Porous Nano-Fiber Structure of Modified Electrospun Chitosan GBR Membranes Improve Osteoblast Calcium Phosphate Deposition in Osteoblast-Fibroblast Co-Cultures.

Desirable characteristics of electrospun chitosan membranes (ESCM) for guided bone regeneration are their nanofiber structure that mimics the extracellular fiber matrix and porosity for the exchange of signals between bone and soft tissue compartments. However, ESCM are susceptible to swelling and loss of nanofiber and porous structure in physiological environments. A novel post-electrospinning method using di-tert-butyl dicarbonate (tBOC) prevents swelling and loss of nanofibrous structure better than sodium carbonate treatments. This study aimed to evaluate the hypothesis that retention of nanofiber morphology and high porosity of tBOC-modified ESCM (tBOC-ESCM) would support more bone mineralization in osteoblast-fibroblast co-cultures compared to Na2CO3 treated membranes (Na2CO3-ESCM) and solution-cast chitosan solid films (CM-film). The results showed that only the tBOC-ESCM retained the nanofibrous structure and had approximately 14 times more pore volume than Na2CO3-ESCM and thousands of times more pore volume than CM-films, respectively. In co-cultures, the tBOC-ESCM resulted in a significantly greater calcium-phosphate deposition by osteoblasts than either the Na2CO3-ESCM or CM-film (p < 0.05). This work supports the study hypothesis that tBOC-ESCM with nanofiber structure and high porosity promotes the exchange of signals between osteoblasts and fibroblasts, leading to improved mineralization in vitro and thus potentially improved bone healing and regeneration in guided bone regeneration applications.

Regulation of microtubule nucleation in mouse bone marrow-derived mast cells by ARF GTPase-activating protein GIT2.

Aggregation of high-affinity IgE receptors (FcϵRIs) on granulated mast cells triggers signaling pathways leading to a calcium response and release of inflammatory mediators from secretory granules. While microtubules play a role in the degranulation process, the complex molecular mechanisms regulating microtubule remodeling in activated mast cells are only partially understood. Here, we demonstrate that the activation of bone marrow mast cells induced by FcϵRI aggregation increases centrosomal microtubule nucleation, with G protein-coupled receptor kinase-interacting protein 2 (GIT2) playing a vital role in this process. Both endogenous and exogenous GIT2 were associated with centrosomes and γ-tubulin complex proteins. Depletion of GIT2 enhanced centrosomal microtubule nucleation, and phenotypic rescue experiments revealed that GIT2, unlike GIT1, acts as a negative regulator of microtubule nucleation in mast cells. GIT2 also participated in the regulation of antigen-induced degranulation and chemotaxis. Further experiments showed that phosphorylation affected the centrosomal localization of GIT2 and that during antigen-induced activation, GIT2 was phosphorylated by conventional protein kinase C, which promoted microtubule nucleation. We propose that GIT2 is a novel regulator of microtubule organization in activated mast cells by modulating centrosomal microtubule nucleation.

A Self-Controlled and Self-Healing Model of Bacterial Cells.

A new kind of self-assembly model, morphogenetic (M) systems, assembles spatial units into larger structures through local interactions of simpler components and enables discovery of new principles for cellular membrane assembly, development, and its interface function. The model is based on interactions among three kinds of constitutive objects such as tiles and protein-like elements in discrete time and continuous 3D space. It was motivated by achieving a balance between three conflicting goals: biological, physical-chemical, and computational realism. A recent example is a unified model of morphogenesis of a single biological cell, its membrane and cytoskeleton formation, and finally, its self-reproduction. Here, a family of dynamic M systems (Mbac) is described with similar characteristics, modeling the process of bacterial cell formation and division that exhibits bacterial behaviors of living cells at the macro-level (including cell growth that is self-controlled and sensitive to the presence/absence of nutrients transported through membranes), as well as self-healing properties. Remarkably, it consists of only 20 or so developmental rules. Furthermore, since the model exhibits membrane formation and septic mitosis, it affords more rigorous definitions of concepts such as injury and self-healing that enable quantitative analyses of these kinds of properties. Mbac shows that self-assembly and interactions of living organisms with their environments and membrane interfaces are critical for self-healing, and that these properties can be defined and quantified more rigorously and precisely, despite their complexity.

Optimal structural and physical properties of aerogels for promoting robust neurite extension in vitro.

