Aligned Polyhydroxyalkanoate Blend Electrospun Fibers as Intraluminal Guidance Scaffolds for Peripheral Nerve Repair.

The use of nerve guidance conduits (NGCs) to treat peripheral nerve injuries is a favorable approach to the current "gold standard" of autografting. However, as simple hollow tubes, they lack specific topographical and mechanical guidance cues present in nerve grafts and therefore are not suitable for treating large gap injuries (30-50 mm). The incorporation of intraluminal guidance scaffolds, such as aligned fibers, has been shown to increase neuronal cell neurite outgrowth and Schwann cell migration distances. A novel blend of PHAs, P(3HO)/P(3HB) (50:50), was investigated for its potential as an intraluminal aligned fiber guidance scaffold. Aligned fibers of 5 and 8 µm diameter were manufactured by electrospinning and characterized using SEM. Fibers were investigated for their effect on neuronal cell differentiation, Schwann cell phenotype, and cell viability in vitro. Overall, P(3HO)/P(3HB) (50:50) fibers supported higher neuronal and Schwann cell adhesion compared to PCL fibers. The 5 µm PHA blend fibers also supported significantly higher DRG neurite outgrowth and Schwann cell migration distance using a 3D ex vivo nerve injury model.

Bioresorbable and Mechanically Optimized Nerve Guidance Conduit Based on a Naturally Derived Medium Chain Length Polyhydroxyalkanoate and Poly(ε-Caprolactone) Blend.

Severe peripheral nerve injuries represent a large clinical problem with relevant challenges such as the development of successful synthetic scaffolds as substitutes to autologous nerve grafting. Numerous studies have reported the use of polyesters and type I collagen-based nerve guidance conduits (NGCs) to promote nerve regeneration through critical nerve defects while providing protection from external factors. However, none of the commercially available hollow bioresorbable NGCs have demonstrated superior clinical outcomes to an autologous nerve graft. Hence, new materials and NGC geometries have been explored in the literature to mimic the native nerve properties and architecture. Here, we report a novel blend of a natural medium chain length polyhydroxyalkanoate (MCL-PHA) with a synthetic aliphatic polyester, poly(ε-caprolactone) (PCL), suitable for extrusion-based high-throughput manufacturing. The blend was designed to combine the excellent ability of PHAs to support the growth and proliferation of mammalian cells with the good processability of PCL. The material exhibited excellent neuroregenerative properties and a good bioresorption rate, while the extruded porous tubes exhibited similar mechanical properties to the rat sciatic nerve. The NGCs were implanted to treat a 10 mm long sciatic nerve defect in rats, where significant differences were found between thin and thick wall thickness implants, and both electrophysiological and histological data, as well as the number of recovered animals, provided superior outcomes than the well-referenced synthetic Neurolac NGC.

UV-Casting on Methacrylated PCL for the Production of a Peripheral Nerve Implant Containing an Array of Porous Aligned Microchannels.

Peripheral nerves are basic communication structures guiding motor and sensory information from the central nervous system to receptor units. Severed peripheral nerve injuries represent a large clinical problem with relevant challenges to successful synthetic nerve repair scaffolds as substitutes to autologous nerve grafting. Numerous studies reported the use of hollow tubes made of synthetic polymers sutured between severed nerve stumps to promote nerve regeneration while providing protection for external factors, such as scar tissue formation and inflammation. Few approaches have described the potential use of a lumen structure comprised of microchannels or microfibers to provide axon growth avoiding misdirection and fostering proper healing. Here, we report the use of a 3D porous microchannel-based structure made of a photocurable methacrylated polycaprolactone, whose mechanical properties are comparable to native nerves. The neuro-regenerative properties of the polymer were assessed in vitro, prior to the implantation of the 3D porous structure, in a 6-mm rat sciatic nerve gap injury. The manufactured implants were biocompatible and able to be resorbed by the host's body at a suitable rate, allowing the complete healing of the nerve. The innovative design of the highly porous structure with the axon guiding microchannels, along with the observation of myelinated axons and Schwann cells in the in vivo tests, led to a significant progress towards the standardized use of synthetic 3D multichannel-based structures in peripheral nerve surgery.

