Capturing the nanoscale complexity of cellular membranes in supported lipid bilayers

The lateral mobility of cell membranes plays an important role in cell signaling, governing the rate at which embedded proteins can interact with other biomolecules. The past two decades have seen a dramatic transformation in understanding of this environment, as the mechanisms and potential implications of nanoscale structure of these systems has become accessible to theoretical and experimental investigation. In particular, emerging micro- and nano-scale fabrication techniques have made possible the direct manipulation of model membranes at the scales relevant to these biological processes. This review focuses on recent advances in nanopatterning of supported lipid bilayers, capturing the impact of membrane nanostructure on molecular diffusion and providing a powerful platform for further investigation of the role of this spatial complexity on cell signaling.     

Biomimetic and bioactive nanofibrous scaffolds from electrospun composite nanofibers

Electrospinning is an enabling technology that can architecturally (in terms of geometry, morphology or topography) and biochemically fabricate engineered cellular scaffolds that mimic the native extracellular matrix (ECM). This is especially important and forms one of the essential paradigms in the area of tissue engineering. While biomimesis of the physical dimensions of native ECM's major constituents (eg, collagen) is no longer a fabrication-related challenge in tissue engineering research, conveying bioactivity to electrospun nanofibrous structures will determine the efficiency of utilizing electrospun nanofibers for regenerating biologically functional tissues. This can certainly be achieved through developing composite nanofibers. This article gives a brief overview on the current development and application status of employing electrospun composite nanofibers for constructing biomimetic and bioactive tissue scaffolds. Considering that composites consist of at least two material components and phases, this review details three different configurations of nanofibrous composite structures by using hybridizing basic binary material systems as example. These are components blended composite nanofiber, core-shell structured composite nanofiber, and nanofibrous mingled structure.     

Fiber alignment directs cell motility over chemotactic gradients

The ability of tissue engineered scaffolds to direct cell behavior is paramount for scaffold design. Cell migration can be directed by various methods including chemical, adhesive, mechanical, and topographical cues. Electrospinning has emerged as a popular method to control topography and create fibrous scaffolds similar to that found in extracellular matrix. One major hurdle is limited cell infiltration and several studies have explored methods to alter electrospun materials to increase scaffold porosity; however, uniform cell distributions within scaffolds is still limited. Towards this, we investigated the motility of HUVECs on a model system of electrospun hyaluronic acid fibers under a gradient of VEGF and found that topographical cues dominate cell motility direction. Using time‐lapse microscopy, cell aspect ratio, and migration angle were measured; cells were directed in a chemical gradient and/or on aligned electrospun fibers. Measurements of the persistence time demonstrated an additive effect of the chemical gradient and fiber alignment. However, when fibers were aligned perpendicular to a chemical gradient, cells were directed by fiber alignment and there was no effect of the chemical gradient. These results suggest that topographical cues may be more influential than chemical cues in directing cell motility and should be considered in material design. Biotechnol. Bioeng. 2013; 110: 1249–1254. © 2012 Wiley Periodicals, Inc.     

Assessment of Spirulina-PCL nanofiber for the regeneration of dermal fibroblast layers

Skin is a barrier which protects injured tissues, and thus, skin regeneration is one of many important medical issues. Tissue engineering is an attractive approach to make artificial tissue or regenerate lost tissues. While constituting artificial tissues, cells must infiltrate through scaffolds, maintaining viability and proliferation. However, a three-dimensional tissue culture involves stressful environments due to several reasons such as mass or gas transport and high cell density. Once stressed, cells produce reactive oxygen species, resulting in alleviating cellular viability and activity. Spirulina is well known to have antioxidant molecules, which have been known to modulate oxidative stress to cells. Electrospun nanofiber has widely been used as a scaffold to mimic natural extracellular matrix. In this research, we assessed Spirulina extract-imbedded nanofiber as a scaffold for an artificial skin tissue. Spirulina extract was proven to positively affect viability and proliferation of mouse fibroblasts. In addition, fibroblasts infiltrated through Spirulina extract-imbedded electrospun nanofiber without cytotoxicity.     

