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Collagen makes up a large proportion of the human body, particularly the skin. As the body ages, collagen content decreases, resulting in wrinkled skin and decreased wound healing capabilities. This paper presents a method of delivering type I collagen into porcine and human skin utilizing a polyvinylpyrrolidone microneedle delivery system. The microneedle patches were made with concentrations of 1, 2, 4, and 8% type I collagen (w/w). Microneedle structures and the distribution of collagen were characterized using scanning electron microscopy and confocal microscopy. Patches were then applied on the porcine and human skin, and their effectiveness was examined using fluorescence microscopy. The results illustrate that this microneedle delivery system is effective in delivering collagen I into the epidermis and dermis of porcine and human skin. Since the technique presented in this paper is quick, safe, effective and easy, it can be considered as a new collagen delivery method for cosmetic and therapeutic applications.

Biofilms-communities of bacteria encased in a polymer-rich matrix-confer bacteria with the ability to persist in pathologic host contexts, such as the cystic fibrosis (CF) airways. How bacteria assemble polymers into biofilms is largely unknown. We find that the extracellular matrix produced by Pseudomonas aeruginosa self-assembles into a liquid crystal through entropic interactions between polymers and filamentous Pf bacteriophages, which are long, negatively charged filaments. This liquid crystalline structure enhances biofilm function by increasing adhesion and tolerance to desiccation and antibiotics. Pf bacteriophages are prevalent among P. aeruginosa clinical isolates and were detected in CF sputum. The addition of Pf bacteriophage to sputum polymers or serum was sufficient to drive their rapid assembly into viscous liquid crystals. Fd, a related bacteriophage of Escherichia coli, has similar biofilm-building capabilities. Targeting filamentous bacteriophage or the liquid crystalline organization of the biofilm matrix may represent antibacterial strategies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A versatile approach for the design and fabrication of multilayer magnetic scaffolds with tunable magnetic gradients is described. Multilayer magnetic gelatin membrane scaffolds with intrinsic magnetic gradients were designed to encapsulate magnetized bioagents under an externally applied magnetic field for use in magnetic-field-assisted tissue engineering. The temperature of the individual membranes increased up to 43.7 °C under an applied oscillating magnetic field for 70 s by magnetic hyperthermia, enabling the possibility of inducing a thermal gradient inside the final 3D multilayer magnetic scaffolds. On the basis of finite element method simulations, magnetic gelatin membranes with different concentrations of magnetic nanoparticles were assembled into 3D multilayered scaffolds. A magnetic-gradient-controlled distribution of magnetically labeled stem cells was demonstrated in vitro. This magnetic biomaterial-magnetic cell strategy can be expanded to a number of different magnetic biomaterials for various tissue engineering applications.

Vascularisation is often deemed the holy grail of tissue engineering because it is one of the key preconditions that determine the in vivo viability of tissue constructs. Given that a well-developed vascular network allows greater complexity in tissue design and helps regulate tissue metabolism, it appears that the overall outcome of engineered tissue implants depends on the success of microvessel formation, maturation and patterning. Current approaches to vascularising tissue include both in vivo and ex vivo techniques, where blood vessel formation is either spontaneous or guided by physical or biochemical factors. The success of these strategies can then be monitored and evaluated for clinical benefit through numerous standard and novel strategies. Despite the impressive progress in the field of tissue engineering in recent times, there are still numerous technical, immunological, surgical and ethical challenges to overcome. Future prospects in this field are likely to depend on the adoption of a wide-ranging approach incorporating a combination of salient themes such as genetic manipulation, modular assembly and bioreactor coupling. Where applicable, the potential contributions of nanobiotechnology to tissue vascularisation will be discussed as appropriate., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: Using TCGA database, we had demonstrated that aberrantly activated Forkhead box M1 (FOXM1) correlates to worse overall survival in a subgroup of platinum resistant patients. Application of thiostrepton, a natural thiazole antibiotics that inhibits FOXM1 transcription activity in the clinic is hampered by difficulties in synthesis, degradation potential, and solubility. In this study, we aim to identify potential FOXM1 small molecule inhibitors to develop a new class of therapeutic agents to address the challenges in treating chemotherapy resistant EOC., Methods: We used in silico screening of compounds against a solved structure of FOXM1 and subsequently to derive a list of possible compounds that could inhibit FOXM1. Three compounds were tested for in vitro cytotoxicity and FOXM1 expression level was confirmed by RT-PCR and Western blot in EOC cell lines., Results: The FOXM1 structure obtained from 3G73 represented the DNA binding region of FOXM1 and possessed the winged helix fold representative of the Forkhead family of enzymes with two wings in direct contact with DNA. For ease of representation, we described both wings as a dimer and a single wing as a monomer. From this structure, we hypothesized two main models of how thiostrepton binding to FOXM1 could possibly curtail its transcriptional activity. In the first model thiostrepton could bind either of the wings or both wings and prevent association to DNA. In the second model thiostrepton bind the FOXM1/DNA complex and weaken association of FOXM1 to DNA. Subsequently, small molecular inhibitors could also use either of the models to inhibit transcription. To account for both models, the NCI diversity set was screened against the FOXM1 dimer:DNA complex (39 hits), dimer (11 hits) and monomer (14 hits). Those hits were further classified by chemical structure, biological function and chemical similarities to known molecules that target FOXM1. In cellular cytotoxicity assays, N-phenylphenanthren-9-amine (related to hit #225) successfully showed cytotoxicity to all three cell lines with IC50 around 1μM, and downregulate FOXM1 and transcription of its downstream molecules such as CCNB1., Conclusion: By a combination of in silico screening coupled to cellular cytotoxicity studies, we have taken the first step towards identifying potential inhibitors of FOXM1 that can replace thiostrepton., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Biomaterials are extensively used to restore damaged tissues, in the forms of implants (e.g. tissue engineered scaffolds) or biomedical devices (e.g. pacemakers). Once in contact with the physiological environment, nanostructured biomaterials undergo modifications as a result of endogenous proteins binding to their surface. The formation of this macromolecular coating complex, known as 'protein corona', onto the surface of nanoparticles and its effect on cell-particle interactions are currently under intense investigation. In striking contrast, protein corona constructs within nanostructured porous tissue engineering scaffolds remain poorly characterized. As organismal systems are highly dynamic, it is conceivable that the formation of distinct protein corona on implanted scaffolds might itself modulate cell-extracellular matrix interactions. Here, we report that corona complexes formed onto the fibrils of engineered collagen scaffolds display specific, distinct, and reproducible compositions that are a signature of the tissue microenvironment as well as being indicative of the subject's health condition. Protein corona formed on collagen matrices modulated cellular secretome in a context-specific manner ex-vivo , demonstrating their role in regulating scaffold-cellular interactions. Together, these findings underscore the importance of custom-designing personalized nanostructured biomaterials, according to the biological milieu and disease state. We propose the use of protein corona as in situ biosensor of temporal and local biomarkers.