Broadly Applicable Stereoselective Syntheses of Serrulatane, Amphilectane Diterpenes, and Their Diastereoisomeric Congeners Using Asymmetric Hydrovinylation for Absolute Stereochemical Control.

A stereogenic center, placed at an exocyclic location next to a chiral carbon in a ring to which it is attached, is a ubiquitous structural motif seen in many bioactive natural products, including di- and triterpenes and steroids. Installation of these centers has been a long-standing problem in organic chemistry. Few classes of compounds illustrate this problem better than serrulatanes and amphilectanes, which carry multiple methyl-bearing exocyclic chiral centers. Nickel-catalyzed asymmetric hydrovinylation (AHV) of vinylarenes and 1,3-dienes such as 1-vinylcycloalkenes provides an exceptionally facile way of introducing these chiral centers. This Article documents our efforts to demonstrate the generality of AHV to access not only the natural products but also their various diastereoisomeric derivatives. Key to success here is the availability of highly tunable phosphoramidite Ni(II) complexes useful for overcoming the inherent selectivity of the chiral intermediates. The yields for hydrovinylation (HV) reactions are excellent, and selectivities are in the range of 92-99% for the desired isomers. Discovery of novel, configurationally fluxional, yet sterically less demanding 2,2'-biphenol-derived phosphoramidite Ni complexes (fully characterized by X-ray) turned out to be critical for success in several HV reactions. We also report a less spectacular yet equally important role of solvents in a metal-ammonia reduction for the installation of a key benzylic chiral center. Starting with simple oxygenated styrene derivatives, we iteratively install the various exocyclic chiral centers present in typical serrulatane [e.g., a (+)- p-benzoquinone natural product, elisabethadione, nor-elisabethadione, helioporin D, a known advanced intermediate for the synthesis of colombiasin and elisapterosin] and amphilectane [e.g., A-F, G-J, and K,L pseudopterosins] derivatives. A concise table showing various synthetic approaches to these molecules is included in the Supporting Information. Our attempts to synthesize a hitherto elusive target, elisabethin A, led to a stereoselective, biomimetic route to pseudopterosin A-F aglycones.

Label-free quantitative chemical imaging and classification analysis of adipogenesis using mouse embryonic stem cells.

Stem cells have received much attention recently for their potential utility in regenerative medicine. The identification of their differentiated progeny often requires complex staining procedures, and is challenging for intermediary stages which are a priori unknown. In this work, the ability of label-free quantitative coherent anti-Stokes Raman scattering (CARS) micro-spectroscopy to identify populations of intermediate cell states during the differentiation of murine embryonic stem cells into adipocytes is assessed. Cells were imaged at different days of differentiation by hyperspectral CARS, and images were analysed with an unsupervised factorization algorithm providing Raman-like spectra and spatially resolved maps of chemical components. Chemical decomposition combined with a statistical analysis of their spatial distributions provided a set of parameters that were used for classification analysis. The first 2 principal components of these parameters indicated 3 main groups, attributed to undifferentiated cells, cells differentiated into committed white pre-adipocytes, and differentiating cells exhibiting a distinct protein globular structure with adjacent lipid droplets. An unsupervised classification methodology was developed, separating undifferentiated cell from cells in other stages, using a novel method to estimate the optimal number of clusters. The proposed unsupervised classification pipeline of hyperspectral CARS data offers a promising new tool for automated cell sorting in lineage analysis.

Pre-clinical evaluation of advanced nerve guide conduits using a novel 3D in vitro testing model.