Tissue scaffold surface patterning for clinical applications

Patterned scaffold surfaces provide a platform for highly defined cellular interactions, and have recently taken precedence in tissue engineering. Despite advances in patterning techniques and improved tissue growth, no clinical studies have been conducted for implantation of patterned biomaterials. Four major clinical application fields where patterned materials hold great promise are antimicrobial surfaces, cardiac constructs, neurite outgrowth, and stem cell differentiation. Specific examples include applications of patterned materials to (i) counter infection by antibiotic resistant bacteria, (ii) establish proper alignment and contractile force of regrown cardiac cells for repairing tissue damaged by cardiac infarction, (iii) increase neurite outgrowth for central nervous system wound repair, and (iv) host differentiated stem cells while preventing reversion to a pluripotent state. Moreover, patterned materials offer unique advantages for artificial implants which other constructs cannot. For example, by inducing selective cell adhesion using topographical cues, patterned surfaces present cellular orientation signals that lead to functional tissue architectures. Mechanical stimuli such as modulus, tension, and material roughness are known to influence tissue growth, as are chemical stimuli for cell adhesion. Scaffold surface patterns allow for control of these mechanical and chemical factors. This review identifies research advances in scaffold surface patterning, in light of pressing clinical needs requiring organization of cellular interactions.     

Fibroblast response is enhanced by poly(L-lactic acid) nanotopography edge density and proximity

The development of scaffolds for use in tissue engineering applications requires careful choice of macroscale properties, such as mechanical characteristics, porosity and biodegradation. The micro- and nano-scale properties of the scaffold surface are also an important design criterion as these influence cell adhesion, proliferation, and differentiation. The cellular response is known to be affected by surface topography but the mechanisms governing this remain unclear. Homogenous poly(L-lactic acid) was textured with surface nanotopographies by two-stage replication molding of heterogeneous demixed polymer films. Initial cell adhesion was improved on nanotextured surfaces compared with smooth controls, but subsequent cell density was significantly reduced on the roughest surfaces. Improvements in cell response were found to correlate with focal contact and actin microfilament development. Cell response was found to trend both with the surface density of topography edges and with inter-topography spacing, indicating possible roles for edges stimulating cell adhesion/proliferation or for spacing to modulate the ability of integrin-ligand bonds to cluster and form focal adhesions. This study furthers understanding of the geometric properties of surface nanotopographies that affect cellular response. It is hoped that identification of the mechanisms governing cell-topography interactions will allow rule-based design of biomaterial surface to engineer specific cellular responses.     

Coating electrospun collagen and gelatin fibers with perlecan domain I for increased growth factor binding

Electrospun natural polymer membranes were fabricated from collagen or gelatin coated with a bioactive recombinant fragment of perlecan, a natural heparan sulfate proteoglycan. The electrospinning process allowed the facile processing of a three-dimensional, porous fibril (2-6 microm in diameter) matrix suitable for tissue engineering. Laser scanning confocal microscopy revealed that osteoblast-like MG63 cells infiltrated the depth of the electrospun membrane evenly without visible apoptosis. Tissue engineering scaffolds ideally mimic the extracellular matrix; therefore, the electrospun membrane must contain both structural and functional matrix features. Fibers were coated, after processing, with perlecan domain I (PlnDI) to improve binding of basic fibroblast growth factor (FGF-2), which binds to native heparan sulfate chains on PlnDI. PlnDI-coated electrospun collagen fibers were ten times more effective than heparin-BSA collagen fibers at binding FGF-2. Because FGF-2 modulates cell growth, differentiation, migration and survival, the ability to effectively bind FGF-2 to an electrospun matrix is a key improvement in creating a successful tissue engineering scaffold.     

