From Acellular Matrices to Smart Polymers: Degradable Scaffolds that are Transforming the Shape of Urethral Tissue Engineering.

Several congenital and acquired conditions may result in severe narrowing of the urethra in men, which represent an ongoing surgical challenge and a significant burden on both health and quality of life. In the field of urethral reconstruction, tissue engineering has emerged as a promising alternative to overcome some of the limitations associated with autologous tissue grafts. In this direction, preclinical as well as clinical studies, have shown that degradable scaffolds are able to restore the normal urethral architecture, supporting neo-vascularization and stratification of the tissue. While a wide variety of degradable biomaterials are under scrutiny, such as decellularized matrices, natural, and synthetic polymers, the search for scaffold materials that could fulfill the clinical performance requirements continues. In this article, we discuss the design requirements of the scaffold that appear to be crucial to better resemble the structural, physical, and biological properties of the native urethra and are expected to support an adequate recovery of the urethral function. In this context, we review the biological performance of the degradable polymers currently applied for urethral reconstruction and outline the perspectives on novel functional polymers, which could find application in the design of customized urethral constructs.

Trilayer composite scaffold for urethral reconstruction:in vitroevaluation of mechanical, biological, and angiogenic properties.

Regeneration of damaged urethral tissue remains a major challenge in the field of lower urinary tract reconstruction. To address this issue, various synthetic and natural biodegradable biomaterials are currently being explored for the fabrication of scaffolds that promote urethral regeneration and healing. In this study, we present an approach to fabricate a trilayer hybrid scaffold comprising a central layer of poly(lactic acid) (PLA) between two layers of chitosan. The chitosan/PLA/chitosan (CPC) scaffolds were fabricated by a sequential electrospinning process and their properties were evaluated for their suitability for urethral tissue engineering. The physical and biological properties of the CPC scaffolds were evaluated in comparison to electrospun PLA scaffolds and acellular dermis (Alloderm) as controls for a synthetic and a natural scaffold, respectively. Compared to the controls, the CPC scaffolds exhibited higher elastic modulus and ultimate tensile strength, while maintaining extensibility and suture retention strength appropriate for clinical use. The CPC scaffolds displayed significant hydrophilicity, which was associated with a higher water absorption capacity of the chitosan nanofibres. The degradation products of the CPC scaffolds did not exhibit cytotoxicity and promoted wound closure by fibroblastsin vitro. In addition, CPC scaffolds showed increased growth of smooth muscle cells, an essential component for functional regeneration of urethral tissue. Furthermore, in a chicken embryo-based assay, CPC scaffolds demonstrated significantly higher angiogenic potential, indicating their ability to promote vascularisation, a crucial aspect for successful urethral reconstruction. Overall, these results suggest that CPC hybrid scaffolds containing both natural and synthetic components offer significant advantages over conventional acellular or synthetic materials alone. CPC scaffolds show promise as potential candidates for further research into the reconstruction of the urethrain vivo.

Synthesis, Self-Assembly and In Vitro Cellular Uptake Kinetics of Nanosized Drug Carriers Based on Aggregates of Amphiphilic Oligomers of N-Vinyl-2-pyrrolidone.

Development of nanocarrier-based drug delivery systems is a major breakthrough in pharmacology, promising targeted delivery and reduction in drug toxicity. On the cellular level, encapsulation of a drug substantially affects the endocytic processes due to nanocarrier-membrane interaction. In this study we synthesized and characterized nanocarriers assembled from amphiphilic oligomers of N-vinyl-2-pyrrolidone with a terminal thiooctadecyl group (PVP-OD). It was found that the dissolution free energy of PVP-OD depends linearly on the molecular mass of its hydrophilic part up to M¯n = 2 × 104, leading to an exponential dependence of critical aggregation concentration (CAC) on the molar mass. A model hydrophobic compound (DiI dye) was loaded into the nanocarriers and exhibited slow release into the aqueous phase on a scale of 18 h. Cellular uptake of the loaded nanocarriers and that of free DiI were compared in vitro using glioblastoma (U87) and fibroblast (CRL2429) cells. While the uptake of both DiI/PVP-OD nanocarriers and free DiI was inhibited by dynasore, indicating a dynamin-dependent endocytic pathway as a major mechanism, a decrease in the uptake rate of free DiI was observed in the presence of wortmannin. This suggests that while macropinocytosis plays a role in the uptake of low-molecular components, this pathway might be circumvented by incorporation of DiI into nanocarriers.