Systemic therapies for inflammatory bowel disease are associated with an increased risk of infections and malignancies. Topical therapies reduce systemic exposure, but can be difficult to retain or have limited proximal distribution. To mitigate these issues, we developed a thermo-sensitive platform, using a polymer-based system that is liquid at room temperature but turns into a viscous gel on reaching body temperature. After rectal administration to mice with dextran sulfate sodium-induced colitis, the platform carrying budesonide or mesalamine becomes more viscoelastic near body temperature. Mice given the drug-containing platform gained more weight and had reduced histologic and biologic features of colitis than mice given the platform alone or liquid drugs via enema. Image analysis showed that enemas delivered with and without the platform reached similar distances in the colons of mice, but greater colonic retention was achieved by using the platform., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Introduction: Cardiovascular diseases such as coronary heart disease often necessitate the surgical repair using conduits. Although autografts still remain the gold standard, the inconvenience of harvesting and/or insufficient availability in patients with atherosclerotic disease has given impetus to look into alternative sources for vascular grafts., Areas Covered: There are four main techniques to produce tissue-engineered vascular grafts (TEVGs): i) biodegradable synthetic scaffolds; ii) gel-based scaffolds; iii) decellularised scaffolds and iv) self-assembled cell-sheet-based techniques. The first three techniques can be grouped together as scaffold-guided approach as it involves the use of a construct to function as a supportive framework for the vascular graft. The most significant advantages of TEVGs are that it possesses the ability to grow, remodel and respond to environmental factors. Cell sources for TEVGs include mature somatic cells, stem cells, adult progenitor cells and pluripotent stem cells., Expert Opinion: TEVG holds great promise with advances in nanotechnology, coupled with important refinements in tissue engineering and decellularisation techniques. This will undoubtedly be an important milestone for cardiovascular medicine when it is eventually translated to clinical use.

There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation.

Nanoparticle-mediated sustained delivery of therapeutics is one of the highly effective and increasingly utilized applications of nanomedicine. Here, we report the development and application of a drug delivery system consisting of polyethylene glycol (PEG)-conjugated liposomal nanoparticles as an efficient in vivo delivery approach for [Pyr1]-apelin-13 polypeptide. Apelin is an adipokine that regulates a variety of biological functions including cardiac hypertrophy and hypertrophy-induced heart failure. The clinical use of apelin has been greatly impaired by its remarkably short half-life in circulation. Here, we investigate whether [Pyr1]-apelin-13 encapsulation in liposome nanocarriers, conjugated with PEG polymer on their surface, can prolong apelin stability in the blood stream and potentiate apelin beneficial effects in cardiac function. Atomic force microscopy and dynamic light scattering were used to assess the structure and size distribution of drug-laden nanoparticles. [Pyr1]-apelin-13 encapsulation in PEGylated liposomal nanocarriers resulted in sustained and extended drug release both in vitro and in vivo. Moreover, intraperitoneal injection of [Pyr1]-apelin-13 nanocarriers in a mouse model of pressure-overload induced heart failure demonstrated a sustainable long-term effect of [Pyr1]-apelin-13 in preventing cardiac dysfunction. We concluded that this engineered nanocarrier system can serve as a delivery platform for treating heart injuries through sustained bioavailability of cardioprotective therapeutics., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Magnetic resonance imaging (MRI) has come to be known as a unique radiological imaging modality because of its ability to perform tomographic imaging of body without the use of any harmful ionizing radiation. The radiologists use MRI to gain insight into the anatomy of organs, including the brain, while biomedical researchers explore the modality to gain better understanding of the brain structure and function. However, due to limited resolution and contrast, the conventional MRI fails to show the brain microstructure. Diffusion tensor imaging (DTI) harnesses the power of conventional MRI to deduce the diffusion dynamics of water molecules within the tissue and indirectly create a three-dimensional sketch of the brain anatomy. DTI enables visualization of brain tissue microstructure, which is extremely helpful in understanding various neuropathologies and neurodegenerative disorders. In this review, we briefly discuss the background and operating principles of DTI, followed by current trends in DTI applications for biomedical and clinical investigation of various brain diseases and disorders.

Infrared (IR) imaging is a collection of non-invasive imaging techniques that utilize the IR domain of the electromagnetic spectrum for tissue assessment. A subset of these techniques construct images using back-reflected light, while other techniques rely on detection of IR radiation emitted by the tissue as a result of its temperature. Modern IR detectors sense thermal emissions and produce a heat map of surface temperature distribution in tissues. Thus, the IR spectrum offers a variety of imaging applications particularly useful in clinical diagnostic area, ranging from high-resolution, depth-resolved visualization of tissue to temperature variation assessment. These techniques have been helpful in the diagnosis of many medical conditions including skin/breast cancer, arthritis, allergy, burns, and others. In this review, we discuss current roles of IR-imaging techniques for diagnostic applications in dermatology with an emphasis on skin cancer, allergies, blisters, burns and wounds.

Effective skin regeneration therapies require a successful interface between progenitor cells and biocompatible delivery systems. We previously demonstrated the efficiency of a biomimetic pullulan-collagen hydrogel scaffold for improving bone marrow-derived mesenchymal stem cell survival within ischemic skin wounds by creating a "stem cell niche" that enhances regenerative cytokine secretion. Adipose-derived mesenchymal stem cells (ASCs) represent an even more appealing source of stem cells because of their abundance and accessibility, and in this study we explored the utility of ASCs for hydrogel-based therapies. To optimize hydrogel cell seeding, a rapid, capillary force-based approach was developed and compared with previously established cell seeding methods. ASC viability and functionality following capillary hydrogel seeding were then analyzed in vitro and in vivo. In these experiments, ASCs were seeded more efficiently by capillary force than by traditional methods and remained viable and functional in this niche for up to 14 days. Additionally, hydrogel seeding of ASCs resulted in the enhanced expression of multiple stemness and angiogenesis-related genes, including Oct4, Vegf, Mcp-1, and Sdf-1. Moving in vivo, hydrogel delivery improved ASC survival, and application of both murine and human ASC-seeded hydrogels to splinted murine wounds resulted in accelerated wound closure and increased vascularity when compared with control wounds treated with unseeded hydrogels. In conclusion, capillary seeding of ASCs within a pullulan-collagen hydrogel bioscaffold provides a convenient and simple way to deliver therapeutic cells to wound environments. Moreover, ASC-seeded constructs display a significant potential to accelerate wound healing that can be easily translated to a clinical setting., (©AlphaMed Press.)

Introduction: Pathophysiologic changes associated with diabetes impair new blood vessel formation and wound healing. Mesenchymal stem cells derived from adipose tissue (ASCs) have been used clinically to promote healing, although it remains unclear whether diabetes impairs their functional and therapeutic capacity., Methods: In this study, we examined the impact of diabetes on the murine ASC niche as well as on the potential of isolated cells to promote neovascularization in vitro and in vivo. A novel single-cell analytical approach was used to interrogate ASC heterogeneity and subpopulation dynamics in this pathologic setting., Results: Our results demonstrate that diabetes alters the ASC niche in situ and that diabetic ASCs are compromised in their ability to establish a vascular network both in vitro and in vivo. Moreover, these diabetic cells were ineffective in promoting soft tissue neovascularization and wound healing. Single-cell transcriptional analysis identified a subpopulation of cells which was diminished in both type 1 and type 2 models of diabetes. These cells were characterized by the high expression of genes known to be important for new blood vessel growth., Conclusions: Perturbations in specific cellular subpopulations, visible only on a single-cell level, represent a previously unreported mechanism for the dysfunction of diabetic ASCs. These data suggest that the utility of autologous ASCs for cell-based therapies in patients with diabetes may be limited and that interventions to improve cell function before application are warranted.

