The effects of different concentrations of silk solution on the mechanical and physical properties of the scaffolds were also comprehensively examined. Our results showed that using silk only (irrespective of concentration) for the modification of ceramic scaffolds could drastically reduce the compressive strength of the modified scaffolds in aqueous media, and the modification made a limited contribution to improving scaffold toughness. Using PCL/nanostructured silk the compressive strength and modulus of the modified scaffolds reached 0.42 MPa (compared with 0.07 MPa for BCP) and ∼25 MPa (compared with 5 MPa for BCP), respectively. The failure strain of the modified scaffold increased more than 6% compared with a BCP scaffold (failure strain of less than 1%), indicating a transformation from brittle to elastic behavior. The cytocompatibility of ECM-like composite scaffolds was investigated by studying the attachment, morphology, proliferation and bone-related gene expression of primary human bone-derived cells. Cells cultured on the developed scaffolds for 7 days had significant up-regulation of cell proliferation (∼1.6-fold higher, P<0.001) and osteogenic gene expression levels (collagen type I, osteocalcin and bone sialoprotein) compared with the other groups tested.

Carbon nanotubes: Their potential and pitfalls for bone tissue regeneration and engineering

UNLABELLED The extracellular environment which supports cell life is composed of a hierarchy of maintenance, force and regulatory systems which integrate from the nano- through to macroscale. For this reason, strategies to recreate cell supporting environments have been investigating the use of nanocomposite biomaterials. Here, we review the use of carbon nanotubes as part of a bottom-up approach for use in bone tissue engineering. We evaluate the properties of carbon nanotubes in the context of synthetic tissue substrates and contrast them with the nanoscale features of the extracellular environment. Key studies are evaluated with an emphasis on understanding the mechanisms through which carbon nanotubes interact with biological systems. This includes an examination of how the different properties of carbon nanotubes affect tissue growth, how these properties and variation to them might be leveraged in regenerative tissue therapies and how impurities or contaminates affect their toxicity and biological interaction. FROM THE CLINICAL EDITOR In this comprehensive review, the authors describe the status and potential applications of carbon nanotubes in bone tissue engineering.

The Spider Effect: Morphological and Orienting Classification of Microglia in Response to Stimuli in Vivo

The different morphological stages of microglial activation have not yet been described in detail. We transected the olfactory bulb of rats and examined the activation of the microglial system histologically. Six stages of bidirectional microglial activation (A) and deactivation (R) were observed: from stage 1A to 6A, the cell body size increased, the cell process number decreased, and the cell processes retracted and thickened, orienting toward the direction of the injury site; until stage 6A, when all processes disappeared. In contrast, in deactivation stages 6R to 1R, the microglia returned to the original site exhibiting a stepwise retransformation to the original morphology. Thin highly branched processes re-formed in stage 1R, similar to those in stage 1A. This reverse transformation mirrored the forward transformation except in stages 6R to 1R: cells showed multiple nuclei which were slowly absorbed. Our findings support a morphologically defined stepwise activation and deactivation of microglia cells.

CNS regeneration after chronic injury using a self-assembled nanomaterial and MEMRI for real-time in vivo monitoring

UNLABELLED To speed up the process of central nervous system (CNS) recovery after injury, the need for real-time measurement of axon regeneration in vivo is essential to assess the extent of injury, as well as the optimal timing and delivery of therapeutics and rehabilitation. It was necessary to develop a chronic animal model with an in vivo measurement technique to provide a real-time monitoring and feedback system. Using the framework of the 4 Ps of CNS regeneration (Preserve, Permit, Promote and Plasticity) as a guide, combined with noninvasive manganese-enhanced magnetic resonance imaging (MEMRI), we show a successful chronic injury model to measure CNS regeneration, combined with an in vivo measurement system to provide real-time feedback during every stage of the regeneration process. We also show that a chronic optic tract (OT) lesion is able to heal, and axons are able to regenerate, when treated with a self-assembling nanofiber peptide scaffold (SAPNS). FROM THE CLINICAL EDITOR The authors of this study demonstrate the development of a chronic injury model to measure CNS regeneration, combined with an in vivo measurement system to provide real-time feedback during every stage of the regeneration process. In addition, they determined that chronic optic tract lesions are able to heal with axonal regeneration when treated with a self-assembling nanofiber peptide scaffold (SAPNS).

Ultrasound-enhanced intrascleral delivery of protein

We aim to investigate ultrasound on enhancing protein penetration into the sclera, a non-invasive method to overcome the first barrier in taking the transscleral route for delivering therapeutics. Rabbit eyes were immersed in a fluorescein isothiocyanate conjugated bovine serum albumin solution. The distances of protein penetration, with and without ultrasound (30s continuous wave, 1MHz, 0.05W/cm(2)) applied on the sclera, and at different immersion time intervals (0, 5, 15, 30 and 60min), were measured by examining the cryo-sectioned tissues under fluorescence microscope (≥60 measurements from 3 eyes for each condition). Retina was examined for structural damage by histology. It was found that ultrasound enhances the intrascleral penetration of protein, increasing the diffusivity by 1.6-folds while causing no damage to the retinal tissues. This physical modulation of the sclera is temporary, as evident by the restoration of the diffusional resistance at 15min after ultrasound treatment. The negligible effect of ultrasound-induced convection and the minimal temperature rise (<0.5°C), together with cavitation detected by acoustic emission and a decreased penetration distance at higher ultrasound frequency (30s continuous wave, 3MHz, 0.05W/cm(2)), suggest that cavitation is a possible mechanism for increasing the permeability of the sclera for diffusive transport.

