WorldWideScience

Sample records for 3d pulp cells

  1. The Effect of 5 Proteins On DPPSC (Dental Pulp Pluripotent Stem Cells) For Osteoblast Differentiation Proliferation In 3D

    OpenAIRE

    Chatakun, Punjamaporn

    2014-01-01

    Bone-tissue engineering is a therapeutic target in the field of dental implant and orthopaedic surgery. It is therefore essential to find a microenvironment that enhances the growth and differentiation of osteoblasts both from mesenchymal stem cells (MSCs) and those derived from dental pulp (DPSC). The aim of this research is to determine the relationship among the proteins fibronectin (FN), bone morphogenetic protein 2 (BMP2) osteopontin (OPN), tenascin C (TNC) and bone sialoprotein (BSP) an...

  2. Human tooth pulp anatomy visualization by 3D magnetic resonance microscopy

    International Nuclear Information System (INIS)

    Precise assessment of dental pulp anatomy is of an extreme importance for a successful endodontic treatment. As standard radiographs of teeth provide very limited information on dental pulp anatomy, more capable methods are highly appreciated. One of these is 3D magnetic resonance (MR) microscopy of which diagnostic capabilities in terms of a better dental pulp anatomy assessment were evaluated in the study. Twenty extracted human teeth were scanned on a 2.35 T MRI system for MR microscopy using the 3D spin-echo method that enabled image acquisition with isotropic resolution of 100 μm. The 3D images were then post processed by ImageJ program (NIH) to obtain advanced volume rendered views of dental pulps. MR microscopy at 2.35 T provided accurate data on dental pulp anatomy in vitro. The data were presented as a sequence of thin 2D slices through the pulp in various orientations or as volume rendered 3D images reconstructed form arbitrary view-points. Sequential 2D images enabled only an approximate assessment of the pulp, while volume rendered 3D images were more precise in visualization of pulp anatomy and clearly showed pulp diverticles, number of pulp canals and root canal anastomosis. This in vitro study demonstrated that MR microscopy could provide very accurate 3D visualization of dental pulp anatomy. A possible future application of the method in vivo may be of a great importance for the endodontic treatment

  3. Dental pulp stem cells

    DEFF Research Database (Denmark)

    Ashri, N. Y.; Ajlan, S. A.; Aldahmash, Abdullah M.

    2015-01-01

    Inflammatory periodontal disease is a major cause of loss of tooth-supporting structures. Novel approaches for regeneration of periodontal apparatus is an area of intensive research. Periodontal tissue engineering implies the use of appropriate regenerative cells, delivered through a suitable...... scaffold, and guided through signaling molecules. Dental pulp stem cells have been used in an increasing number of studies in dental tissue engineering. Those cells show mesenchymal (stromal) stem cell-like properties including self-renewal and multilineage differentiation potentials, aside from...... an updated review on dental pulp stem cells and their applications in periodontal regeneration, in combination with different scaffolds and growth factors....

  4. 3D culture for cardiac cells.

    Science.gov (United States)

    Zuppinger, Christian

    2016-07-01

    This review discusses historical milestones, recent developments and challenges in the area of 3D culture models with cardiovascular cell types. Expectations in this area have been raised in recent years, but more relevant in vitro research, more accurate drug testing results, reliable disease models and insights leading to bioartificial organs are expected from the transition to 3D cell culture. However, the construction of organ-like cardiac 3D models currently remains a difficult challenge. The heart consists of highly differentiated cells in an intricate arrangement.Furthermore, electrical “wiring”, a vascular system and multiple cell types act in concert to respond to the rapidly changing demands of the body. Although cardiovascular 3D culture models have been predominantly developed for regenerative medicine in the past, their use in drug screening and for disease models has become more popular recently. Many sophisticated 3D culture models are currently being developed in this dynamic area of life science. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

  5. A microfluidic device for 2D to 3D and 3D to 3D cell navigation

    International Nuclear Information System (INIS)

    Microfluidic devices have received wide attention and shown great potential in the field of tissue engineering and regenerative medicine. Investigating cell response to various stimulations is much more accurate and comprehensive with the aid of microfluidic devices. In this study, we introduced a microfluidic device by which the matrix density as a mechanical property and the concentration profile of a biochemical factor as a chemical property could be altered. Our microfluidic device has a cell tank and a cell culture chamber to mimic both 2D to 3D and 3D to 3D migration of three types of cells. Fluid shear stress is negligible on the cells and a stable concentration gradient can be obtained by diffusion. The device was designed by a numerical simulation so that the uniformity of the concentration gradients throughout the cell culture chamber was obtained. Adult neural cells were cultured within this device and they showed different branching and axonal navigation phenotypes within varying nerve growth factor (NGF) concentration profiles. Neural stem cells were also cultured within varying collagen matrix densities while exposed to NGF concentrations and they experienced 3D to 3D collective migration. By generating vascular endothelial growth factor concentration gradients, adult human dermal microvascular endothelial cells also migrated in a 2D to 3D manner and formed a stable lumen within a specific collagen matrix density. It was observed that a minimum absolute concentration and concentration gradient were required to stimulate migration of all types of the cells. This device has the advantage of changing multiple parameters simultaneously and is expected to have wide applicability in cell studies. (paper)