Background: Exosomes are small biological membrane vesicles that measure 30 to 100 nm in diameter. They are involved in a wide array of biological activities, such as cell-cell communication, signal transduction, transport of genetic materials, and modulation of immune response. Evidence indicates that they can be used as not only therapeutic agents targeted against disease but also diagnostic biomarkers for pathologic conditions., Objective: In this review, we endeavor to present exosomes as immunologic agents that can be used as pioneering cancer vaccines to prime the immune system and explicate their therapeutic and diagnostic capabilities., Methods: An extensive literature search for studies that involved the use of exosomes as immunotheranostic nanoparticles was conducted using PubMed, ISI Web of Knowledge, and Google Scholar. Clinical trials that involved exosomes were also compiled by searching the clinicaltrials.gov database., Results: In its therapeutic facet of application, exosomes can be used as vehicles for drug or gene delivery. These biological vesicles have been found to have excellent host biodistribution and biocompatibility, issues often presented with gene delivery vehicles. Diagnostically, exosomes may prove to be useful biomarkers that are able to surpass current setbacks of modern diagnostic testing, which include invasive methods. Finally, current evidence has implied that the use of exosomes could form the basis for the development of future cell-free cancer vaccines., Conclusion: Exosomes have numerous functions, and their double-edged features make the scope of their clinical applications, as both a diagnostic and therapeutic tool, immense., (Crown Copyright © 2014. Published by EM Inc USA. All rights reserved.)

Unlabelled: Zinc protoporphyrin (ZnPP) is a promising metalloporphyrin with sufficient potency, but has poor solubility and is not absorbed well orally. Intragastric administration of ZnPP microparticles (30 μmol/kg) to 3-day-old mice resulted in a twofold increase in potency and no signs of phototoxicity., Conclusion: The use of polymeric particulate delivery systems can improve the stability and enhance intestinal absorption of ZnPP, while retaining HO inhibitory potency without photosensitising effects, and thus is potentially useful in treating neonatal hyperbilirubinemia., (©2014 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.)

Due to the limited self-renewal capacity of cardiomyocytes, the mammalian heart exhibits impaired regeneration and insufficient ability to restore heart function after injury. Cardiovascular tissue engineering is currently considered as a promising alternative therapy to restore the structure and function of the failing heart. Recent evidence suggests that the epicardium may play critical roles in regulation of myocardial development and regeneration. One of the mechanisms that has been proposed for the restorative effect of the epicardium is the specific physiomechanical cues that this layer provides to the cardiac cells. In this article we explore whether a new generation of epicardium-mimicking, acellular matrices can be utilized to enhance cardiac healing after injury. The matrix consists of a dense collagen scaffold with optimized biomechanical properties approaching those of embryonic epicardium. Grafting the epicardial patch onto the ischemic myocardium--promptly after the incidence of infarct--resulted in preserved contractility, attenuated ventricular remodeling, diminished fibrosis, and vascularization within the injured tissue in the adult murine heart.

Skin cancer is the leading cause of malignancy in the United States, with Basal Cell Carcinoma, Squamous Cell Carcinoma , and Melanoma being the three most common diagnoses, respectively. Squamous Cell Carcinoma (SCC) is a particular concern for patients suffering from Dystrophic Epidermolysis Bullosa (DEB), a disease that affects the production and function of collagen VII, a protein that forms the anchoring fibrils which bind the epidermis to the dermis. Patients with DEB suffer from chronic blistering and wounds that have impaired healing capabilities, often leading to the development of SCC and eventual mortality. Nanomedicine is playing an increasing role in the delivery of effective therapeutics to combat a wide range of diseases, including the imaging and treatment of SCC. In this review, we discuss the role of nanoparticles in the treatment of SCC with an emphasis on PLGA nanoparticles and SCCs found in patients suffering from DEB, and address recent patents that are pertinent to the development of novel nanomedical therapeutics.

Airway tissue ischemia and hypoxia in human lung transplantation is a consequence of the sacrifice of the bronchial circulation during the surgical procedure and is a major risk factor for the development of airway anastomotic complications. Augmented expression of hypoxia-inducible factor (HIF)-1α promotes microvascular repair and alleviates allograft ischemia and hypoxia. Deferoxamine mesylate (DFO) is an FDA-approved iron chelator which has been shown to upregulate cellular HIF-1α. Here, we developed a nanoparticle formulation of DFO that can be topically applied to airway transplants at the time of surgery. In a mouse orthotopic tracheal transplant (OTT) model, the DFO nanoparticle was highly effective in enhancing airway microvascular perfusion following transplantation through the production of the angiogenic factors, placental growth factor (PLGF) and stromal cell-derived factor (SDF)-1. The endothelial cells in DFO treated airways displayed higher levels of p-eNOS and Ki67, less apoptosis, and decreased production of perivascular reactive oxygen species (ROS) compared to vehicle-treated airways. In summary, a DFO formulation topically-applied at the time of surgery successfully augmented airway anastomotic microvascular regeneration and the repair of alloimmune-injured microvasculature. This approach may be an effective topical transplant-conditioning therapy for preventing airway complications following clinical lung transplantation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The secondary structures of amyloidogenic proteins are largely influenced by various intra and extra cellular microenvironments and metal ions that govern cytotoxicity. The secondary structure of a prion fragment, PrP(111-126), was determined using circular dichroism (CD) spectroscopy in various microenvironments. The conformational preferences of the prion peptide fragment were examined by changing solvent conditions and pH, and by introducing external stress (sonication). These physical and chemical environments simulate various cellular components at the water-membrane interface, namely differing aqueous environments and metal chelating ions. The results show that PrP(111-126) adopts different conformations in assembled and non-assembled forms. Aging studies on the PrP(111-126) peptide fragment in aqueous buffer demonstrated a structural transition from random coil to a stable β-sheet structure. A similar, but significantly accelerated structural transition was observed upon sonication in aqueous environment. With increasing TFE concentrations, the helical content of PrP(111-126) increased persistently during the structural transition process from random coil. In aqueous SDS solution, PrP(111-126) exhibited β-sheet conformation with greater α-helical content. No significant conformational changes were observed under various pH conditions. Addition of Cu(2+) ions inhibited the structural transition and fibril formation of the peptide in a cell free in vitro system. The fact that Cu(2+) supplementation attenuates the fibrillar assemblies and cytotoxicity of PrP(111-126) was witnessed through structural morphology studies using AFM as well as cytotoxicity using MTT measurements. We observed negligible effects during both physical and chemical stimulation on conformation of the prion fragment in the presence of Cu(2+) ions. The toxicity of PrP(111-126) to cultured astrocytes was reduced following the addition of Cu(2+) ions, owing to binding affinity of copper towards histidine moiety present in the peptide.