Graphene Functionalized Scaffolds Reduce the Inflammatory Response and Supports Endogenous Neuroblast Migration when Implanted in the Adult Brain

Electroactive materials have been investigated as next-generation neuronal tissue engineering scaffolds to enhance neuronal regeneration and functional recovery after brain injury. Graphene, an emerging neuronal scaffold material with charge transfer properties, has shown promising results for neuronal cell survival and differentiation in vitro. In this in vivo work, electrospun microfiber scaffolds coated with self-assembled colloidal graphene, were implanted into the striatum or into the subventricular zone of adult rats. Microglia and astrocyte activation levels were suppressed with graphene functionalization. In addition, self-assembled graphene implants prevented glial scarring in the brain 7 weeks following implantation. Astrocyte guidance within the scaffold and redirection of neuroblasts from the subventricular zone along the implants was also demonstrated. These findings provide new functional evidence for the potential use of graphene scaffolds as a therapeutic platform to support central nervous system regeneration.     

Development of a 3D cell culture system for investigating cell interactions with electrospun fibers

There are many variables to be considered in studying how cells interact with 3D scaffolds used in tissue engineering. In this study we investigated the influence of the fiber diameter and interfiber spaces of 3D electrospun fiber scaffolds on the behavior of human dermal fibroblasts. Fibers of two dissimilar model materials, polystyrene and poly-L-lactic acid, with a broad range of diameters were constructed in a specifically developed 3D cell culture system. When fibroblasts were introduced to freestanding fibers, and encouraged to "walk the plank," a minimum fiber diameter of 10 microm was observed for cell adhesion and migration, irrespective of fiber material chemistry. A distance between fibers of up to 200 microm was also observed to be the maximum gap that could be bridged by cell aggregates--a behavior not seen in conventional 2D culture. This approach has identified some basic micro-architectural parameters for electrospun scaffold design and some key differences in fibroblast growth in 3D. We suggest the findings will be of value for optimizing the integration of cells in these scaffolds for skin tissue engineering.     

Preparation and Evaluation of Electrospun Zein/HA Fibers Based on Two Methods of Adding HA Nanoparticles

Zein/HA fibrous membranes were successfully prepared by electrospinning the zein/HA solution mixed by magnetic stirrer （Method I） or ultrasonic power （Method Ⅱ）. The morphology of zeirdHA nanocomposite fibers and the distribution of HA within the fibers electrospun by two methods were researched by Scanning Electron Microscopy （SEM） and Energy-dispersive X-ray spectroscopy （EDX）. In Method I, the distribution of HA nanoparticles is not homogeneous and HA particles tend to agglom- erate. The relatively homogeneous HA distribution can be observed in the membranes electrospun by Method Ⅱ. Using mag- netic stirrer to prepare the electrospinning solution improves the wettability of zein/HA membranes. From the viewpoint of application, electrospun zein/HA membranes fabricated by the solution mixed via Methods I and II both possessed reasonable tensile strength and elongation at break for both handling and sterilization. Considering two aspects of strength and elongation, electrospun zein/HA membranes fabricated by Method I are more balanced than those fabricated by Method Ⅱ. Biological performances of the control zein and zein/HA membranes were assessed by in vitro culture of hMSCs. Results show that both types of the membranes can support cell proliferation. The cells cultured on the zein/HA membranes electrospun by Method I with 5 wt% HA （on weight ofzein） show significantly higher proliferation than those cultured on the control zein membranes on the seventh day. The electrospun zein/HA fibrous membranes show promises for bone tissue engineering applications.     