Autografts are the current gold standard for large peripheral nerve defects in clinics despite the frequently occurring side effects like donor site morbidity. Hollow nerve guidance conduits (NGC) are proposed alternatives to autografts, but failed to bridge gaps exceeding 3 cm in humans. Internal NGC guidance cues like microfibres are believed to enhance hollow NGCs by giving additional physical support for directed regeneration of Schwann cells and axons. In this study, we report a new 3D in vitro model that allows the evaluation of different intraluminal fibre scaffolds inside a complete NGC. The performance of electrospun polycaprolactone (PCL) microfibres inside 5 mm long polyethylene glycol (PEG) conduits were investigated in neuronal cell and dorsal root ganglion (DRG) cultures in vitro. Z-stack confocal microscopy revealed the aligned orientation of neuronal cells along the fibres throughout the whole NGC length and depth. The number of living cells in the centre of the scaffold was not significantly different to the tissue culture plastic (TCP) control. For ex vivo analysis, DRGs were placed on top of fibre-filled NGCs to simulate the proximal nerve stump. In 21 days of culture, Schwann cells and axons infiltrated the conduits along the microfibres with 2.2 ± 0.37 mm and 2.1 ± 0.33 mm, respectively. We conclude that this in vitro model can help define internal NGC scaffolds in the future by comparing different fibre materials, composites and dimensions in one setup prior to animal testing.

Nerve guides manufactured from photocurable polymers to aid peripheral nerve repair.

The peripheral nervous system has a limited innate capacity for self-repair following injury, and surgical intervention is often required. For injuries greater than a few millimeters autografting is standard practice although it is associated with donor site morbidity and is limited in its availability. Because of this, nerve guidance conduits (NGCs) can be viewed as an advantageous alternative, but currently have limited efficacy for short and large injury gaps in comparison to autograft. Current commercially available NGC designs rely on existing regulatory approved materials and traditional production methods, limiting improvement of their design. The aim of this study was to establish a novel method for NGC manufacture using a custom built laser-based microstereolithography (µSL) setup that incorporated a 405 nm laser source to produce 3D constructs with ∼ 50 µm resolution from a photocurable poly(ethylene glycol) resin. These were evaluated by SEM, in vitro neuronal, Schwann and dorsal root ganglion culture and in vivo using a thy-1-YFP-H mouse common fibular nerve injury model. NGCs with dimensions of 1 mm internal diameter × 5 mm length with a wall thickness of 250 µm were fabricated and capable of supporting re-innervation across a 3 mm injury gap after 21 days, with results close to that of an autograft control. The study provides a technology platform for the rapid microfabrication of biocompatible materials, a novel method for in vivo evaluation, and a benchmark for future development in more advanced NGC designs, biodegradable and larger device sizes, and longer-term implantation studies.

Time-lapse analysis of human embryonic stem cells reveals multiple bottlenecks restricting colony formation and their relief upon culture adaptation.

Using time-lapse imaging, we have identified a series of bottlenecks that restrict growth of early-passage human embryonic stem cells (hESCs) and that are relieved by karyotypically abnormal variants that are selected by prolonged culture. Only a minority of karyotypically normal cells divided after plating, and these were mainly cells in the later stages of cell cycle at the time of plating. Furthermore, the daughter cells showed a continued pattern of cell death after division, so that few formed long-term proliferating colonies. These colony-forming cells showed distinct patterns of cell movement. Increasing cell density enhanced cell movement facilitating cell:cell contact, which resulted in increased proportion of dividing cells and improved survival postplating of normal hESCs. In contrast, most of the karyotypically abnormal cells reentered the cell cycle on plating and gave rise to healthy progeny, without the need for cell:cell contacts and independent of their motility patterns.

Quantitative chemical imaging and unsupervised analysis using hyperspectral coherent anti-Stokes Raman scattering microscopy.

In this work, we report a method to acquire and analyze hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy images of organic materials and biological samples resulting in an unbiased quantitative chemical analysis. The method employs singular value decomposition on the square root of the CARS intensity, providing an automatic determination of the components above noise, which are retained. Complex CARS susceptibility spectra, which are linear in the chemical composition, are retrieved from the CARS intensity spectra using the causality of the susceptibility by two methods, and their performance is evaluated by comparison with Raman spectra. We use non-negative matrix factorization applied to the imaginary part and the nonresonant real part of the susceptibility with an additional concentration constraint to obtain absolute susceptibility spectra of independently varying chemical components and their absolute concentration. We demonstrate the ability of the method to provide quantitative chemical analysis on known lipid mixtures. We then show the relevance of the method by imaging lipid-rich stem-cell-derived mouse adipocytes as well as differentiated embryonic stem cells with a low density of lipids. We retrieve and visualize the most significant chemical components with spectra given by water, lipid, and proteins segmenting the image into the cell surrounding, lipid droplets, cytosol, and the nucleus, and we reveal the chemical structure of the cells, with details visualized by the projection of the chemical contrast into a few relevant channels.

iTRAQ identification of candidate serum biomarkers associated with metastatic progression of human prostate cancer.

