A heparin-mimicking polymer conjugate stabilizes basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a protein that plays a crucial role in diverse cellular functions, from wound healing to bone regeneration. However, a major obstacle to the widespread application of bFGF is its inherent instability during storage and delivery. Here, we describe the stabilization of bFGF by covalent conjugation with a heparin-mimicking polymer, a copolymer consisting of styrene sulfonate units and methyl methacrylate units bearing poly(ethylene glycol) side chains. The bFGF conjugate of this polymer retained bioactivity after synthesis and was stable to a variety of environmentally and therapeutically relevant stressors--such as heat, mild and harsh acidic conditions, storage and proteolytic degradation--unlike native bFGF. Following the application of stress, the conjugate was also significantly more active than the control conjugate system in which the styrene sulfonate units were omitted from the polymer structure. This research has important implications for the clinical use of bFGF and for the stabilization of heparin-binding growth factors in general

Macro-Architectures in Spinal Cord Scaffold Implants Influence Regeneration

Abstract Biomaterial scaffold architecture has not been investigated as a tunable source of influence on spinal cord regeneration. This study compared regeneration in a transected spinal cord within various designed-macro-architecture scaffolds to determine if these architectures alone could enhance regeneration. Three-dimensional (3-D) designs were created and molds were built on a 3-D printer. Salt-leached porous poly(ε-caprolactone) was cast in five different macro-architectures: cylinder, tube, channel, open-path with core, and open-path without core. The two open-path designs were created in this experiment to compare different supportive aspects of architecture provided by scaffolds and their influence on regeneration. Rats received T8 transections and implanted scaffolds for 1 and 3 months. Overall morphology and orientation of sections were characterized by H&E, luxol fast blue, and cresyl violet staining. Borders between intact gray matter and non-regenerated defect were observed from GFAP immunolabeling. Nerve fibers and regenerating axons were identified with Tuj-1 immunolabeling. The open-path designs allowed extension of myelinated fibers along the length of the defect both exterior to and inside the scaffolds and maintained their original defect length up to 3 months. In contrast, the cylinder, tube, and channel implants had a doubling of defect length from secondary damage and large scar and cyst formation with no neural tissue bridging. The open-path scaffold architectures enhanced spinal cord regeneration compared to the three other designs without the use of biological factors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63279/1/neu.2007.0473.pd

Toward a biomarker panel measured in CNS-originating extracellular vesicles for improved differential diagnosis of Parkinson’s disease and multiple system atrophy

Synucleinopathies are neurodegenerative diseases characterized by accumulation of misfolded α-synuclein (α-syn) inclusions in neuronal and/or glial cells. Despite differences in the underlying pathophysiology, synucleinopathies often are misdiagnosed, especially in early stages, due to the overlapping clinical symptoms [1]

Low-Dose, Long-Wave UV Light Does Not Affect Gene Expression of Human Mesenchymal Stem Cells

<div><p>Light is a non-invasive tool that is widely used in a range of biomedical applications. Techniques such as photopolymerization, photodegradation, and photouncaging can be used to alter the chemical and physical properties of biomaterials in the presence of live cells. Long-wave UV light (315 nm–400 nm) is an easily accessible and commonly used energy source for triggering biomaterial changes. Although exposure to low doses of long-wave UV light is generally accepted as biocompatible, most studies employing this wavelength only establish cell viability, ignoring other possible (non-toxic) effects. Since light exposure of wavelengths longer than 315 nm may potentially induce changes in cell behavior, we examined changes in gene expression of human mesenchymal stem cells exposed to light under both 2D and 3D culture conditions, including two different hydrogel fabrication techniques, decoupling UV exposure and radical generation. While exposure to long-wave UV light did not induce significant changes in gene expression regardless of culture conditions, significant changes were observed due to scaffold fabrication chemistry and between cells plated in 2D versus encapsulated in 3D scaffolds. In order to facilitate others in searching for more specific changes between the many conditions, the full data set is available on Gene Expression Omnibus for querying.</p></div

Principle components analysis shows tightly segregated clustering based on culture condition, not by UV exposure.