Regeneration of the damaged myocardium is one of the most challenging fronts in the field of tissue engineering due to the limited capacity of adult heart tissue to heal and to the mechanical and structural constraints of the cardiac tissue. In this study we demonstrate that an engineered acellular scaffold comprising type I collagen, endowed with specific physiomechanical properties, improves cardiac function when used as a cardiac patch following myocardial infarction. Patches were grafted onto the infarcted myocardium in adult murine hearts immediately after ligation of left anterior descending artery and the physiological outcomes were monitored by echocardiography, and by hemodynamic and histological analyses four weeks post infarction. In comparison to infarcted hearts with no treatment, hearts bearing patches preserved contractility and significantly protected the cardiac tissue from injury at the anatomical and functional levels. This improvement was accompanied by attenuated left ventricular remodeling, diminished fibrosis, and formation of a network of interconnected blood vessels within the infarct. Histological and immunostaining confirmed integration of the patch with native cardiac cells including fibroblasts, smooth muscle cells, epicardial cells, and immature cardiomyocytes. In summary, an acellular biomaterial with specific biomechanical properties promotes the endogenous capacity of the infarcted myocardium to attenuate remodeling and improve heart function following myocardial infarction., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

An unmet need exists for the development of next-generation multifunctional nanocomposite materials for biomedical applications, particularly in the field of cardiovascular regenerative biology. Herein, we describe the preparation and characterization of a novel polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU) nanocomposite polymer with covalently attached anti-CD34 antibodies to enhance capture of circulating endothelial progenitor cells (EPC). This material may be used as a new coating for bare metal stents used after balloon angioplasty to improve re-endothelialization. Biophysical characterization techniques were used to assess POSS-PCU and its subsequent functionalization with anti-CD34 antibodies. Results indicated successful covalent attachment of anti-CD34 antibodies on the surface of POSS-PCU leading to an increased propensity for EPC capture, whilst maintaining in vitro biocompatibility and hemocompatibility. POSS-PCU has already been used in 3 first-in-man studies, as a bypass graft, lacrimal duct and a bioartificial trachea. We therefore postulate that its superior biocompatibility and unique biophysical properties would render it an ideal candidate for coating medical devices, with stents as a prime example. Taken together, anti-CD34 functionalized POSS-PCU could form the basis of a nano-inspired polymer platform for the next generation stent coatings.

In situ endothelialization of cardiovascular implants has emerged in recent years as an attractive means of targeting the persistent problems of thrombosis and intimal hyperplasia. This study aimed to investigate the efficacy of immobilizing anti-CD34 antibodies onto a POSS-PCU nanocomposite polymer surface to sequester endothelial progenitor cells (EPCs) from human blood, and to characterize the surface properties and hemocompatibility of this surface. Amine-functionalized fumed silica was used to covalently conjugate anti-CD34 to the polymer surface. Water contact angle, fluorescence microscopy, and scanning electron microscopy were used for surface characterization. Peripheral blood mononuclear cells (PBMCs) were seeded on modified and pristine POSS-PCU polymer films. After 7 days, adhered cells were immunostained for the expression of EPC and endothelial cell markers, and assessed for the formation of EPC colonies. Hemocompatibility was assessed by thromboelastography, and platelet activation and adhesion assays. The number of EPC colonies formed on anti-CD34-coated POSS-PCU surfaces was not significantly higher than that of POSS-PCU (5.0±1.0 vs. 1.7±0.6, p>0.05). However, antibody conjugation significantly improved hemocompatibility, as seen from the prolonged reaction and clotting times, decreased angle and maximum amplitude (p<0.05), as well as decreased platelet adhesion (76.8±7.8 vs. 8.4±0.7, p<0.05) and activation. Here, we demonstrate that POSS-PCU surface immobilized anti-CD34 antibodies selectively captured CD34+ cells from peripheral blood, although only a minority of these were EPCs. Nevertheless, antibody conjugation significantly improves the hemocompatibility of POSS-PCU, and should therefore continue to be explored in combination with other strategies to improve the specificity of EPC capture to promote in situ endothelialization.

Pulmonary hypertension (PH) is a serious condition that affects mainly young and middle-aged women, and its etiology is poorly understood. A prominent pathological feature of PH is accumulation of macrophages near the arterioles of the lung. In both clinical tissue and the SU5416 (SU)/athymic rat model of severe PH, we found that the accumulated macrophages expressed high levels of leukotriene A4 hydrolase (LTA4H), the biosynthetic enzyme for leukotriene B4 (LTB4). Moreover, macrophage-derived LTB4 directly induced apoptosis in pulmonary artery endothelial cells (PAECs). Further, LTB4 induced proliferation and hypertrophy of human pulmonary artery smooth muscle cells. We found that LTB4 acted through its receptor, BLT1, to induce PAEC apoptosis by inhibiting the protective endothelial sphingosine kinase 1 (Sphk1)-endothelial nitric oxide synthase (eNOS) pathway. Blocking LTA4H decreased in vivo LTB4 levels, prevented PAEC apoptosis, restored Sphk1-eNOS signaling, and reversed fulminant PH in the SU/athymic rat model of PH. Antagonizing BLT1 similarly reversed established PH. Inhibition of LTB4 biosynthesis or signal transduction in SU-treated athymic rats with established disease also improved cardiac function and reopened obstructed arterioles; this approach was also effective in the monocrotaline model of severe PH. Human plexiform lesions, one hallmark of PH, showed increased numbers of macrophages, which expressed LTA4H, and patients with connective tissue disease-associated pulmonary arterial hypertension exhibited significantly higher LTB4 concentrations in the systemic circulation than did healthy subjects. These results uncover a possible role for macrophage-derived LTB4 in PH pathogenesis and identify a pathway that may be amenable to therapeutic targeting.