  6. A microfluidic device for 2D to 3D and 3D to 3D cell navigation

    Science.gov (United States)

    Shamloo, Amir; Amirifar, Leyla

    2016-01-01

    Microfluidic devices have received wide attention and shown great potential in the field of tissue engineering and regenerative medicine. Investigating cell response to various stimulations is much more accurate and comprehensive with the aid of microfluidic devices. In this study, we introduced a microfluidic device by which the matrix density as a mechanical property and the concentration profile of a biochemical factor as a chemical property could be altered. Our microfluidic device has a cell tank and a cell culture chamber to mimic both 2D to 3D and 3D to 3D migration of three types of cells. Fluid shear stress is negligible on the cells and a stable concentration gradient can be obtained by diffusion. The device was designed by a numerical simulation so that the uniformity of the concentration gradients throughout the cell culture chamber was obtained. Adult neural cells were cultured within this device and they showed different branching and axonal navigation phenotypes within varying nerve growth factor (NGF) concentration profiles. Neural stem cells were also cultured within varying collagen matrix densities while exposed to NGF concentrations and they experienced 3D to 3D collective migration. By generating vascular endothelial growth factor concentration gradients, adult human dermal microvascular endothelial cells also migrated in a 2D to 3D manner and formed a stable lumen within a specific collagen matrix density. It was observed that a minimum absolute concentration and concentration gradient were required to stimulate migration of all types of the cells. This device has the advantage of changing multiple parameters simultaneously and is expected to have wide applicability in cell studies.

  7. Fabrication of Nanostructured Poly-ε-caprolactone 3D Scaffolds for 3D Cell Culture Technology

    KAUST Repository

    Schipani, Rossana

    2015-04-21

    Tissue engineering is receiving tremendous attention due to the necessity to overcome the limitations related to injured or diseased tissues or organs. It is the perfect combination of cells and biomimetic-engineered materials. With the appropriate biochemical factors, it is possible to develop new effective bio-devices that are capable to improve or replace biological functions. Latest developments in microfabrication methods, employing mostly synthetic biomaterials, allow the production of three-dimensional (3D) scaffolds that are able to direct cell-to-cell interactions and specific cellular functions in order to drive tissue regeneration or cell transplantation. The presented work offers a rapid and efficient method of 3D scaffolds fabrication by using optical lithography and micro-molding techniques. Bioresorbable polymer poly-ε-caprolactone (PCL) was the material used thanks to its high biocompatibility and ability to naturally degrade in tissues. 3D PCL substrates show a particular combination in the designed length scale: cylindrical shaped pillars with 10μm diameter, 10μm height, arranged in a hexagonal lattice with spacing of 20μm were obtained. The sidewalls of the pillars were nanostructured by attributing a 3D architecture to the scaffold. The suitability of these devices as cell culture technology supports was evaluated by plating NIH/3T3 mouse embryonic fibroblasts and human Neural Stem Cells (hNSC) on them. Scanning Electron Microscopy (SEM) analysis was carried out in order to examine the micro- and nano-patterns on the surface of the supports. In addition, after seeding of cells, SEM and immunofluorescence characterization of the fabricated systems were performed to check adhesion, growth and proliferation. It was observed that cells grow and develop healthy on the bio-polymeric devices by giving rise to well-interconnected networks. 3D PCL nano-patterned pillared scaffold therefore may have considerable potential as effective tool for

  8. Multizone paper platform for 3D cell cultures.

    Directory of Open Access Journals (Sweden)

    Ratmir Derda

    Full Text Available In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc. The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously ("cells-in-gels-in-paper" or CiGiP, this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, "sections" all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures.

  9. Stem cell reprogramming: A 3D boost

    Science.gov (United States)

    Abilez, Oscar J.; Wu, Joseph C.

    2016-03-01

    Biophysical factors in an optimized three-dimensional microenvironment enhance the reprogramming efficiency of human somatic cells into pluripotent stem cells when compared to traditional cell-culture substrates.

