Evaluation of osteogenic design factors in electrospun poly(ε-caprolactone) nanofiber scaffolds

Includes bibliographical references.Biodegradable bone tissue scaffolds have the potential to impact patients with numerous ailments. Starting with fabrication techniques that produce nano-scale features, the ability to manipulate architecture, alter surface chemistry, and deliver biological molecules allows for the design of elegant and highly effective bone scaffolds. This work aimed to develop a porous, nanofiber scaffold with osteogenic design features the capability to deliver an antibiotic molecule from within the nanofibers. Two osteogenic design factors with unique mechanisms of action were selected; hydroxyapatite nanoparticles and oleic acid. Hydroxyapatite (HAp) is the primary inorganic phase of natural bone tissue and has been used to more closely mimic the extracellular environment of synthetic bone tissue scaffolds. Oleic acid (OLA) is an ω-9 fatty acid with suspected osteogenic effects due to activation of peroxisome proliferator-activator receptors (PPARs). In separate in vitro evaluations, OLA significantly increased osteoblast phenotypic behaviors and led to differential expression of the three PPAR isoforms, suggesting that the OLA is activating its anticipated receptor. HAp produced mixed results by inducing a small increase in alkaline phosphatase activity, but decreasing expression levels of bone matrix proteins. An in vivo evaluation of biocompatibility revealed that neither design factor increased the inflammatory response over control nanofiber scaffolds in paravertebral muscle pouches. However, both factors separately increased new osteoid production. Scaffolds with both HAp and OLA elicited the greatest osteogenic response in vivo, suggesting positive synergy between the two design factors. Finally, rifampicin (RIF), an antibiotic molecule was loaded into the nanofibers, and its release into static bacterial culture was effective in inhibiting bacterial population growth for both a Gram-positive and Gram-negative bacterial strain, separately. Overall, these nanofiber scaffolds were demonstrated to be effective carriers of soluble (OLA, RIF) and insoluble signals (HAp) which can modulate cell behaviors. Future work will aim to incorporate additional osteogenic features into the scaffolds and to develop multiple antibiotic release mechanisms from the nanofibers

Insufficient OPC migration into demyelinated lesions is a cause of poor remyelination in MS and mouse models

Failure of remyelination of multiple sclerosis (MS) lesions contributes to neurodegeneration that correlates with chronic disability in patients. Currently, there are no available treatments to reduce neurodegeneration, but one therapeutic approach to fill this unmet need is to promote remyelination. As many demyelinated MS lesions contain plentiful oligodendrocyte precursor cells (OPCs), but no mature myelinating oligodendrocytes, research has previously concentrated on promoting OPC maturation. However, some MS lesions contain few OPCs, and therefore, remyelination failure may also be secondary to OPC recruitment failure. Here, in a series of MS samples, we determined how many lesions contained few OPCs, and correlated this to pathological subtype and expression of the chemotactic molecules Semaphorin (Sema) 3A and 3F. 37 % of MS lesions contained low numbers of OPCs, and these were mostly chronic active lesions, in which cells expressed Sema3A (chemorepellent). To test the hypothesis that differential Sema3 expression in demyelinated lesions alters OPC recruitment and the efficiency of subsequent remyelination, we used a focal myelinotoxic mouse model of demyelination. Adding recombinant (r)Sema3A (chemorepellent) to demyelinated lesions reduced OPC recruitment and remyelination, whereas the addition of rSema3F (chemoattractant), or use of transgenic mice with reduced Sema3A expression increased OPC recruitment and remyelination. We conclude that some MS lesions fail to remyelinate secondary to reduced OPC recruitment, and that chemotactic molecules are involved in the mechanism, providing a new group of drug targets to improve remyelination, with a specific target in the Sema3A receptor neuropilin-1. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00401-013-1112-y) contains supplementary material, which is available to authorized users

Optical Drug Monitoring: Photoacoustic Imaging of Nanosensors to Monitor Therapeutic Lithium in Vivo