We present a method of fabricating microneedles from polyvinylpyrrolidone (PVP) that enables delivery of intact proteins (or peptides) to the dermal layers of the skin. PVP is known to self-assemble into branched hollow fibers in aqueous and alcoholic solutions; we utilized this property to develop dissolvable patches of microneedles. Proteins were dissolved in concentrated PVP solution in both alcohol and water, poured into polydimethylsiloxane templates shaped as microneedles and, upon evaporation of solvent, formed into concentric, fibrous, layered structures. This approach of making PVP microneedles overcomes problems in dosage, uniform delivery and stability of protein formulation as compared to protein-coated metallic microneedles or photopolymerized PVP microneedles. Here we characterize the PVP microneedles and measure the delivery of proteins into skin. We show that our method of fabrication preserves the protein conformation. These microneedles can serve as a broadly useful platform for delivering protein antigens and therapeutic proteins to the skin, for example for allergen skin testing or immunotherapy., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The advent of optogenetics provides a new direction for the field of neuroscience and biotechnology, serving both as a refined investigative tool and as potential cure for many medical conditions via genetic manipulation. Although still in its infancy, recent advances in optogenetics has made it possible to remotely manipulate in vivo cellular functions using light. Coined Nature Methods' 'Method of the Year' in 2010, the optogenetic toolbox has the potential to control cell, tissue and even animal behaviour. This optogenetic toolbox consists of light-sensitive proteins that are able to modulate membrane potential in response to light. Channelrhodopsins (ChR) are light-gated microbial ion channels, which were first described in green algae. ChR2 (a subset of ChR) is a seven transmembrane α helix protein, which evokes membrane depolarization and mediates an action potential upon photostimulation with blue (470 nm) light. By contrast to other seven-transmembrane proteins that require second messengers to open ion channels, ChR2 form ion channels themselves, allowing ultrafast depolarization (within 50 milliseconds of illumination). It has been shown that integration of ChR2 into various tissues of mice can activate neural circuits, control heart muscle contractions, and even restore breathing after spinal cord injury. More compellingly, a plethora of evidence has indicated that artificial expression of ChR2 in retinal ganglion cells can reinstate visual perception in mice with retinal degeneration., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cardiovascular disease is one of the major causes of death in the world. Coronary stenting in percutaneous coronary intervention (PCI) has revolutionized the field of cardiology. Coronary stenting is seen as a less invasive procedure compared to coronary artery bypass graft (CABG) surgery. Two main types of stents currently exist in the market: bare-metal stents (BMS) and drug-eluting stents (DES). DES were developed in response to problems associated with BMS use, like neointimal hyperplasia leading to restenosis. However, the use of DES engendered other problems as well, like late stent thrombosis (ST), which is a serious and lethal complication. Gene-eluting stents (GES) have recently been proposed as a novel method of circumventing problems seen in BMS and DES. Utilizing nanotechnology, sustained and localized delivery of genes can mitigate problems of restenosis and late ST by accelerating the regenerative capacity of re-endothelialization. Therefore this review seeks to explore the realm of GES as a novel alternative to BMS and DES, and its potential implications in the field of nanotechnology and regenerative medicine.

Percutaneous coronary intervention (PCI) is used to treat blocked coronary arteries. Bare-metal stents (BMS) were first used in PCI but often necessitated repair procedures due to in-stent restenosis. Drug-eluting stents (DES) were developed to address this problem as the stent-incorporated anti-proliferative drugs prevented restenosis. However late-stent thrombosis arose with the use of DES due to polymer hypersensitivity and impaired re-endothelialization. Evidence suggests that using a combination of biofunctionalized polymers and antibody/peptide motifs can prevent thrombosis while ensuring in situ endothelialization. The advent of nanotechnology has engendered techniques like layer-by-layer self-assembly, and localized drug and gene delivery using nanoparticles. Therefore, this review seeks to explore the convergence of biotechnology and nanotechnology for the next generation coronary stent coatings, with an emphasis on its development from bench to beside., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Exosomes are biological membrane vesicles measuring 30 to 100 nm. They contain an abundance of small molecules like tetraspanins, receptors for targeting and adhesion, lipids, and RNA. They are secreted by most biological cells, and are involved in a plethora of physiological functions including, but not limited to, transport of genetic material, modulation of the immune system, and cell-to-cell communication. It has been further reported that exosomes utilize a mechanism similar to that of viruses for gaining entry into cells. Due to their viral-like transfection efficiency and inherent biological function, compelling evidence indicates that exosomes can be used as novel delivery platforms for gene therapy. Furthermore, RNA-containing exosomes derived from cells can serve as functional genetic biomarkers for diseases. This twin modality of therapeutic and diagnostic is termed theranostics in the emerging field of nanomedicine. Hence in this review, we seek to expound on the various facets of exosomes, highlighting their significance in and relevance to nano-theranostic platforms for gene therapy., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The soluble Abeta oligomers in brain are highly correlated with memory related synaptic dysfunctions in Alzheimer's disease (AD). However, more recent studies implicate the involvement of Abeta dimers and trimers in memory related AD pathology. Apparently, Abeta oligomers can bind with cellular prion protein at the membrane receptors, forming annular amyloid pores and membrane ion channels to induce aberrant spine cytoskeletal changes. Hence synapse targeting of Abeta oligomers involves activation of many receptors such as N-Methyl-D-aspartate (NMDA), alpha-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), nicotinic acetylcholine (nAChRs), p75 neurotrophin (p75NTR) following aberrant clustering of metabotropic glutamate receptors (mGluR5) leading to neuronal loss and LTP failure. In particular, NMDA and AMPA receptor activation by soluble amyloid oligomers involves calcium mediated mitochondrial dysfunction, decreased Ca((2+))/calmodulin-dependent protein kinase II (CaMKII) levels at the synapses accompanying dramatic loss of synaptic proteins such as postsynaptic density-95 (PSD-95), dynamin-1 and synaptophysin. This kind of receptor-Abeta oligomer interaction might eventually affect the neuronal membrane integrity by altering dielectric barrier, various synaptic proteins, spine morphology and density and P/Q calcium currents that might provoke a cascade of events leading to neuronal loss and memory failure. In this review, we try to explain in detail the various possible mechanisms that connect Abeta oligomers with synapse damage and memory failure.

Amyloid accumulation in the brain of Alzheimer's patients results from altered processing of the 39- to 43-amino acid amyloid β protein (Aβ). The mechanisms for the elevated amyloid (Aβ(1-42)) are considered to be over-expression of the amyloid precursor protein (APP), enhanced cleavage of APP to Aβ, and decreased clearance of Aβ from the central nervous system (CNS). We report herein studies of Aβ stimulated effects on endothelial cells. We observe an interesting and as yet unprecedented feedback effect involving Aβ(1-42) fibril-induced synthesis of APP by Western blot analysis in the endothelial cell line Hep-1. We further observe an increase in the expression of Aβ(1-40) by flow cytometry and fluorescence microscopy. This phenomenon is reproducible for cultures grown both in the presence and absence of serum. In the former case, flow cytometry reveals that Aβ(1-40) accumulation is less pronounced than under serum-free conditions. Immunofluorescence staining further corroborates these observations. Cellular responses to fibrillar Aβ(1-42) treatment involving eNOS upregulation and increased autophagy are also reported.

Peripheral nerve injury is a debilitating condition. The gold standard for treatment is surgery, requiring an autologous nerve graft. Grafts are harvested from another part of the body (a secondary site) to treat the affected primary area. However, autologous nerve graft harvesting is not without risks, with associated problems including injury to the secondary site. Research into biomaterials has engendered the use of bioartificial nerve conduits as an alternative to autologous nerve grafts. These include synthetic and artificial materials, which can be manufactured into nerve conduits using techniques inspired by nanotechnology. Recent evidence indicates that peptide amphiphiles (PAs) are promising candidates for use as materials for bioengineering nerve conduits. PAs are biocompatible and biodegradable protein-based nanomaterials, capable of self-assembly in aqueous solutions. Their self-assembly system, coupled with their intrinsic capacity for carrying bioactive epitopes for tissue regeneration, form particularly novel attributes for biochemically-engineered materials. Furthermore, PAs can function as biomimetic materials and advanced drug delivery platforms for sustained and controlled release of a plethora of therapeutic agents. Here we review the realm of nerve conduit tissue engineering and the potential for PAs as viable materials in this exciting and rapidly advancing field., (Copyright © 2012 Elsevier B.V. All rights reserved.)

