Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone,Elastin, and Collagen: A Preliminary Study

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 - 2.8 % / 100 mmHg, while uniaxial results demonstrated an average modulus range of 2.0 - 11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties.     

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.     

应用于组织工程支架制备的电纺技术

组织工程技术为修复病损的组织和器官提供了一种新的途径,在组织工程中,细胞支架起着支撑细胞生长、引导组织再生、控制组织结构和释放活性因子等作用。针对电纺技术的新发展和细胞支架的新理念,综述了国内外利用电纺技术制备细胞支架的工艺条件、制备方法、组织细胞培养等方面的研究进展,并结合作者所在研究团队的研究工作提出了对未来电纺技术在组织工程中应用的研究重点和发展方向的认识。     

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.     

Electrospinning preparation and photoluminescence properties of Y3Al5O12:Tb3+ nanostructures

Novel nanostructures of Y₃Al₅O₁₂:Tb³⁺ (denoted as YAG:Tb³⁺ for short) nanobelts and nanofibers were fabricated by calcination of the respective electrospun PVP/[Y(NO₃)₃ + Tb(NO₃)₃ + Al(NO₃)₃] composite nanobelts and nanofibers. YAG:Tb³⁺ nanostructures are cubic in structure with a space group of I_a3d. The thickness and width of the YAG:7%Tb³⁺ nanobelts are respectively ca. 125 nm and 5.9 ± 0.3 µm, and the diameter of YAG:7%Tb³⁺ nanofibers is 166.0 ± 20 nm (95% confidence level). The YAG:Tb³⁺ nanostructures emit predominantly at 544 nm from the energy levels transition of ⁵D₄ → ⁷ F₅ of Tb³⁺ ions under the excitation of 274‐nm ultraviolet light. It was found that the optimum doping molar concentration of Tb³⁺ ions for YAG:Tb³⁺ nanostructures was 7%. Compared with YAG:7%Tb³⁺ nanofibers, YAG:7%Tb³⁺ nanobelts exhibit a stronger photoluminescence (PL) intensity under the same doping concentration. Commission International de l'Eclairage (CIE) analysis demonstrates that the emitting colors of YAG:Tb³⁺ nanostructures are located in the green region and color‐tuned luminescence can be obtained by changing the doping concentration of Tb³⁺ and morphologies of the nanostructures, which could be applied in the field of optical telecommunication and optoelectronic devices. The possible formation mechanisms of YAG:Tb³⁺ nanobelts and nanofibers are also proposed. Copyright © 2014 John Wiley & Sons, Ltd.     

Electrospun Scaffold of Collagen and Polycaprolactone Containing ZnO Quantum Dots for Skin Wound Regeneration

Nanofibers(NFs)have been widely used in tissue engineering such as wound healing.In this work,the antibacterial ZnO quantum dots(ZnO QDs)have been incorporated into the biocompatible poly(ε-caprolactone)/collagen(PCL/Col)fibrous scaffolds for wound healing.The as-fabricated PCL-Col/ZnO fibrous scaffolds exhibited good swelling,antibacterial activity,and biodegradation behaviors,which were beneficial for the applications as a wound dressing.Moreover,the PCL-Col/ZnO fibrous scaffolds showed excellent cytocompatibility for promoting cell proliferation.The resultant PCL-Col/ZnO fibrous scaffolds containing vascular endothelial growth factor(VEGF)also exhibited promoted wound-healing effect through promoting expression of transforming growth factor-β(TGF-β)and the vascular factor(CD31)in tissues in the early stages of wound healing.This new electrospun fibrous scaffolds with wound-healing promotion and antibacterial property should be convenient for treating wound healing.     

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the subsequent attachment and activation of platelets, monocyte/macrophage adhesion, and inflammatory cell signaling events, leading to post-procedural complications. The Chandler Loop Apparatus is an experimental system that allows researchers to study the molecular and cellular interactions that occur when large volumes of blood are perfused over polymeric conduits. To that end, this apparatus has been used as an ex vivo model allowing the assessment of the anti-inflammatory properties of various polymer surface modifications. Our laboratory has shown that blood conduits, covalently modified via photoactivation chemistry with recombinant CD47, can confer biocompatibility to polymeric surfaces. Appending CD47 to polymeric surfaces could be an effective means to promote the efficacy of polymeric blood conduits. Herein is the methodology detailing the photoactivation chemistry used to append recombinant CD47 to clinically relevant polymeric blood conduits and the use of the Chandler Loop as an ex vivo experimental model to examine blood interactions with the CD47 modified and control conduits.     