A major challenge in the management of patients with prostate cancer is identifying those individuals at risk of developing metastatic disease, as in most cases the disease will remain indolent. We analyzed pooled serum samples from 4 groups of patients (n = 5 samples/group), collected prospectively and actively monitored for a minimum of 5 yrs. Patients groups were (i) histological diagnosis of benign prostatic hyperplasia with no evidence of cancer 'BPH', (ii) localised cancer with no evidence of progression, 'non-progressing' (iii) localised cancer with evidence of biochemical progression, 'progressing', and (iv) bone metastasis at presentation 'metastatic'. Pooled samples were immuno-depleted of the 14 most highly abundant proteins and analysed using a 4-plex iTRAQ approach. Overall 122 proteins were identified and relatively quantified. Comparisons of progressing versus non-progressing groups identified the significant differential expression of 25 proteins (p<0.001). Comparisons of metastatic versus progressing groups identified the significant differential expression of 23 proteins. Mapping the differentially expressed proteins onto the prostate cancer progression pathway revealed the dysregulated expression of individual proteins, pairs of proteins and 'panels' of proteins to be associated with particular stages of disease development and progression. The median immunostaining intensity of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), one of the candidates identified, was significantly higher in osteoblasts in close proximity to metastatic tumour cells compared with osteoblasts in control bone (p = 0.0353, Mann Whitney U). Our proteomic approach has identified leads for potentially useful serum biomarkers associated with the metastatic progression of prostate cancer. The panels identified, including eEF1A1 warrant further investigation and validation.

Eight-plex iTRAQ analysis of variant metastatic human prostate cancer cells identifies candidate biomarkers of progression: An exploratory study.

BACKGROUND: Due to the heterogeneity in the biological behavior of prostate cancer, biomarkers that can reliably distinguish indolent from aggressive disease are urgently needed to inform treatment choices. METHODS: We employed 8-plex isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to profile the proteomes of two distinct panels of isogenic prostate cancer cells with varying growth and metastatic potentials, in order to identify novel biomarkers associated with progression. The LNCaP, LNCaP-Pro5, and LNCaP-LN3 panel of cells represent a model of androgen-responsive prostate cancer, while the PC-3, PC-3M, and PC-3M-LN4 panel represent a model of androgen-insensitive disease. RESULTS: Of the 245 unique proteins identified and quantified (>or=95% confidence; >or=2 peptides/protein), 17 showed significant differential expression (>or=+/-1.5), in at least one of the variant LNCaP cells relative to parental cells. Similarly, comparisons within the PC-3 panel identified 45 proteins to show significant differential expression in at least one of the variant PC-3 cells compared with parental cells. Differential expression of selected candidates was verified by Western blotting or immunocytochemistry, and corresponding mRNA expression was determined by quantitative real-time PCR (qRT-PCR). Immunostaining of prostate tissue microarrays for ERp5, one of the candidates identified, showed a significant higher immunoexpression in pre-malignant lesions compared with non-malignant epithelium (P < 0.0001, Mann-Whitney U-test), and in high Gleason grade (4-5) versus low grade (2-3) cancers (P < 0.05). CONCLUSIONS: Our study provides proof of principle for the application of an 8-plex iTRAQ approach to uncover clinically relevant candidate biomarkers for prostate cancer progression.

Novel regulators of stem cell fates identified by a multivariate phenotype screen of small compounds on human embryonic stem cell colonies.