Personalized medicine could revolutionize how primary care physicians treat chronic disease and how researchers study fundamental biological questions. To realize this goal, we need to develop more robust, modular tools and imaging approaches for in vivo monitoring of analytes. In this report, we demonstrate that synthetic nanosensors can measure physiologic parameters with photoacoustic contrast, and we apply that platform to continuously track lithium levels in vivo. Photoacoustic imaging achieves imaging depths that are unattainable with fluorescence or multiphoton microscopy. We validated the photoacoustic results that illustrate the superior imaging depth and quality of photoacoustic imaging with optical measurements. This powerful combination of techniques will unlock the ability to measure analyte changes in deep tissue and will open up photoacoustic imaging as a diagnostic tool for continuous physiological tracking of a wide range of analytes

M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination

The lack of therapies for progressive multiple sclerosis highlights the need to understand the regenerative process of remyelination that can follow CNS demyelination. This involves an innate immune response consisting of microglia/macrophages, which can be polarized to distinct functional phenotypes: proinflammatory (M1) or anti-inflammatory/immunoregulatory (M2). Here we show that a switch from an M1- to M2-dominant response occurred within microglia and peripherally-derived macrophages as remyelination started. Oligodendrocyte differentiation was enhanced in vitro with M2 conditioned media, and impaired in vivo following intra-lesional M2 depletion. M2 densities were increased in lesions of aged mice in which remyelination was enhanced by parabiotic coupling to a younger animal, and in MS lesions that normally show remyelination. Blocking M2-derived activin-A inhibited oligodendrocyte differentiation during remyelination in cerebellar slice cultures. Our results therefore show that M2 polarization is essential for efficient remyelination and identify activin-A as a novel therapeutic target for CNS regeneration

Regeneration of myelin sheaths of normal length and thickness in the zebrafish CNS correlates with growth of axons in caliber

Demyelination is observed in numerous diseases of the central nervous system, including multiple sclerosis (MS). However, the endogenous regenerative process of remyelination can replace myelin lost in disease, and in various animal models. Unfortunately, the process of remyelination often fails, particularly with ageing. Even when remyelination occurs, it is characterised by the regeneration of myelin sheaths that are abnormally thin and short. This imperfect remyelination is likely to have implications for the restoration of normal circuit function and possibly the optimal metabolic support of axons. Here we describe a larval zebrafish model of demyelination and remyelination. We employ a drug-inducible cell ablation system with which we can consistently ablate 2/3rds of oligodendrocytes in the larval zebrafish spinal cord. This leads to a concomitant demyelination of 2/3rds of axons in the spinal cord, and an innate immune response over the same time period. We find restoration of the normal number of oligodendrocytes and robust remyelination approximately two weeks after induction of cell ablation, whereby myelinated axon number is restored to control levels. Remarkably, we find that myelin sheaths of normal length and thickness are regenerated during this time. Interestingly, we find that axons grow significantly in caliber during this period of remyelination. This suggests the possibility that the active growth of axons may stimulate the regeneration of myelin sheaths of normal dimensions

Zebrafish regenerate full thickness optic nerve myelin after demyelination, but this fails with increasing age

INTRODUCTION: In the human demyelinating central nervous system (CNS) disease multiple sclerosis, remyelination promotes recovery and limits neurodegeneration, but this is inefficient and always ultimately fails. Furthermore, these regenerated myelin sheaths are thinner and shorter than the original, leaving the underlying axons potentially vulnerable. In rodent models, CNS remyelination is more efficient, so that in young animals (but not old) the number of myelinated axons is efficiently restored to normal, but in both young and old rodents, regenerated myelin sheaths are still short and thin. The reasons for these differences in remyelination efficiency, the thinner remyelinated myelin sheaths compared to developmental myelin and the subsequent effect on the underlying axon are unclear. We studied CNS remyelination in the highly regenerative adult zebrafish (Danio rerio), to better understand mechanisms of what we hypothesised would be highly efficient remyelination, and to identify differences to mammalian CNS remyelination, as larval zebrafish are increasingly used for high throughput screens to identify potential drug targets to improve myelination and remyelination. RESULTS: We developed a novel method to induce a focal demyelinating lesion in adult zebrafish optic nerve with no discernible axonal damage, and describe the cellular changes over time. Remyelination is indeed efficient in both young and old adult zebrafish optic nerves, and at 4 weeks after demyelination, the number of myelinated axons is restored to normal, but internode lengths are short. However, unlike in rodents or in humans, in young zebrafish these regenerated myelin sheaths were of normal thickness, whereas in aged zebrafish, they were thin, and remained so even 3 months later. This inability to restore normal myelin thickness in remyelination with age was associated with a reduced macrophage/microglial response. CONCLUSION: Zebrafish are able to efficiently restore normal thickness myelin around optic nerve axons after demyelination, unlike in mammals. However, this fails with age, when only thin myelin is achieved. This gives us a novel model to try and dissect the mechanism for restoring myelin thickness in CNS remyelination. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40478-014-0077-y) contains supplementary material, which is available to authorized users