  10. Cyto-3D-print to attach mitotic cells.

    Science.gov (United States)

    Castroagudin, Michelle R; Zhai, Yujia; Li, Zhi; Marnell, Michael G; Glavy, Joseph S

    2016-08-01

    The Cyto-3D-print is an adapter that adds cytospin capability to a standard centrifuge. Like standard cytospinning, Cyto-3D-print increases the surface attachment of mitotic cells while giving a higher degree of adaptability to other slide chambers than available commercial devices. The use of Cyto-3D-print is cost effective, safe, and applicable to many slide designs. It is durable enough for repeated use and made of biodegradable materials for environment-friendly disposal.

  11. Laser printing of cells into 3D scaffolds

    International Nuclear Information System (INIS)

    One of the most promising approaches in tissue engineering is the application of 3D scaffolds, which provide cell support and guidance in the initial tissue formation stage. The porosity of the scaffold and internal pore organization influence cell migration and play a major role in its biodegradation dynamics, nutrient diffusion and mechanical stability. In order to control cell migration and cellular interactions within the scaffold, novel technologies capable of producing 3D structures in accordance with predefined design are required. The two-photon polymerization (2PP) technique, used in this report for the fabrication of scaffolds, allows the realization of arbitrary 3D structures with submicron spatial resolution. Highly porous 3D scaffolds, produced by 2PP of acrylated poly(ethylene glycol), are seeded with cells by means of laser-induced forward transfer (LIFT). In this laser printing approach, a propulsive force, resulting from laser-induced shock wave, is used to propel individual cells or cell groups from a donor substrate towards the receiver substrate. We demonstrate that with this technique printing of multiple cell types into 3D scaffolds is possible. Combination of LIFT and 2PP provides a route for the realization of 3D multicellular tissue constructs and artificial ECM engineered on the microscale.

  12. Cyto-3D-print to attach mitotic cells.

    Science.gov (United States)

    Castroagudin, Michelle R; Zhai, Yujia; Li, Zhi; Marnell, Michael G; Glavy, Joseph S

    2016-08-01

    The Cyto-3D-print is an adapter that adds cytospin capability to a standard centrifuge. Like standard cytospinning, Cyto-3D-print increases the surface attachment of mitotic cells while giving a higher degree of adaptability to other slide chambers than available commercial devices. The use of Cyto-3D-print is cost effective, safe, and applicable to many slide designs. It is durable enough for repeated use and made of biodegradable materials for environment-friendly disposal. PMID:26464272

  13. Dental pulp stem cells and bone regeneration.

    Directory of Open Access Journals (Sweden)

    Amalia KAPAROU

    2015-04-01

    Full Text Available SUMMARY: Dental pulp, a soft tissue of mesenchymal origin, contains stem cells derived from cranial neural crest cells. Dental pulp stem cells (DPSCs reside into special anatomic locations of dental pulp, the so called “niches”. Stem cell niches are located predominately, but not exclusively, in the perivascular regions of the pulpal cavity. DPSCs exhibit clonogenic and high proliferative activity and are capable of differentiating into several cell types. The main function of these cells is the production of tertiary/reparative dentine following trauma or caries of dental crown. Previous studies have shown that DPSCs can differentiate into osteoblast-like cells that secrete abundant extracellular matrix and can build a woven bone in vitro. Moreover, DPSCs are capable of forming a complete and well-vascularised lamellar bone after grafting ectopically into immunocompromised rats. The in vivo transplantation of DPSCs into critical-sized bone defects in animal models has been shown to promote and/or accelerate bone regeneration. These results are clearly encouraging and stress the need of further research for the potential clinical use of DPSCs in bone tissue engineering.

  14. Molecular predictors of 3D morphogenesis by breast cancer cell lines in 3D culture.

    Directory of Open Access Journals (Sweden)

    Ju Han

    2010-02-01

    Full Text Available Correlative analysis of molecular markers with phenotypic signatures is the simplest model for hypothesis generation. In this paper, a panel of 24 breast cell lines was grown in 3D culture, their morphology was imaged through phase contrast microscopy, and computational methods were developed to segment and represent each colony at multiple dimensions. Subsequently, subpopulations from these morphological responses were identified through consensus clustering to reveal three clusters of round, grape-like, and stellate phenotypes. In some cases, cell lines with particular pathobiological phenotypes clustered together (e.g., ERBB2 amplified cell lines sharing the same morphometric properties as the grape-like phenotype. Next, associations with molecular features were realized through (i differential analysis within each morphological cluster, and (ii regression analysis across the entire panel of cell lines. In both cases, the dominant genes that are predictive of the morphological signatures were identified. Specifically, PPARgamma has been associated with the invasive stellate morphological phenotype, which corresponds to triple-negative pathobiology. PPARgamma has been validated through two supporting biological assays.