Nanofiber‐Based Bulk‐Heterojunction Organic Solar Cells Using Coaxial Electrospinning

Nanofibers consisting of the bulk heterojunction organic photovoltaic (BHJ–OPV) electron donor–electron acceptor pair poly(3‐hexylthiophene):phenyl‐C₆₁‐butyric acid methyl ester (P3HT:PCBM) are produced through a coaxial electrospinning process. While P3HT:PCBM blends are not directly electrospinnable, P3HT:PCBM‐containing fibers are produced in a coaxial fashion by utilizing polycaprolactone (PCL) as an electrospinnable sheath material. Pure P3HT:PCBM fibers are easily obtained after electrospinning by selectively removing the PCL sheath with cyclopentanone (average diameter 120 ± 30 nm). These fibers are then incorporated into the active layer of a BHJ–OPV device, which results in improved short‐circuit current densities, fill factors, and power‐conversion efficiencies (PCE) as compared to thin‐film devices of identical chemical composition. The best‐performing fiber‐based devices exhibit a PCE of 4.0%, while the best thin‐film devices have a PCE of 3.2%. This increase in device performance is attributed to the increased in‐plane alignment of P3HT polymer chains on the nanoscale, caused by the electrospun fibers, which leads to increased optical absorption and subsequent exciton generation. This methodology for improving device performance of BHJ–OPVs could also be implemented for other electron donor–electron acceptor systems, as nanofiber formation is largely independent of the PV material.     

静电纺丝的主要参数对ＰＬＧＡ纤维支架形貌和纤维直径的影响

目的以聚乳酸-羟基乙酸共聚物(PLGA)为材料,采用静电纺丝方法制备纤维支架,考察制备参数对纤维支架结构及纤维直径的影响规律。方法以四氢呋喃(THF)和N,N-二甲基甲酰胺(DMF)的混合液为溶剂,调节PLGA溶液浓度、流量及电场强度分别制备了具有不同表面形貌的纤维支架。通过扫描电镜(SEM)观察了纺丝溶液的浓度、流量及电场强度对纤维形貌和直径的影响。同时在制备的PLGA纤维支架上接种了人的真皮成纤维细胞,并对细胞在PLGA支架上的黏附和增殖情况进行了研究,从而来评价支架材料的细胞相容性。结论结果表明,随着纺丝溶液浓度的增加,纤维直径逐渐增大,纤维直径的分布也随之增大。随着流量的增加,纤维直径略有增大。随着电场强度的增大,纤维直径没有明显的变化。但是电压和浓度的增大有助于减少珠丝的产生。体外细胞培养实验证明,制备的PLGA纤维支架能支持细胞正常的黏附和增殖活动。     

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.     

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions ¹. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA ² and PLGA ³ or a number of naturally occurring proteins such as collagen ⁴, hyaluronic acid ⁵ or fibrin ^6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body''s natural wound healing processes ⁸. Fibrin is cell-degradable and potentially autologous ⁹, making it an ideal temporary scaffold for tissue engineering.Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering ⁴. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers ¹⁰. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries ^11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture ¹³.After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions ⁶. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.     

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol−ene and thiol−yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol−X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol−ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.     

Electrospun Nanomaterials for Supercapacitor Electrodes: Designed Architectures and Electrochemical Performance

Electrospinning is the most facile and highly versatile approach to produce 1D polymeric, inorganic, and hybrid nanomaterials with a small diameter, controllable dimensions, and designed architectures. In particular, with large surface area, high porosity, low density, good directionality, and tunable composition, electrospun nanofibers and mats are regarded as ideal candidates for various kinds of electrochemical energy storage devices such as supercapacitors (SCs). In this review, the recent progress in electrospun electrode materials for SCs is presented, covering the architecture design and their electrochemical performance. After a brief introduction about SCs, the basic principles of the electrospinning technique are discussed. Following, attention is paid to the discussion of various electrospun nanofibers and mats including 1D carbons, metal oxides, metal sulfides, metal nitrides, conducting polymers and composite nanomaterials with various types of architectures as electrodes for SCs. The relationship between the composition, architecture, and the electrochemical performance is discussed in detail. Finally, some challenges and perspectives of future research of the electrospun nanofibers and mats for high performance SCs are highlighted. It is anticipated that this review would provide the researchers some inspiration for constructing new types of energy storage devices.     

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ ¹. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture ^2-5, in excised ⁶ and living animal tissues ^2,5, and in human tissues in vivo ^2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine ^2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) ⁵, and fluoroscein ^6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.     

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular entanglement of the constituent polymers in the spinning dope was found to be an essential prerequisite for successful electrospinning. Rheology is a powerful tool to probe the molecular conformation and interaction of biopolymers. In this report, we demonstrate the protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan, from their dimethyl sulfoxide (DMSO)/water dispersions. Well-formed starch and pullulan fibers with average diameters in the submicron to micron range were obtained. Electrospinnability was evaluated by visual and microscopic observation of the fibers formed. By correlating the rheological properties of the dispersions to their electrospinnability, we demonstrate that molecular conformation, molecular entanglement, and shear viscosity all affect electrospinning. Rheology is not only useful in solvent system selection and process optimization, but also in understanding the mechanism of fiber formation on a molecular level.     

Fabrication of a Bioactive,PCL-based Self-fitting Shape Memory Polymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect “self-fitting.” In this way, following exposure to warm saline (~60 ºC), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 ºC) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.     

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH₂O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.