To determine whether the therapeutic activity of αB crystallin, small heat shock protein B5 (HspB5), was shared with other human sHsps, a set of seven human family members, a mutant of HspB5 G120 known to exhibit reduced chaperone activity, and a mycobacterial sHsp were expressed and purified from bacteria. Each of the recombinant proteins was shown to be a functional chaperone, capable of inhibiting aggregation of denatured insulin with varying efficiency. When injected into mice at the peak of disease, they were all effective in reducing the paralysis in experimental autoimmune encephalomyelitis. Additional structure activity correlations between chaperone activity and therapeutic function were established when linear regions within HspB5 were examined. A single region, corresponding to residues 73-92 of HspB5, forms amyloid fibrils, exhibited chaperone activity, and was an effective therapeutic for encephalomyelitis. The linkage of the three activities was further established by demonstrating individual substitutions of critical hydrophobic amino acids in the peptide resulted in the loss of all of the functions.

Background: The application of nanotechnology in biology and medicine represents a significant paradigm shift in the approach to the treatment of cancer. Evidence suggests that when exposed to near-infrared radiation (NIR), carbon nanotubes (CNTs) dissipate a substantial amount of heat energy. We have developed a novel nanocomposite polymer, polyhedral oligomeric silsesquioxane poly (carbonate-urea) urethane (POSS-PCU). POSS-PCU displays excellent biocompatibility and has been used in making artificial organs as well as protective coatings for medical devices., Results: Functionalizing (or "coating") CNTs with POSS-PCU confers biocompatibility and increase the amount of heat energy generated, by enhancing dispersion. Here we demonstrate that POSS-PCU-functionalized multi-walled CNTs (MWNTs) act synergistically together when exposed to NIR to thermally ablate cancer cells., Conclusion: Given that POSS-PCU has already been used in human in first-in-man studies as trachea, lacrimal duct, bypass graft and other organs, our long-term goal is to take POSS-PCU coated CNTs to clinical studies to address the treatment of cancer by optimizing its therapeutic index and increasing its specificity via antibody conjugation.

Alzheimer's disease (AD) is the most common neurodegenerative disease and the leading cause of dementia in the elderly. Accumulating evidence supports soluble amyloid-β (Aβ) oligomers as the leading candidate for the causative agent in AD and synapses as the primary site of Aβ oligomer action. However, the molecular and cellular mechanisms by which Aβ oligomers cause synaptic dysfunction and cognitive impairments remain poorly understood. Using primary cultures of rat hippocampal neurons as a model system, we show that the partitioning defective-1 (PAR-1)/microtubule affinity-regulating kinase (MARK) family kinases act as critical mediators of Aβ toxicity on synapses and dendritic spines. Overexpression of MARK4 led to tau hyperphosphorylation, reduced expression of synaptic markers, and loss of dendritic spines and synapses, phenotypes also observed after Aβ treatment. Importantly, expression of a non-phosphorylatable form of tau with the PAR-1/MARK site mutated blocked the synaptic toxicity induced by MARK4 overexpression or Aβ treatment. To probe the involvement of endogenous MARK kinases in mediating the synaptic toxicity of Aβ, we employed a peptide inhibitor capable of effectively and specifically inhibiting the activities of all PAR-1/MARK family members. This inhibitor abrogated the toxic effects of Aβ oligomers on dendritic spines and synapses as assayed at the morphological and electrophysiological levels. Our results reveal a critical role for PAR-1/MARK kinases in AD pathogenesis and suggest PAR-1/MARK inhibitors as potential therapeutics for AD and possibly other tauopathies where aberrant tau hyperphosphorylation is involved.

Stem cell-based therapies offer tremendous potential for skin regeneration following injury and disease. Functional stem cell units have been described throughout all layers of human skin and the collective physical and chemical microenvironmental cues that enable this regenerative potential are known as the stem cell niche. Stem cells in the hair follicle bulge, interfollicular epidermis, dermal papillae, and perivascular space have been closely investigated as model systems for niche-driven regeneration. These studies suggest that stem cell strategies for skin engineering must consider the intricate molecular and biologic features of these niches. Innovative biomaterial systems that successfully recapitulate these microenvironments will facilitate progenitor cell-mediated skin repair and regeneration.

In this study, we examined the capacity of a biomimetic pullulan-collagen hydrogel to create a functional biomaterial-based stem cell niche for the delivery of mesenchymal stem cells (MSCs) into wounds. Murine bone marrow-derived MSCs were seeded into hydrogels and compared to MSCs grown in standard culture conditions. Hydrogels induced MSC secretion of angiogenic cytokines and expression of transcription factors associated with maintenance of pluripotency and self-renewal (Oct4, Sox2, Klf4) when compared to MSCs grown in standard conditions. An excisonal wound healing model was used to compare the ability of MSC-hydrogel constructs versus MSC injection alone to accelerate wound healing. Injection of MSCs did not significantly improve time to wound closure. In contrast, wounds treated with MSC-seeded hydrogels showed significantly accelerated healing and a return of skin appendages. Bioluminescence imaging and FACS analysis of luciferase+/GFP+ MSCs indicated that stem cells delivered within the hydrogel remained viable longer and demonstrated enhanced engraftment efficiency than those delivered via injection. Engrafted MSCs were found to differentiate into fibroblasts, pericytes and endothelial cells but did not contribute to the epidermis. Wounds treated with MSC-seeded hydrogels demonstrated significantly enhanced angiogenesis, which was associated with increased levels of VEGF and other angiogenic cytokines within the wounds. Our data suggest that biomimetic hydrogels provide a functional niche capable of augmenting MSC regenerative potential and enhancing wound healing., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Substantial evidence suggests that the aggregation of amyloid-β (Aβ) peptide into fibrillar structures that is rich in β-sheets is implicated as the cause of Alzheimer's disease. Therefore, an attractive therapeutic strategy is to prevent or alter Aβ aggregation. Phenolic compounds are natural substances that are composed of one or more aromatic phenolic rings and present in wine, tea, fruits, vegetables and a wide variety of plants. In this work, we investigated the effects of ferulic acid, morin, quercetin and gossypol against Aβ aggregation. From the ThT and turbidity assays, it is observed that in addition to the fibril aggregate, another type of aggregate is formed in the presence of morin, quercetin, and gossypol. On the other hand, ferulic acid did not prevent fibril formation, but it did appear to reduce the average length of fibrils compared to Aβ alone. To study the protective effects of phenolic compounds on Aβ-induced toxicity, we utilized the nematode Caenorhabditis elegans (C. elegans) as an in vivo model organism, human Aβ is expressed intracellularly in the body wall muscle. We found that exposure of Caenorhabditis elegans to ferulic acid give more protection against Aβ toxicity than morin, quercetin and gossypol.