Forever young: how to control the elongation, differentiation, and proliferation of cells using nanotechnology.

Within the emerging field of stem cells there is a need for an environment that can regulate cell activity, to slow down differentiation or proliferation, in vitro or in vivo while remaining invisible to the immune system. By creating a nanoenvironment surrounding PC12 cells, Schwann cells, and neural precursor cells (NPCs), we were able to control the proliferation, elongation, differentiation, and maturation in vitro. We extended the method, using self-assembling nanofiber scaffold (SAPNS), to living animals with implants in the brain and spinal cord. Here we show that when cells are placed in a defined system we can delay their proliferation, differentiation, and maturation depending on the density of the cell population, density of the matrix, and the local environment. A combination of SAPNS and young cells can be implanted into the central nervous system (CNS), eliminating the need for immunosuppressants.

A self-assembling nanomaterial reduces acute brain injury and enhances functional recovery in a rat model of intracerebral hemorrhage

There is no effective treatment for intracerebral hemorrhage (ICH). Intracerebral delivery of nanomaterials into the hemorrhagic lesion may be a new therapeutic strategy. In a rat model of ICH plus ultra-early hematoma aspiration, we found that locally delivered self-assembling peptide nanofiber scaffold (SAPNS) replaced the hematoma, reduced acute brain injury and brain cavity formation, and improved sensorimotor functional recovery. SAPNS serves as biocompatible material in the hemorrhagic brain cavity. Local delivery of this nanomaterial may facilitate the repair of ICH related brain injury and functional recovery. From the clinical editor: In a rat model of intracranial hemorrhage, these authors demonstrate that following ultra-early hematoma aspiration, local delivery of a self-assembling peptide nanofiber scaffold replaces the hematoma, reduces brain cavity formation, and improves sensorimotor functional recovery. Similar approaches would be welcome additions to the clinical treatment of this often devastating condition.

Impediments to Eye Transplantation: Ocular Viability Following Optic Nerve Transection or Enucleation

Maintenance of ocular viability is one of the major impediments to successful whole-eye transplantation. This review provides a comprehensive understanding of the current literature to help guide future studies in order to overcome this hurdle. A systematic multistage review of published literature was performed. Three specific questions were addressed: (1) Is recovery of visual function following eye transplantation greater in cold-blooded vertebrates when compared with mammals? (2) Is outer retina function following enucleation and reperfusion improved compared with enucleation alone? (3) Following optic-nerve transection, is there a correlation between retinal ganglion cell (RGC) survival and either time after transection or proximity of the transection to the globe? In a majority of the studies performed in the literature, recovery of visual function can occur after whole-eye transplantation in cold-blooded vertebrates. Following enucleation (and reperfusion), outer retinal function is maintained from 4 to 9 h. RGC survival following optic-nerve transection is inversely related to both the time since transection and the proximity of transection to the globe. Lastly, neurotrophins can increase RGC survival following optic-nerve transection. This review of the literature suggests that the use of a donor eye is feasible for whole-eye transplantation.

Redefining tissue engineering for nanomedicine in ophthalmology

Working at the nanoscale means to completely rethink how to approach engineering in the body in general and in the eye in particular. In nanomedicine, tissue engineering is the ability to influence an environment either by adding, subtracting or manipulating that environment to allow it to be more conducive for its purpose. The goal is to function at the optimum state, or to return to that optimum state. Additive tissue engineering replaces cells or tissue, or tries to get something to grow that is no longer there. Arrestive tissue engineering tries to stop aberrant growth which, if left uncontrolled, would result in a decrease in function. Nano delivery of therapeutics can perform both additive and arrestive functions influencing the environment either way, depending on the targeting. By manipulating the environment at the nanoscale, the rate and distribution of healing can be controlled. It infers that potential applications of nanomedicine in ophthalmology include procedures, such as corneal endothelial cell transplantation, single retinal ganglion cell repair, check of retinal ganglion cell viability, building of nanofibre scaffolds, such as self‐assembling peptides, to create a scaffold‐like tissue‐bridging structure to provide a framework for axonal regeneration in the case of optic nerve reconnection or eye transplantation, and ocular drug delivery. Examples of potential arrestive therapies include gene‐related treatment modalities to inhibit intraocular neovascularization and to block retinal cell apoptosis. Looking towards the future, this review focuses on how nanoscale tissue engineering can be and is being used to influence that local environment.