Cell behaviour is influenced by external factors including the physical properties of the substrate such as its surface topography and stiffness. Recent studies have demonstrated the potential of aerogels as biomaterials and specifically as neural scaffolds. The 3-D structure inherent to aerogels offers an advantage over other biocompatible substrates which lack the dimensionality needed to mimic the in vivo topography of tissues. Here, we used a variety of aerogel types to correlate the extension of neurites by neuronal cells with surface roughness ranging from 0 to 3 µm and stiffness 10 kPa-4 MPa. This investigation reveals that the optimal surface features for neurite extension are a surface roughness of 0.5 µm and a Young's modulus between 1 and 3.5 MPa. The significance of these findings to optimize materials for nerve repair is discussed.

Nematic Templated Complex Nanofiber Structures by Projection Display.

Oriented arrays of nanofibers are ubiquitous in nature and have been widely used in recreation of the biological functions such as bone and muscle tissue regenerations. However, it remains a challenge to produce nanofiber arrays with a complex organization by using current fabrication techniques such as electrospinning and extrusion. In this work, we propose a method to fabricate the complex organization of nanofiber structures templated by a spatially varying ordered liquid crystal host, which follows the pattern produced by a maskless projection display system. By programming the synchronization of the rotated polarizer and projected segments with different shapes, various configurations of nanofiber organization ranging from a single to two-dimensional lattice of arbitrary topological defects are created in a deterministic manner. The nanofiber arrays can effectively guide and promote neurite outgrowth. The application of nanofibers with arced profiles and topological defects on neural tissue organization is also demonstrated. This finding, combined with the versatility and programmability of nanofiber structures, suggests that they will help solve challenges in nerve repair, neural regeneration, and other related tissue engineering fields.

Cutaneous Effects of In Utero and Lactational Exposure of C57BL/6J Mice to 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

To determine the cutaneous effects of in utero and lactational exposure to the AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), pregnant C57BL/6J mice were exposed by gavage to a vehicle or 5 µg TCDD/kg body weight at embryonic day 12 and epidermal barrier formation and function were studied in their offspring from postnatal day 1 (P1) through adulthood. TCDD-exposed pups were born with acanthosis. This effect was AHR-dependent and subsided by P6 with no evidence of subsequent inflammatory dermatitis. The challenge of adult mice with MC903 showed similar inflammatory responses in control and treated animals, indicating no long-term immunosuppression to this chemical. Chloracne-like sebaceous gland hypoplasia and cyst formation were observed in TCDD-exposed P21 mice, with concomitant microbiome dysbiosis. These effects were reversed by P35. CYP1A1 and CYP1B1 expression in the skin was increased in the exposed mice until P21, then declined. Both CYP proteins co-localized with LRIG1-expressing progenitor cells at the infundibulum. CYP1B1 protein also co-localized with a second stem cell niche in the isthmus. These results indicate that this exposure to TCDD causes a chloracne-like effect without inflammation. Transient activation of the AhR, due to the shorter half-life of TCDD in mice, likely contributes to the reversibility of these effects.

Enhanced neurite outgrowth on electrically conductive carbon aerogel substrates in the presence of an external electric field.

Previous works from our laboratory have firmly established that aerogels are a suitable substrate to elicit accelerated neurite extension. On non-conducting aerogels, in the presence of an externally-applied DC bias, neurons extended neurites which were preferentially aligned towards the anode. In this investigation, we sought to determine whether electrically-conductive carbon aerogels elicited a more robust alignment of neurites toward the anode than non-conductive aerogels due to the capacity of conductive aerogels to sustain a current, thereby providing a direct interface between neurons and the external electrical stimulus. To determine if this was the case, we plated PC12 neuronal cells on electrically conductive carbon aerolges derived from acetic acid-catalized resorcinol formaldehyde aerogels (ARF-CA) and subjected them to an external electric field. The voltages applied at the electrodes of the custom-built electro-stimulation chamber were 0 V, 15 V, and 30 V. For each voltage, the directionality and length of the neurites extended by PC12 cells were determined and compared to those observed when PC12 cells were plated on non-conductive aerogels subjected to the same voltage. The results show that the directionality of neurite extension was similar between conductive and non-conductive aerogels. A higher neurite length difference was observed on conductive aerogels with increasing voltage, 43% and 106% for 0-15 V and 0-30 V respectively, compared to non-conductive aerogels, 12% and 20%. These findings indicate that conductive carbon aerogels have a greater potential as scaffolds for nerve regeneration than non-conductive ones.