<p>(alternate viewing angle and axis values available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139307#pone.0139307.s002" target="_blank">S2 Fig</a>). PCA is a statistical analysis tool which reduces the dimensionality of data by determining the key variables resulting in differences seen between samples[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139307#pone.0139307.ref025" target="_blank">25</a>]. Each axis of this PCA map represents a linear combination of expression levels from many thousands of gene transcripts such that, combined, the maximum variation among all data points is achieved on only three axes. The result gives a visual representation of which samples behave similarly to each other by their physical closeness in three dimensions, while including information from many thousands of variables (gene expression levels). This is a bird’s eye view of the entire set of gene array data, for which absolute units and values can be considered arbitrary. The following in-depth analysis of pathway enrichment and specific gene expression provide insight into how each group differs from the others in their gene expression.</p

UV effects in 2D groups are insignificant.

<p>(a) This Venn diagram shows that whether combining raw data across months or using repetition power between months, no significant pathways or biological functions are found in the differentially expressed genes from the Kyoto Encyclopedia of Genes and Genomes (KEGG) or Gene Ontology (GO) except for a slight change in cell cycle. (b) The most extreme gene changes for each comparison have relatively weak fold changes, and a heat map (unsupervised hierarchical clustering) of the 148 gene transcripts (103 unique genes) differentially expressed between 2D₂UV vs. 2D₂ shows that none of the other UV comparisons in 3D have any differences in expression level for these genes. (red is upregulated, blue is downregulated, genes included in the heat map are those differentially expressed between denoted * groups) Also of note is the stark similarity between samples 2D₁ and 2D₂. The 3D groups look very similar to each other, while they are very different from the 2D groups. The most repeated changes of significance were in the genes DHRS3 and FOXQ1. Their individual scatter plots, (c) and (d), reinforce the similarities between the months as well as the direction of change. However, for so few genes and so small a change, the effects could easily be found by chance. (RMA = robust multi-array average).</p

Experimental groups illustrated.

<p>A single vial of P0 hMSCs were expanded at low density to greater than 25 million cells at P1. These cells were trypsinized and a portion of them replated at 500,000 cells / 75cm² flask for 2D samples. Another portion of those cells was encapsulated in poly(ethylene glycol) diacrylate (MW 4000 Da). Two encapsulation methods were used, radical polymerization with ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), and conjugate addition with a pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) crosslinker. Several samples of each type were created and half from each type were irradiated with a Black Ray UV bench lamp, peak wavelength 365nm.</p

IPA summary for 3D_R±UV vs. 3D_C±UV, (>2fold, p<0.05).

<p>^aIPA analysis returned several categories of known networks, molecular and cellular functions, and upstream regulators that could be considered important differences between radical polymerization and conjugate addition. This table summarizes the specific changes between these two polymerization groups at each level of evaluation (whole network activity, group function, specific protein activation) and gives the relative strengths of these components in the relevant statistical measure.</p><p>^bIPA network score: For networks, the IPA network score is a relative measure of relevance, with the two highest scores reported.</p><p>^c<u>p-value range (# molecules):</u> For molecular and cellular functions, each function, such as “Cellular Development”, represents a combination of lower level functions, each with a p-value. Thus the significance of these higher level functions is given as a range that covers the p-values of the lower level functions. The # molecules is the number of user input dataset molecules associated with that higher level function.</p><p>^d<u>p-value (activation z-score, prediction):</u> For individual upstream regulators, the expected cascade of transcriptional changes in downstream molecules is evaluated to determine if a particular upstream regulator is acting, and directions and magnitudes of particular changes can be used to determine the activation z-score. Positive z-scores above 2 predict activation while negative z-scores below -2 predict inhibition.</p><p>IPA summary for 3D_R±UV vs. 3D_C±UV, (>2fold, p<0.05).</p

A heparin-mimicking polymer conjugate stabilizes basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a protein that plays a crucial role in diverse cellular functions, from wound healing to bone regeneration. However, a major obstacle to the widespread application of bFGF is its inherent instability during storage and delivery. Here, we describe the stabilization of bFGF by covalent conjugation with a heparin-mimicking polymer, a copolymer consisting of styrene sulfonate units and methyl methacrylate units bearing poly(ethylene glycol) side chains. The bFGF conjugate of this polymer retained bioactivity after synthesis and was stable to a variety of environmentally and therapeutically relevant stressors--such as heat, mild and harsh acidic conditions, storage and proteolytic degradation--unlike native bFGF. Following the application of stress, the conjugate was also significantly more active than the control conjugate system in which the styrene sulfonate units were omitted from the polymer structure. This research has important implications for the clinical use of bFGF and for the stabilization of heparin-binding growth factors in general