Cell-based therapy for the treatment of female stress urinary incontinence: an early cost-effectiveness analysis.

AIM: To perform an early cost-effectiveness analysis of in vitro expanded myoblasts (IVM) and minced myofibers versus midurethral slings (MUS) for surgical treatment of female stress urinary incontinence. METHODS: Cost-effectiveness and sensitivity analyses were performed using a decision tree comprising previously published data and expert opinions. RESULTS & CONCLUSION: In the base case scenario, MUS was the cost-effective strategy with a negative incremental cost-effectiveness ratio compared with IVM and a positive incremental cost-effectiveness ratio compared with minced myofibers. However, the sensitivity analysis indicates that IVM may become an alternative providing greater effect at a higher cost. With the possibility of becoming more effective, IVM treatment would be advantageous over MUS given its reduced invasiveness and lower risks of complications.

Diamond/Porous Titanium Nitride Electrodes With Superior Electrochemical Performance for Neural Interfacing.

Robust devices for chronic neural stimulation demand electrode materials which exhibit high charge injection (Q inj) capacity and long-term stability. Boron-doped diamond (BDD) electrodes have shown promise for neural stimulation applications, but their practical applications remain limited due to the poor charge transfer capability of diamond. In this work, we present an attractive approach to produce BDD electrodes with exceptionally high surface area using porous titanium nitride (TiN) as interlayer template. The TiN deposition parameters were systematically varied to fabricate a range of porous electrodes, which were subsequently coated by a BDD thin-film. The electrodes were investigated by surface analysis methods and electrochemical techniques before and after BDD deposition. Cyclic voltammetry (CV) measurements showed a wide potential window in saline solution (between -1.3 and 1.2 V vs. Ag/AgCl). Electrodes with the highest thickness and porosity exhibited the lowest impedance magnitude and a charge storage capacity (CSC) of 253 mC/cm2, which largely exceeds the values previously reported for porous BDD electrodes. Electrodes with relatively thinner and less porous coatings displayed the highest pulsing capacitances (C pulse), which would be more favorable for stimulation applications. Although BDD/TiN electrodes displayed a higher impedance magnitude and a lower C pulse as compared to the bare TiN electrodes, the wider potential window likely allows for higher Q inj without reaching unsafe potentials. The remarkable reduction in the impedance and improvement in the charge transfer capacity, together with the known properties of BDD films, makes this type of coating as an ideal candidate for development of reliable devices for chronic neural interfacing.

Nanosized carriers based on amphiphilic poly-N-vinyl-2-pyrrolidone for intranuclear drug delivery.

AIM: Ability to deliver drugs into the cell nuclei can significantly increase the efficacy of cancer therapies, in particular in the case of multidrug-resistant cancer Results: Polymer nanocarriers based on amphiphilic thiooctadecyl-terminated poly-N-vinyl-2-pyrrolidone were produced and loaded with a model hydrophobic drug, curcumin. Two commonly used loading approaches - emulsification and ultrasonic dispersion - were found to lead to two different size distributions with distinctively different biological effect. While nanocarriers produced via the emulsion method penetrated cells by dynamin-dependent endocytic mechanisms, sub-100 nm dispersion-produced nanocarriers were capable of crossing the membranes via biologically independent mechanisms. CONCLUSION: This finding opens an intriguing possibility of intranuclear delivery by merely tailoring the size of polymeric carriers, thus promising a new approach for cancer therapies.

Hypoxia enhances the wound-healing potential of adipose-derived stem cells in a novel human primary keratinocyte-based scratch assay.