Understanding the complex mechanisms that govern the fate decisions of human embryonic stem cells (hESCs) is fundamental to their use in cell replacement therapies. The progress of dissecting these mechanisms will be facilitated by the availability of robust high-throughput screening assays on hESCs. In this study, we report an image-based high-content assay for detecting compounds that affect hESC survival or pluripotency. Our assay was designed to detect changes in the phenotype of hESC colonies by quantifying multiple parameters, including the number of cells in a colony, colony area and shape, intensity of nuclear staining, and the percentage of cells in the colony that express a marker of pluripotency (TRA-1-60), as well as the number of colonies per well. We used this assay to screen 1040 compounds from two commercial compound libraries, and identified 17 that promoted differentiation, as well as 5 that promoted survival of hESCs. Among the novel small compounds we identified with activity on hESC are several steroids that promote hESC differentiation and the antihypertensive drug, pinacidil, which affects hESC survival. The analysis of overlapping targets of pinacidil and the other survival compounds revealed that activity of PRK2, ROCK, MNK1, RSK1, and MSK1 kinases may contribute to the survival of hESCs.

Prostate cancer proteomics: The urgent need for clinically validated biomarkers.

Prostate cancer (PCa) is the most common cancer diagnosis and the second most common cause of cancer-related deaths in men. Currently, serum prostate-specific antigen (PSA) is the only biomarker widely used in the diagnosis and management of patients with PCa. However, PSA lacks diagnostic sensitivity and specificity, leading to false-negative and false-positive test results. PSA cannot distinguish indolent from aggressive disease, leading to many patients being over-treated with associated side-effects. Furthermore, PSA is unable to identify which tumors are likely to become unresponsive to treatment at an early stage. Thus, there is an urgent need for clinically validated biomarkers which will improve the diagnosis and management of PCa. Given the heterogeneity of PCa it is likely that a panel of biomarkers will be required. In the quest for PCa biomarkers, a wide range of samples including urine, serum, tissues, and cell lines have been studied using proteomic approaches such as 2-DE, SELDI-TOF, SILAC, ICAT, iTRAQ, and MALDI-IMS. The value of these technologies, and other emerging platforms such as selected reaction monitoring (SRM) and multiple reaction monitoring (MRM), are discussed in the context of biomarker discovery, validation and addressing the "bottle-necks" that exist prior to clinical translation.

iTRAQ-facilitated proteomic analysis of human prostate cancer cells identifies proteins associated with progression.

The unpredictable behavior of prostate cancer presents a major clinical challenge during patient management. In order to gain an insight into the molecular mechanisms associated with prostate cancer progression, we employed the shot-gun proteomic approach of isobaric tags for relative and absolute quantitation (iTRAQ), followed by 2D-LC-MS/MS, using the poorly metastatic LNCaP cell line and its highly metastatic variant LNCaP-LN3 cell line as a model. A total number of 280 unique proteins were identified (> or =95% confidence), and relative expression data was obtained for 176 of these. Ten proteins were found to be significantly up-regulated (> or =1.50 fold), while 4 proteins were significantly down-regulated (> or = -1.50 fold), in LNCaP-LN3 cells. Differential expression of brain creatine kinase (CKBB), soluble catechol-O-methyltransferase (S-COMT), tumor rejection antigen (gp96), and glucose regulated protein, 78 kDa (grp78), was confirmed by Western blotting or independent 2D-PAGE analysis. Additionally, iTRAQ analysis identified absence of the lactate dehydrogenase-B (LDH-B) subunit in LNCaP-LN3 cells, confirming our published data. The clinical relevance of gp96 was assessed by immunohistochemistry using prostate tissues from benign ( n = 95), malignant ( n = 66), and metastatic cases ( n = 3). Benign epithelium showed absent/weak gp96 expression in the basal cells, in contrast to the moderate/strong expression seen in malignant epithelium. Furthermore, there was a statistically significant difference in the intensity of gp96 expression between benign and malignant cases ( p < 0.0005, Mann-Whitney U). Our study is the first to report the application of iTRAQ technology and its potential for the global proteomic profiling of prostate cancer cells, including the identification of absent protein expression.