TEM study of chronic alcoholism effects on early carcinogenesis by probing the nanoscale structural alterations of cell nuclei.

Nanoscale structural alteration in the nuclei of cells with the progression of carcinogenesis is due to the rearrangements of the basic building blocks in the cell such as DNA, RNA, lipids, etc. Although epigenetic modifications underlie the development of cancer, exposure to carcinogenic chemicals such as alcohol also enhances the development of cancer. We report the effects of chronic alcoholism on early-carcinogenesis based on changes in the degree of nanoscale structural alterations (L d) in nuclei. For this, transmission electron microscopy (TEM) imaging of the nuclei of colonic cells is performed for the following four mouse models: control mice; chronic alcoholic mice treated with ethanol (i.e., EtOH mice); mice treated with colonic carcinogen azoxymethane (AOM) and dextran sulfate sodium (DSS) that induced colitis (i.e., AOM + DSS mice); and chronic alcoholic or EtOH treated mice, together with AOM and DSS treatment (i.e., AOM + DSS + EtOH mice). The disordered optical lattices are constructed from their respective TEM images of thin colonic cell nuclei and the L d values are calculated using the inverse participation ratio (IPR) technique from the spatially localized eigenfunctions of these lattices. Results show no significant difference in the average L d value of the colon cell nuclei of alcohol treated mice relative to its control [i.e., L d(C) ∼ L d(EtOH)]; however, an increase in the L d value of alcohol treated precancerous cells [i.e., L d(AOM + DSS + EtOH) > L d(AOM + DSS)], indicating that alcohol accelerates the early carcinogenic process.

Nerve Response to Superelastic Shape Memory Polyurethane Aerogels.

We have previously shown the suitability of aerogels as scaffolds for neuronal cells. Here, we report on the use of superelastic shape memory polyurethane aerogels (SSMPA). SSMPA have a distinctly different stiffness than previously reported aerogels. The soft and deformable nature of SSMPA allowed for radial compression of the aerogel induced by a custom designed apparatus. This radial compression changed the pore diameter and surface roughness (Sa) of SSMPA, while maintaining similar stiffness. Two varieties of SSMPA were used, Mix-14 and Mix-18, with distinctly different pore diameters and Sa. Radial compression led to a decreased pore diameter, which, in turn, decreased the Sa. The use of custom designed apparatus and two types of SSMPA allowed us to examine the influence of stiffness, pore size, and Sa on the extension of processes (neurites) by PC12 neuronal cells. PC12 cells plated on SSMPA with a higher degree of radial compression extended fewer neurites per cell when compared to other groups. However, the average length of the neurites was significantly longer when compared to the unrestricted group and to those extended by cells plated on SSMPA with less radial compression. These results demonstrate that SSMPA with 1.9 µm pore diameter, 1.17 µm Sa, and 203 kPa stiffness provides the optimum combination of physical parameters for nerve regeneration.

Advantages of Fresnel biprism-based digital holographic microscopy in quantitative phase imaging.

SIGNIFICANCE: The hallmarks of digital holographic microscopy (DHM) compared with other quantitative phase imaging (QPI) methods are high speed, accuracy, spatial resolution, temporal stability, and polarization-sensitivity (PS) capability. The above features make DHM suitable for real-time quantitative PS phase imaging in a broad number of biological applications aimed at understanding cell growth and dynamic changes occurring during physiological processes and/or in response to pharmaceutical agents. AIM: The insertion of a Fresnel biprism (FB) in the image space of a light microscope potentially turns any commercial system into a DHM system enabling QPI with the five desired features in QPI simultaneously: high temporal sensitivity, high speed, high accuracy, high spatial resolution, and PS. To the best of our knowledge, this is the first FB-based DHM system providing these five features all together. APPROACH: The performance of the proposed system was calibrated with a benchmark phase object. The PS capability has been verified by imaging human U87 glioblastoma cells. RESULTS: The proposed FB-based DHM system provides accurate phase images with high spatial resolution. The temporal stability of our system is in the order of a few nanometers, enabling live-cell studies. Finally, the distinctive behavior of the cells at different polarization angles (e.g., PS capability) can be observed with our system. CONCLUSIONS: We have presented a method to turn any commercial light microscope with monochromatic illumination into a PS QPI system. The proposed system provides accurate quantitative PS phase images in a new, simple, compact, and cost-effective format, thanks to the low cost (a few hundred dollars) involved in implementing this simple architecture, enabling the use of this QPI technique accessible to most laboratories with standard light microscopes.