Preclinical studies have suggested that paracrine factors from adipose-derived stem cells (ASCs) promote the healing of chronic wounds, and that the exposure of ASCs to hypoxia enhances their wound healing effect. To aid the translation of these findings into clinical use, robust wound models are necessary to explore each aspect of wound healing. The aspect of re-epithelization is often studied in a scratch assay based on transformed keratinocytes. However, there are concerns regarding the validity of this model, since these cell lines differ from normal keratinocytes, both in terms of proliferative capacity and differentiation, and sensitivity to environmental cues. In this study, the main challenge of using primary keratinocytes to examine the effects of ASCs was identified to be their different requirements for calcium in the culture media. We confirmed that a high calcium content led to morphological and cytoskeletal changes in primary keratinocytes, and demonstrated that a low calcium content compromised the growth of ASCs. We found that it is possible to perform the wound healing assay with primary keratinocytes, if the conditioned media from the ASCs is dialyzed to reduce the calcium concentration. Additionally, using this model of re-epithelization, conditioned media from normoxic ASCs was shown to markedly increase the rate of wound closure by primary keratinocytes, and this effect was significantly enhanced with media from the hypoxia-exposed ASCs. These findings, which are in line with the observations from previous in vivo studies, highlight the validity of this modified assay to investigate the wound healing properties of ASCs in vitro.

Boron-Doped Nanocrystalline Diamond Electrodes for Neural Interfaces: In vivo Biocompatibility Evaluation.

Boron-doped nanocrystalline diamond (BDD) electrodes have recently attracted attention as materials for neural electrodes due to their superior physical and electrochemical properties, however their biocompatibility remains largely unexplored. In this work, we aim to investigate the in vivo biocompatibility of BDD electrodes in relation to conventional titanium nitride (TiN) electrodes using a rat subcutaneous implantation model. High quality BDD films were synthesized on electrodes intended for use as an implantable neurostimulation device. After implantation for 2 and 4 weeks, tissue sections adjacent to the electrodes were obtained for histological analysis. Both types of implants were contained in a thin fibrous encapsulation layer, the thickness of which decreased with time. Although the level of neovascularization around the implants was similar, BDD electrodes elicited significantly thinner fibrous capsules and a milder inflammatory reaction at both time points. These results suggest that BDD films may constitute an appropriate material to support stable performance of implantable neural electrodes over time.

Patterned poly(lactic acid) films support growth and spontaneous multilineage gene expression of adipose-derived stem cells.

Conventional culture surfaces do not provide optimal environmental cues for expansion or differentiation of adult stem cells. Aiming to increase the efficiency of the in vitro culture conditions, biocompatible and biodegradable biomaterials such as poly(lactic acid) (PLA) have been proposed to engineer the stem cell microenvironment. In this study, we explored the feasibility of using PLA substrates to control the responses of adipose-derived stem cells (ASCs). The substrates consisted of flat and patterned PLA films fabricated by casting a chloroform-PLA solution on a glass surface. Patterning was achieved through the condensation of nano-sized water droplets during chloroform evaporation, which resulted in films displaying irregularly distributed circular indentations with a mean diameter of 248±65 nm. Both types of PLA substrates were assessed for protein adsorption using fibronectin and in vitro cell culturing. Tissue-culture polystyrene (TCPS) plates were used as control surfaces. The experiments demonstrated that the patterned PLA substrates had a significantly higher fibronectin adsorption capacity when compared with the flat counterparts. For the entire duration of the culture period, there was no significant difference in cell growth rate on the PLA surfaces with respect to TCPS despite signs of reduced adhesion. In addition, the semi-quantitative real-time RT-PCR analysis of a set of 14 lineage-specific genes revealed that the PLA-related transcriptional activity significantly surpassed that of TCPS. Remarkably, when assessing the effect of patterning, the patterned films proved superior regarding the activation of genes involved in the skeletal myogenic, cardiomyogenic, chondrogenic, and adipogenic pathways. Taken together, our data provide evidence that the surface patterning can exert such an influence on the stem cell microenvironment that the differentiation process can be effectively modulated. Consequently, the patterned PLA surfaces could potentially be used as a platform for localized delivery and engraftment of stem cells.

Hypoxia and adipose-derived stem cell-based tissue regeneration and engineering.

INTRODUCTION: Realization that oxygen is one of the key regulators of development and differentiation has a profound significance on how current cell-based and tissue engineering applications using adipose-derived stem cells (ASCs) can be further improved. AREAS COVERED: The article provides an overview of mechanisms of hypoxic responses during physiological adaptations and development. Furthermore, a synopsis of the hypoxic responses of ASCs is provided, and this information is presented in context of their utility as a major source of stem cells across the regenerative applications explored to date. EXPERT OPINION: The reader will obtain insight into a highly specific area of stem cell research focusing on ASCs and hypoxia. In order to enhance the level of comprehension, a broader context with other stem cell and experimental systems is provided. It is emphasized that the pericellular oxygen tension is a critical regulatory factor that should be taken into account when devising novel stem cell-based therapeutic applications along with other parameters, such as biochemical soluble factors and the growth substrates.