Amyloid diseases occur due to conformational change in the native protein. Understanding the amyloid peptide structural stability and conformational preference at the molecular level in membranous environment may lead to advancement in drug design and therapy. The conformational preferences of amyloid peptide fragments, Aβ₁₋₁₁, Aβ₁₂₋₂₂, Aβ₂₃₋₃₃ and Aβ₃₄₋₄₂ was studied in buffers, trifluoroethanol (TFE) and sodium dodecyl sulfate (SDS) micelles using circular dichroism spectroscopy. The fragment, Aβ₁₋₁₁ in TFE adopts a mixture of random coil and turn conformations. Aβ₁₂₋₂₂ and Aβ₂₃₋₃₃ underwent transition from random coil to helix conformation, while Aβ₃₄₋₄₂ exhibited β-sheet conformation in initial stage which was unaltered on complete evaporation of TFE. Addition of SDS to Aβ₁₂₋₂₂ and Aβ₃₄₋₄₂ favors β-sheet structure, which was predominant in the case of Aβ₃₄₋₄₂. However, in Aβ₁₋₁₁ and Aβ₂₃₋₃₃, no secondary structural change was noticed even at high SDS concentrations. On aging, all the peptide fragments showed β-sheet conformational transition. The C-terminal fragment has the ability to adopt predominant β-sheet conformation even in the presence of detergent and membrane mimicking environment. Altogether, the structural information gained from the short fragments could be further used for determining their role in the organization of Aβ peptide in stable fibril form., (Published by Elsevier Ltd.)

Cell-based therapies for wound repair are limited by inefficient delivery systems that fail to protect cells from the acute inflammatory environment. Here, a biomimetic hydrogel system is described that is based on the polymer pullulan, a carbohydrate glucan known to exhibit potent antioxidant capabilities. It is shown that pullulan hydrogels are an effective cell delivery system and improve mesenchymal stem cell survival and engraftment in high-oxidative-stress environments. The results suggest that glucan hydrogel systems may prove beneficial for progenitor-cell-based approaches to skin regeneration., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ischemic heart disease is the leading cause of death worldwide. Recent studies suggest that adipose tissue-derived stem cells (ASCs) can be used as a potential source for cardiovascular tissue engineering due to their ability to differentiate along the cardiovascular lineage and to adopt a proangiogenic phenotype. To understand better ASCs' biology, we used a novel 3D culture device. ASCs' and b.END-3 endothelial cell proliferation, migration, and vessel morphogenesis were significantly enhanced compared to 2D culturing techniques. ASCs were isolated from inguinal fat pads of 6-week-old GFP+/BLI+ mice. Early passage ASCs cells (P3-P4), PKH26-labeled murine b.END-3 cells or a co-culture of ASCs and b.END-3 cells were seeded at a density of 1 × 10(5) on three different surface configurations: (a) a 2D surface of tissue culture plastic, (b) Matrigel, and (c) a highly porous 3D scaffold fabricated from inert polystyrene. VEGF expression, cell proliferation, and tubulization, were assessed using optical microscopy, fluorescence microscopy, 3D confocal microscopy, and SEM imaging (n = 6). Increased VEGF levels were seen in conditioned media harvested from co-cultures of ASCs and b.END-3 on either Matrigel or a 3D matrix. Fluorescence, confocal, SEM, bioluminescence revealed improved cell, proliferation, and tubule formation for cells seeded on the 3D polystyrene matrix. Collectively, these data demonstrate that co-culturing ASCs with endothelial cells in a 3D matrix environment enable us to generate prevascularized tissue-engineered constructs. This can potentially help us to surpass the tissue thickness limitations faced by the tissue engineering community today., (Copyright © 2011 Wiley Periodicals, Inc.)

Vascular anastomosis is the cornerstone of vascular, cardiovascular and transplant surgery. Most anastomoses are performed with sutures, which are technically challenging and can lead to failure from intimal hyperplasia and foreign body reaction. Numerous alternatives to sutures have been proposed, but none has proven superior, particularly in small or atherosclerotic vessels. We have developed a new method of sutureless and atraumatic vascular anastomosis that uses US Food and Drug Administration (FDA)-approved thermoreversible tri-block polymers to temporarily maintain an open lumen for precise approximation with commercially available glues. We performed end-to-end anastomoses five times more rapidly than we performed hand-sewn controls, and vessels that were too small (<1.0 mm) to sew were successfully reconstructed with this sutureless approach. Imaging of reconstructed rat aorta confirmed equivalent patency, flow and burst strength, and histological analysis demonstrated decreased inflammation and fibrosis at up to 2 years after the procedure. This new technology has potential for improving efficiency and outcomes in the surgical treatment of cardiovascular disease.

Cancer is one of the main causes of death in the world, and according to the WHO it is projected to continue rising. Current diagnostic modalities for the detection of cancer include the use of x-rays, magnetic resonance imaging and positron emission tomography, among others. The treatment of cancer often involves the use (or combination) of chemotherapeutic drugs, radiotherapy and interventional surgery (for solid and operable tumors). The application of nanotechnology in biology and medicine is advancing rapidly. Recent evidence suggests that quantum dots (QDs) can be used to image cancer cells as they display superior fluorescent properties compared with conventional chromophores and contrast agents. In addition, carbon nanotubes (CNTs) have emerged as viable candidates for novel chemotherapeutic drug delivery-platforms. The unique photothermal properties of CNTs also allow them to be used in conjunction with near infrared radiation and lasers to thermally ablate cancer cells. Furthermore, mounting evidence indicates that it is possible to conjugate QDs to CNTs, making it possible to exploit their novel attributes in the realm of cancer theranostics (diagnostics and therapy). Here we review the current literature pertaining to the applications of QDs and CNTs in oncology, and also discuss the relevance and implications of nanomedicine in a clinical setting.

Polyethylenimine (PEI) based polymers are efficient agents for cell transfection. However, their use has been hampered due to high cell death associated with transfection thereby resulting in low efficiency of gene delivery within the cells. To circumvent the problem of cellular toxicity, metal binding peptides were linked to PEI. Eight peptide-PEI derivatives were synthesized to improve cell survival and transfection efficiency. TAT linked PEI was used as a control polymer. Peptides linked with PEI amines formed nanogels as shown by electron microscopy and atomic force microscopic measurements. Polymers were characterized by spectroscopic methods and their ability to form complexes with plasmids was tested using electrophoretic studies. These modifications improved polymer biocompatibility as well as cell survival markedly, when compared to PEI alone. A subset of the modified peptide-polymers also showed significantly higher transfection efficiency in primary human cells with respect to the widely used transfection agent, lipofectamine. Study of the underlying mechanism of the observed phenomena revealed lower levels of 'reactive oxygen species' (ROS) in the presence of the peptide-polymers when compared to PEI alone. This was further corroborated with global gene expression analysis which showed upregulation of multiple genes and pathways involved in regulating intracellular oxidative stress., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

New strategies for skin regeneration are needed to address the significant medical burden caused by cutaneous wounds and disease. In this study, pullulan-collagen composite hydrogel matrices were fabricated using a salt-induced phase inversion technique, resulting in a structured yet soft scaffold for skin engineering. Salt crystallization induced interconnected pore formation, and modification of collagen concentration permitted regulation of scaffold pore size. Hydrogel architecture recapitulated the reticular distribution of human dermal matrix while maintaining flexible properties essential for skin applications. In vitro, collagen hydrogel scaffolds retained their open porous architecture and viably sustained human fibroblasts and murine mesenchymal stem cells and endothelial cells. In vivo, hydrogel-treated murine excisional wounds demonstrated improved wound closure, which was associated with increased recruitment of stromal cells and formation of vascularized granulation tissue. In conclusion, salt-induced phase inversion techniques can be used to create modifiable pullulan-collagen composite dermal scaffolds that augment early wound healing. These novel biomatrices can potentially serve as a structured delivery template for cells and biomolecules in regenerative skin applications.