Controlled Dynamics of Neural Tumor Cells by Templated Liquid Crystalline Polymer Networks.

The ability to control the alignment and organization of cell populations has great potential for tissue engineering and regenerative medicine. A variety of approaches such as nano/microtopographical patterning, mechanical loading, and nanocomposite synthesis have been developed to engineer scaffolds able to control cellular properties and behaviors. In this work, a patterned liquid crystal polymer network (LCN) film is synthesized by using a nematic liquid crystal template in which the molecular orientations are predesigned by photopatterning technique. Various configurations of polymer networks such as linear and circular patterns are created. When neural tumor cells are plated onto the templated LCN films, the cell alignment, migration, and proliferation are directed in both linear and curvilinear fashions following the pattern of the aligned polymer chains. A complex LCN pattern with zigzag geometry is also fabricated and found to be capable of controlling cell alignment and collective cellular organization. The demonstrated control of cell dynamics and organization by LCN films with various molecular alignments opens new opportunities to design scaffolds to control cultured cell organization in a manner resembling that found in tissues and to develop novel advanced materials for nerve repair, tissue engineering, and regenerative medicine applications.

Growing Neural PC-12 Cell on Crosslinked Silica Aerogels Increases Neurite Extension in the Presence of an Electric Field.

Externally applied electrical stimulation (ES) has been shown to enhance the nerve regeneration process and to influence the directionality of neurite outgrowth. In addition, the physical and chemical properties of the substrate used for nerve-cell regeneration is critical in fostering regeneration. Previously, we have shown that polyurea-crosslinked silica aerogels (PCSA) exert a positive influence on the extension of neurites by PC-12 cells, a cell-line model widely used to study neurite extension and electrical excitability. In this work, we have examined how an externally applied electric field (EF) influences the extension of neurites in PC-12 cells grown on two substrates: collagen-coated dishes versus collagen-coated crosslinked silica aerogels. The externally applied direct current (DC) bias was applied in vitro using a custom-designed chamber containing polydimethysiloxane (PDMS) embedded copper electrodes to create an electric field across the substrate for the cultured PC-12 cells. Results suggest orientation preference towards the anode, and, on average, longer neurites in the presence of the applied DC bias than with 0 V DC bias. In addition, neurite length was increased in cells grown on silica-crosslinked aerogel when compared to cells grown on regular petri-dishes. These results further support the notion that PCSA is a promising material for nerve regeneration.

Quantification of photonic localization properties of targeted nuclear mass density variations: Application in cancer-stage detection.

Light localization is a phenomenon which arises due to the interference effects of light waves inside a disordered optical medium. Quantification of degree light localization in optical media is widely used for characterizing degree of structural disorder in that media. Recently, this light localization approach was extended to analyze structural changes in biological cell like heterogeneous optical media, with potential application in cancer diagnostics. Confocal fluorescence microscopy was used to construct "optical lattices," which represents 2-dimensional refractive index map corresponding to the spatial mass density distribution of a selected molecule inside the cell. The structural disorder properties of the selected molecules were evaluated numerically using light localization strength in these optical lattices, in a single parameter called "disorder strength." The method showed a promising potential in differentiating cancerous and non-cancerous cells. In this paper, we show that by quantifying submicron scale disorder strength in the nuclear DNA mass density distribution, a wide range of control and cancerous breast and prostate cells at different hierarchy levels of tumorigenicity were correctly distinguished. We also discuss how this photonic technique can be used in examining tumorigenicity level in unknown prostate cancer cells, and potential to generalize the method to other cancer cells.

Investigation of surface topography and stiffness on adhesion and neurites extension of PC12 cells on crosslinked silica aerogel substrates.

Fundamental understanding and characterization of neural response to substrate topography is essential in the development of next generation biomaterials for nerve repair. Aerogels are a new class of materials with great potential as a biomaterial. In this work, we examine the extension of neurites by PC12 cells plated on matrigel-coated and collagen-coated mesoporous aerogel surfaces. We have successfully established the methodology for adhesion and growth of PC12 cells on polyurea crosslinked silica aerogels. Additionally, we have quantified neurite behaviors and compared their response on aerogel substrates with their behavior on tissue culture (TC) plastic, and polydimethylsiloxane (PDMS). We found that, on average, PC12 cells extend longer neurites on crosslinked silica aerogels than on tissue culture plastic, and, that the average number of neurites per cluster is lower on aerogels than on tissue culture plastic. Aerogels are an attractive candidate for future development of smart neural implants and the work presented here creates a platform for future work with this class of materials as a substrate for bioelectronic interfacing.