电纺技术在生物医学中的应用进展

电纺技术已经成为结合多组分化合物与织造技术的关键工具,可改变电纺丝材料的化学、物理和生物特性,使其与不同的应用环境相适应。通过电纺技术制作的功能化纳米电纺丝材料,在组织工程、创伤敷料、酶的固定化和药物（基因）载体等生物医学方面得到了广泛的应用。新型的电纺技术可以进一步优化纳米电纺丝的特性,如同轴电纺、二相电纺技术;电纺丝膜的修饰也为调控电纺丝的各向异性和多孔性提供了有效的方法。该文将概述功能化电纺丝的纺织技术及修饰方法在生物医学领域的研究与应用进展。     

Motif-programmed artificial extracellular matrix

Motif-programming is a method for creating artificial proteins by combining functional peptide motifs in a combinatorial manner. This method is particularly well suited for developing liaison molecules that interface between cells and inorganic materials. Here we describe our creation of artificial proteins through the programming of two motifs, a natural cell attachment motif (RGD) and an artificial Ti-binding motif (minTBP-1). The created proteins were found to reversibly bind Ti and to bind MC3T3-E1 osteoblast-like cells. Moreover, although the interaction with Ti was not covalent, the proteins recapitulated several functions of fibronectin, and thus, could serve as an artificial ECM on Ti materials. Because this motif-programming system could be easily extended to create artificial proteins having other biological functions and material specificities, it should be highly useful for application to tissue engineering and regenerative medicine.     

Cell adhesion on artificial materials for tissue engineering

Advanced interdisciplinary scientific field of tissue engineering has been developed to meet increasing demand for safe, functional and easy available substitutes of irreversibly damaged tissues and organs. First biomaterials were constructed as "two-dimensional" (allowing cell adhesion only on their surface), and durable (non-biodegradable). In contrast, biomaterials of new generation are characterized by so-called three dimensional porous or scaffold-like architecture promoting attachment, growth and differentiation of cells inside the material, accompanied by its gradual removal and replacement with regenerated fully functional tissue. In order to control these processes, these materials are endowed with a defined spectrum of bioactive molecules, such as ligands for adhesion receptors on cells, functional parts of natural growth factors, hormones and enzymes or synthetic regulators of cell behavior, incorporated in defined concentrations and spatial distribution against a bioinert background resistant to uncontrolled protein adsorption and cell adhesion.     

Cellular infiltration on nanofibrous scaffolds using a modified electrospinning technique

Electrospinning is currently used to fabricate nanofibrous scaffolds for tissue engineering applications. The major problem of these scaffolds is their intrinsically two-dimensional nature which inhibits cellular migration and in-growth. In this study, we have introduced a modified setup of electrospinning to produce three-dimensional nanofibrous scaffolds which allows improved infiltration of cells. An array of focused halogen light bulbs was used to localize the heat in the path of electrospun jet near the collector. The fabricated mats were then seeded with cells in order to evaluate migration and infiltration. After 14 days of culture, a homogenous distribution of cells was observed throughout the scaffolds and showed the three-dimensional architecture of nanofibrous mats. By this novel and simple setup, the prepared electrospun mats will allow the seeded cells to obtain a three-dimensional arrangement which is ideal for tissue engineering applications.     

Design concepts and strategies for tissue engineering scaffolds

In the emerging field of tissue engineering and regenerative medicine, new viable and functional tissue is fabricated from living cells cultured on an artificial matrix in a simulated biological environment. It is evident that the specific requirements for the three main components, cells, scaffold materials, and the culture environment, are very different, depending on the type of cells and the organ-specific application. Identifying the variables within each of these components is a complex and challenging assignment, but there do exist general requirements for designing and fabricating tissue engineering scaffolds. Therefore, this review explores one of the three main components, namely, the key concepts, important parameters, and required characteristics related to the development and evaluation of tissue engineering scaffolds. An array of different design strategies will be discussed, which include mimicking the extra cellular matrix, responding to the need for mass transport, predicting the structural architecture, ensuring adequate initial mechanical integrity, modifying the surface chemistry and topography to provide cell signaling, and anticipating the material selection so as to predict the required rate of bioresorption. In addition, this review considers the major challenge of achieving adequate vascularization in tissue engineering constructs, without which no three-dimensional thick tissue such as the heart, liver, and kidney can remain viable.     