Molecular photovoltaic structures for optical activation of excitable cells: current advances and perspectives.

Current neural stimulation devices for the treatment of sensory and motor disorders are based on electrical stimulation. Using this technique, neural activity is triggered by electrical stimuli applied through electrodes in contact with the cells. Due to physical constraints of the electrodes the spatial control of stimulation is limited, which in some cases generates unwanted side effects. In addition, adverse tissue reactions occur after long term contact with the electrodes. A potential solution is the application of methods based on light instead of electrical energy, in which the electrical stimulator and the electrode are replaced by a light source and an optical fiber. Although optical stimulation approaches that allow spatially selective, highly specific and contact-free control of the neural activity have been developed in recent years, their implementation requires genetic manipulation, limiting the perspectives for clinical applications. A molecular photovoltaic structure potentially able to mediate light-induced cellular responses without involving genetic modification is the photosynthetic pigment-protein complex Photosystem I (PSI). In this work, the recent advances on the application of PSI reaction centers for optical control of cellular activity are presented. Perspectives of application of PSI reaction centers in the development of future methods for clinical neural stimulation are also presented.

The influence of surface properties of plasma-etched polydimethylsiloxane (PDMS) on cell growth and morphology.

Polydimethylsiloxane (PDMS) or silicone rubber is a widely used implant material. Approaches to promote tissue integration to PDMS are desirable to avoid clinical problems associated with sliding and friction between tissue and implant. Plasma-etching is a useful way to control cell behavior on PDMS without additional coatings. In this work, different plasma processing conditions were used to modify the surface properties of PDMS substrates. Surface nanotopography and wettability were measured to study their effect on in vitro growth and morphology of fibroblasts. While fluorinated plasma treatments produced nanorough hydrophobic and superhydrophobic surfaces that had negative or little influences on cellular behavior, water vapor/oxygen plasma produced smooth hydrophillic surfaces that enhanced cell growth.

Notch and hedgehog signaling cooperate to maintain self-renewal of human embryonic stem cells exposed to low oxygen concentration.

BACKGROUND AND OBJECTIVES: Expansion and maintenance of human embryonic stem cells (hESCs) in undifferentiated state is influenced by complex signals in the microenvironment, including those contingent upon oxygen availability. Responses mediated by Notch and Hedgehog (Hh) have essential role in the growth and maintenance of hESCs, therefore this study examined their effect on the self-renewal of hESCs exposed to low oxygen. METHODS AND RESULTS: Using potent antagonists γ-secretase inhibitor and cyclopamine, we inhibited Notch and Hh pathways, respectively, in the CLS1 hESC line expanded continuously in a hypoxic atmosphere of 5% oxygen. Immunohistochemical staining and protein assays revealed loss of Oct4 and gain of stage-specific embryonic antigen 1 (SSEA1) markers in the inhibited cells. Semiquantitative real-time RT-PCR, and bromodeoxyuridine and thymidine incorporation assays demonstrated low Oct4 and Nanog mRNA expression, and decreased DNA synthesis, respectively, resulting from the block of each of the pathways. The loss increased significantly with co-inhibition of both pathways. Importantly, Notch and Hh downstream targets, including Hes1, Hey1, GIi1, and Ptc1, were surprisingly suppressed not only by the pathway-specific but also the unrelated inhibitor. CONCLUSIONS: These findings demonstrate complementary effect of Notch and Hh signaling in hypoxia enhanced maintenance of hESCs.

Electrostatics of photosynthetic reaction centers in membranes.

Photosynthetic reaction centers are integral membrane complexes. They have potential application as molecular photovoltaic structures and have been used in diverse technological applications. A three-dimensional electrostatic model of the photosystem I reaction center (PSI) embedded in a lipid membrane is presented. The potential is obtained by solving the Poisson-Boltzmann equation with the finite element method (FEM). Simulations showing the potential distribution in a vesicle containing PSI reaction centers under different conditions are presented. The results of the simulations are compared with previous findings and a possible application of PSI to provide light activation of voltage-gated ion channels is discussed.