Light localization properties of weakly disordered optical media using confocal microscopy: application to cancer detection.

We have developed a novel technique to quantify submicron scale mass density fluctuations in weakly disordered heterogeneous optical media using confocal fluorescence microscopy. Our method is based on the numerical evaluation of the light localization properties of an 'optical lattice' constructed from the pixel intensity distributions of images obtained with confocal fluorescence microscopy. Here we demonstrate that the technique reveals differences in the mass density fluctuations of the fluorescently labeled molecules between normal and cancer cells, and that it has the potential to quantify the degree of malignancy of cancer cells. Potential applications of the technique to other disease situations or characterizing disordered samples are also discussed.

Microtubule actin cross-linking factor 1, a novel target in glioblastoma.

Genetic heterogeneity is recognized as a major contributing factor of glioblastoma resistance to clinical treatment modalities and consequently low overall survival rates. This genetic diversity results in variations in protein expression, both intratumorally and between individual glioblastoma patients. In this regard, the spectraplakin protein, microtubule actin cross-linking factor 1 (MACF1), was examined in glioblastoma. An expression analysis of MACF1 in various types of brain tumor tissue revealed that MACF1 was predominately present in grade III-IV astroctyomas and grade IV glioblastoma, but not in normal brain tissue, normal human astrocytes and lower grade brain tumors. Subsequent genetic inhibition experiments showed that suppression of MACF1 selectively inhibited glioblastoma cell proliferation and migration in cell lines established from patient derived xenograft mouse models and immortalized glioblastoma cell lines that were associated with downregulation of the Wnt-signaling mediators, Axin1 and ß-catenin. Additionally, concomitant MACF1 silencing with the chemotherapeutic agent temozolomide (TMZ) used for the clinical treatment of glioblastomas cooperatively reduced the proliferative capacity of glioblastoma cells. In conclusion, the present study represents the first investigation on the functional role of MACF1 in tumor cell biology, as well as demonstrates its potential as a unique biomarker that can be targeted synergistically with TMZ as part of a combinatorial therapeutic approach for the treatment of genetically multifarious glioblastomas.

Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.

Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes.

Pre-embedding Double-Label Immunoelectron Microscopy of Chemically Fixed Tissue Culture Cells.

Localization of specific proteins within cells at the nanometer level of resolution is central to understanding how these proteins function in cell processes such as motility and intracellular trafficking. Such localization can be achieved by combining transmission electron microscopy (TEM) with immunogold labeling. Here we describe a pre-embedding, indirect gold immunolabeling approach to localize two different proteins of interest with secondary antibodies labeled with gold particles of different sizes in cells grown on cover slips. In this protocol, the cells are immunolabeled prior to being embedded in an epoxy resin for ultrathin sectioning. The protocol also includes strategies for optimizing the balance between ultrastructure and antigen preservation, steps to minimize nonspecific antibody binding, and steps to optimize antibody penetration.

Synemin: Molecular Features and the Use of Proximity Ligation Assay to Study Its Interactions.

Synemin has three splice variants (α, ß, and L) with identical head and rod domains but with tail domains of varying size. α- and ß-Synemin are larger than most intermediate filament proteins (1565 and 1253 amino acids, respectively) but L-synemin is shorter (339 amino acids). Synemin isoforms do not self-assemble into filaments but can copolymerize with vimentin and desmin. Synemin is present in all muscle cell types, in a few neural cell types, and in various other nonepithelial cell types. Synemin expression is regulated, sometimes in an isoform-specific manner, during development of the nervous system, in brain and breast cancer cells and during injuries to the brain and liver. Mice-lacking synemin develop a myopathic phenotype, possibly due to synemin role in linking desmin filaments to costameres and sarcomeres. Synemin may play this role through its demonstrated binding to costameric and sarcolemmal proteins, such as α-actinin, vinculin, and members of the dystroglycan complex. In astrocytoma cells, synemin regulates proliferation by interacting with PP2A to modulate Akt phosphorylation status. Methods to identify synemin binding partners are central to understand the roles of this protein in diverse cell types. Here, we describe how to use proximal ligation assays (PLA) for this purpose. PLA complement biochemical methods such as immunoprecipitation by relying on the use of antibodies conjugated to oligonucleotide probes to visualize by fluorescence microscopy protein-protein interactions in cells and tissues.