Composite materials based on chitosan and montmorillonite: prospects for use as a matrix for stem and regenerative cell cultivation

This paper examines the structural and mechanical properties of composite materials based on chitosan and micro- and nanoparticles of Na-montmorillonite and possibility of application for cultivation and targeted delivery of mesenchymal stem cells and regenerative cells. It's have been shown by addition of Na-montmorillonite biomaterial acquires stability of structural and mechanical properties in the sterilization process the handling of liquid media in cell culture. In vitro studies using dermal fibroblasts and adipose tissue mesenchymal stem cells demonstrated that this material has a set of properties to ensure matrix biocompatibility.     

Engineered living materials (ELMs) design: From function allocation to dynamic behavior modulation

Natural materials possess many distinctive “living” attributes, such as self-growth, self-healing, environmental responsiveness, and evolvability, that are beyond the reach of many existing synthetic materials. The emerging field of engineered living materials (ELMs) takes inspiration from nature and harnesses engineered living systems to produce dynamic and responsive materials with genetically programmable functionalities. Here, we identify and review two main directions for the rational design of ELMs: first, engineering of living materials with enhanced performances by incorporating functional material modules, including engineered biological building blocks (proteins, polysaccharides, and nucleic acids) or well-defined artificial materials; second, engineering of smart ELMs that can sense and respond to their surroundings by programming dynamic cellular behaviors regulated via cell–cell or cell–environment interactions. We next discuss the strengths and challenges of current ELMs and conclude by providing a perspective of future directions in this promising area.     

XPS and AFM analysis of antifouling PEG interfaces for microfabricated silicon biosensors

In the past two decades, the biological and medical fields have seen great advances in the development of biosensors capable of quantifying biomolecules. Many of these biosensors have micro- and nano-scale features, are fabricated using biochip technology, and use silicon as a base material. The creation of antifouling sensor interfaces is critical to avoid serious consequences that arise due to their contact with biological fluids. To this end, we have created thin PEG interfaces of various grafting densities on silicon using a single-step PEG-silane coupling reaction scheme. Initial PEG concentration (5-50 mM) and coupling time (0.5-24 h) were varied to attain different grafting densities, and different PEG interfaces so created were analyzed using XPS and AFM. Furthermore, all the PEG interfaces were evaluated using XPS and AFM for their antifouling abilities using fibrinogen as the model protein. Results indicated that PEG interfaces created in this investigation are appropriate for biosensors with micro- and nano-scale features, and are efficient in controlling protein fouling.     

Electrospun nitrocellulose and nylon: Design and fabrication of novel high performance platforms for protein blotting applications

Background  

Electrospinning is a non-mechanical processing strategy that can be used to process a variety of native and synthetic polymers into highly porous materials composed of nano-scale to micron-scale diameter fibers. By nature, electrospun materials exhibit an extensive surface area and highly interconnected pore spaces. In this study we adopted a biological engineering approach to ask how the specific unique advantages of the electrospinning process might be exploited to produce a new class of research/diagnostic tools.     

Mitochondria in apoptosis and human disease

Apoptosis is a process of cell suicide whereby individual cells are destroyed while preserving the integrity and architecture of surrounding tissue. This targeted cell destruction is critical both in physiological contexts as well as pathological states. It seems increasingly evident that mitochondria play an important role in the regulation of programmed cell death via release of proapoptotic agents and/or disruption of cellular energy metabolism. The mechanisms of mitochondrial involvement are beginning to be elucidated, and may involve the participation of bcl-2 family members, reactive oxygen species, and caspases. As part of a central mechanism of amplification of the apoptotic signal, mitochondria may be an appropriate target for therapeutic agents designed to modulate apoptosis. This review focuses on recent advances in understanding mitochondrial mechanisms in apoptosis and the involvement of these pathways in human disease.