Mineralization Content Alters Osteogenic Responses of Bone Marrow Stromal Cells on Hydroxyapatite/Polycaprolactone Composite Nanofiber Scaffolds

Abstract: Synthetic tissue scaffolds have a high potential impact for patients experiencing osteogenesis imperfecta. Using electrospinning, tissue scaffolds composed of hydroxyapatite/polycaprolactone (HAp/PCL) composite nanofibers were fabricated with two different HAp concentrations—1 % and 10 % of the solid scaffold weight. After physico-chemical scaffold characterization, rat bone marrow stromal cells were cultured on the composite scaffolds in maintenance medium and then in osteogenic medium. Quantitative PCR, colorimetric assays, immunofluorescent labeling, and electron microscopy measured osteogenic cell responses to the HAp/PCL scaffolds. In maintenance conditions, both Hap/PCL scaffolds and control scaffolds supported cell colonization through seven days with minor differences. In osteogenic conditions, the 10 % HAp scaffolds exhibited significantly increased ALP assay levels at week 3, consistent with previous reports. However, qPCR analysis demonstrated an overall decrease in bone matrix-associated genes on Hap/PCL scaffolds. Osteopontin and osteocalcin immunofluorescent microscopy revealed a trend that both mineralized scaffolds had greater amounts of both proteins, though qPCR results indicated the opposite trend for osteopontin. Additionally, type

Mineralization Content Alters Osteogenic Responses of Bone Marrow Stromal Cells on Hydroxyapatite/Polycaprolactone Composite Nanofiber Scaffolds

Synthetic tissue scaffolds have a high potential impact for patients experiencing osteogenesis imperfecta. Using electrospinning, tissue scaffolds composed of hydroxyapatite/polycaprolactone (HAp/PCL) composite nanofibers were fabricated with two different HAp concentrations—1% and 10% of the solid scaffold weight. After physico-chemical scaffold characterization, rat bone marrow stromal cells were cultured on the composite scaffolds in maintenance medium and then in osteogenic medium. Quantitative PCR, colorimetric assays, immunofluorescent labeling, and electron microscopy measured osteogenic cell responses to the HAp/PCL scaffolds. In maintenance conditions, both Hap/PCL scaffolds and control scaffolds supported cell colonization through seven days with minor differences. In osteogenic conditions, the 10% HAp scaffolds exhibited significantly increased ALP assay levels at week 3, consistent with previous reports. However, qPCR analysis demonstrated an overall decrease in bone matrix-associated genes on Hap/PCL scaffolds. Osteopontin and osteocalcin immunofluorescent microscopy revealed a trend that both mineralized scaffolds had greater amounts of both proteins, though qPCR results indicated the opposite trend for osteopontin. Additionally, type I collagen expression decreased on HAp scaffolds. These results indicate that cells are sensitive to minor changes in mineral content within nanofibers, even at just 1% w/w, and elucidating the sensing mechanism may lead to optimized osteogenic scaffold designs

Development of an Optical Nanosensor Incorporating a pH-Sensitive Quencher Dye for Potassium Imaging

One of the key challenges in the design of a sensor for measuring extracellular changes in potassium concentration is selectivity against the competing cation, sodium. Here, we present an optode-based nanosensor selective to potassium ions, owing to the addition of a pH-sensitive quencher molecule paired with a static fluorophore. The nanosensor was fabricated using emulsification and characterized in solution by absorbance and fluorescence spectroscopy. The resulting nanosensor detected potassium with nearly 1 order of magnitude higher selectivity compared to our chromoionophore-based optode nanosensors. In addition to the improved selectivity, the nanosensor has the following properties required for measurements in a biological environment: (1) a physiologically relevant dynamic range, (2) response to potassium ions at a physiological ionic strength, and (3) response to serum potassium in the presence of fouling biological components. The potassium nanosensor described in this study is envisioned to have application in cellular imaging and drug screening