Enhanced production of a 42-residue beta amyloid peptide (Aβ(42)) in affected parts of the brain has been suggested to be the main causative factor for the development of Alzheimer's Disease (AD). The severity of the disease depends not only on the amount of the peptide but also its conformational transition leading to the formation of oligomeric amyloid-derived diffusible ligands (ADDLs) in the brain of AD patients. Despite being significant to the understanding of AD mechanism, no atomic-resolution structures are available for these species due to the evanescent nature of ADDLs that hinders most structural biophysical investigations. Based on our molecular modeling and computational studies, we have designed Met35Nle and G37p mutations in the Aβ(42) peptide (Aβ(42)Nle35p37) that appear to organize Aβ(42) into stable oligomers. 2D NMR on the Aβ(42)Nle35p37 peptide revealed the occurrence of two β-turns in the V24-N27 and V36-V39 stretches that could be the possible cause for the oligomer stability. We did not observe corresponding NOEs for the V24-N27 turn in the Aβ(21-43)Nle35p37 fragment suggesting the need for the longer length amyloid peptide to form the stable oligomer promoting conformation. Because of the presence of two turns in the mutant peptide which were absent in solid state NMR structures for the fibrils, we propose, fibril formation might be hindered. The biophysical information obtained in this work could aid in the development of structural models for toxic oligomer formation that could facilitate the development of therapeutic approaches to AD.

To study the role of cell-extracellular matrix (ECM) interactions, microscale approaches provide the potential to perform high throughput assessment of the effect of the ECM microenvironment on cellular function and phenotype. Using a microscale direct writing (MDW) technique, we characterized the generation of multicomponent ECM microarrays for cellular micropatterning, localization and stem cell fate determination. ECMs and other biomolecules of various geometries and sizes were printed onto epoxide-modified glass substrates to evaluate cell attachment by human endothelial cells. The endothelial cells displayed strong preferential attachment to the ECM patterned regions and aligned their cytoskeleton along the direction of the micropatterns. We next generated ECM microarrays that contained one or more ECM components (namely gelatin, collagen IV and fibronectin) and then cultured murine embryonic stem cell (ESCs) on the microarrays. The ESCs selectively attached to the micropatterned features and expressed markers associated with a pluripotent phenotype, such as E-cadherin and alkaline phosphatase, when maintained in growth medium containing leukemia inhibitory factor. In the presence of the soluble factors retinoic acid and bone morphogenetic protein-4 the ESCs differentiated towards the ectodermal lineage on the ECM microarray with differential ECM effects. The ESCs cultured on gelatin showed significantly higher levels of pan cytokeratin expression, when compared with cells cultured on collagen IV or fibronectin, suggesting that gelatin preferentially promotes ectodermal differentiation. In summary, our results demonstrate that MDW is a versatile approach to print ECMs of diverse geometries and compositions onto surfaces, and it is amenable to the generation of multicomponent ECM microarrays for stem cell fate determination., (Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Folding and self-assembly of amyloid beta (Abeta) peptide are linked to Alzheimer's disease. To understand the initial stage of amyloid-beta peptide aggregation, conformational characteristics of monomers of wild-type (WT) Abeta(28-35) and its mutant peptides A30G and A30I were investigated using density functional theory calculations and experimental studies. Monomeric structures and their relative stabilities were obtained on the basis of systematic structural optimization in the gas-phase and in the aqueous medium. Computations were performed by hybrid Hartree-Fock-Density Functional Theory (HF-DFT) at B3LYP/6-31G * level. Experimentally, the conformational transitions in the early stages of the octapeptide Abeta(28-35) and its mutants A30G and A30I in solution were characterized by CD, Thioflavin assay and FRET spectroscopy. Examination of the secondary structures revealed that Abeta(28-35) and its mutant monomers exist in random coil conformation in the aqueous medium in agreement with the theoretical predictions, which upon aging is transformed to sheet with different kinetics. This study deals with the structurally important intermediates and it may help to understand the mechanism of amyloid fibril aggregation leading to the onset of Alzheimer's disease.

Brain-derived neurotrophic factor (BDNF) activates the receptor tropomyosin-related kinase B (TrkB) with high potency and specificity, promoting neuronal survival, differentiation, and synaptic function. Correlations between altered BDNF expression and/or function and mechanism(s) underlying numerous neurodegenerative conditions, including Alzheimer disease and traumatic brain injury, suggest that TrkB agonists might have therapeutic potential. Using in silico screening with a BDNF loop-domain pharmacophore, followed by low-throughput in vitro screening in mouse fetal hippocampal neurons, we have efficiently identified small molecules with nanomolar neurotrophic activity specific to TrkB versus other Trk family members. Neurotrophic activity was dependent on TrkB and its downstream targets, although compound-induced signaling activation kinetics differed from those triggered by BDNF. A selected prototype compound demonstrated binding specificity to the extracellular domain of TrkB. In in vitro models of neurodegenerative disease, it prevented neuronal degeneration with efficacy equal to that of BDNF, and when administered in vivo, it caused hippocampal and striatal TrkB activation in mice and improved motor learning after traumatic brain injury in rats. These studies demonstrate the utility of loop modeling in drug discovery and reveal what we believe to be the first reported small molecules derived from a targeted BDNF domain that specifically activate TrkB.We propose that these compounds constitute a novel group of tools for the study of TrkB signaling and may provide leads for developing new therapeutic agents for neurodegenerative diseases.

A new method for creating substrates made out of ordered collagen fibers, on which cells in culture can align, is proposed. The substrates can be used for research in cell culture, and this research presents a significant advance in the technology to coat implants in order to improve cell adhesion. In the procedure presented here, a molecular solution of collagen is spread at the interface of a saline solution and air to induce fiber formation, compressed at a high speed to induce orientation and deposited on solid substrates via Langmuir-Blodgett transfer. Several interfacial techniques are employed to investigate the behavior of collagen, which is shown to be dependent on the salt concentration of the subphase as well as the temperature. After Langmuir-Blodgett transfer, primary human fibroblasts and adipose-derived stem cells are cultured on the collagen substrates. Both types of cells respond favorably to the collagen orientation and align with the deposited fibers. The technique presented here provides a simple method to produce well-controlled, oriented collagen substrates that can be used in tissue culture research or scaffolding applications without the use of additives and/or bioincompatible materials.