  15. Molecular Predictors of 3D Morphogenesis by Breast Cancer Cell Lines in 3D Culture

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ju; Chang, Hang; Giricz, Orsi; Lee, Genee; Baehner, Frederick; Gray, Joe; Bissell, Mina; Kenny, Paraic; Parvin, Bahram

    2010-02-01

    Correlative analysis of molecular markers with phenotypic signatures is the simplest model for hypothesis generation. In this paper, a panel of 24 breast cell lines was grown in 3D culture, their morphology was imaged through phase contrast microscopy, and computational methods were developed to segment and represent each colony at multiple dimensions. Subsequently, subpopulations from these morphological responses were identified through consensus clustering to reveal three clusters of round, grape-like, and stellate phenotypes. In some cases, cell lines with particular pathobiological phenotypes clustered together (e.g., ERBB2 amplified cell lines sharing the same morphometric properties as the grape-like phenotype). Next, associations with molecular features were realized through (i) differential analysis within each morphological cluster, and (ii) regression analysis across the entire panel of cell lines. In both cases, the dominant genes that are predictive of the morphological signatures were identified. Specifically, PPAR? has been associated with the invasive stellate morphological phenotype, which corresponds to triple-negative pathobiology. PPAR? has been validated through two supporting biological assays.

  16. Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

    Science.gov (United States)

    Goodwin, Thomas J.

    2010-01-01

    Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that

  17. Extracellular matrix of dental pulp stem cells: Applications in pulp tissue engineering using somatic MSCs

    Directory of Open Access Journals (Sweden)

    Sriram eRavindran

    2014-01-01

    Full Text Available Dental Caries affects approximately 90% of the world’s population. At present, the clinical treatment for dental caries is root canal therapy. This treatment results in loss of tooth sensitivity and vitality. Tissue engineering can potentially solve this problem by enabling regeneration of a functional pulp tissue. Dental pulp stem cells (DPSCs have been shown to be an excellent source for pulp regeneration. However, limited availability of these cells hinders its potential for clinical translation. We have investigated the possibility of using somatic mesenchymal stem cells from other sources for dental pulp tissue regeneration using a biomimetic dental pulp extracellular matrix (ECM incorporated scaffold. Human periodontal ligament stem cells (PDLSCs and human bone marrow stromal cells (HMSCs were investigated for their ability to differentiate towards an odontogenic lineage. In vitro real-time PCR results coupled with histological and immunohistochemical examination of the explanted tissues confirmed the ability of PDLSCs and HMSCs to form a vascularized pulp-like tissue. These findings indicate that the dental pulp stem derived ECM scaffold stimulated odontogenic differentiation of PDLSCs and HMSCs without the need for exogenous addition of growth and differentiation factors. This study represents a translational perspective toward possible therapeutic application of using a combination of somatic stem cells and extracellular matrix for pulp regeneration.

  18. 3D Reconstruction of Coronary Artery Vascular Smooth Muscle Cells.

    Directory of Open Access Journals (Sweden)

    Tong Luo

    Full Text Available The 3D geometry of individual vascular smooth muscle cells (VSMCs, which are essential for understanding the mechanical function of blood vessels, are currently not available. This paper introduces a new 3D segmentation algorithm to determine VSMC morphology and orientation.A total of 112 VSMCs from six porcine coronary arteries were used in the analysis. A 3D semi-automatic segmentation method was developed to reconstruct individual VSMCs from cell clumps as well as to extract the 3D geometry of VSMCs. A new edge blocking model was introduced to recognize cell boundary while an edge growing was developed for optimal interpolation and edge verification. The proposed methods were designed based on Region of Interest (ROI selected by user and interactive responses of limited key edges. Enhanced cell boundary features were used to construct the cell's initial boundary for further edge growing. A unified framework of morphological parameters (dimensions and orientations was proposed for the 3D volume data. Virtual phantom was designed to validate the tilt angle measurements, while other parameters extracted from 3D segmentations were compared with manual measurements to assess the accuracy of the algorithm. The length, width and thickness of VSMCs were 62.9±14.9 μm, 4.6±0.6 μm and 6.2±1.8 μm (mean±SD. In longitudinal-circumferential plane of blood vessel, VSMCs align off the circumferential direction with two mean angles of -19.4±9.3° and 10.9±4.7°, while an out-of-plane angle (i.e., radial tilt angle was found to be 8±7.6° with median as 5.7°.A 3D segmentation algorithm was developed to reconstruct individual VSMCs of blood vessel walls based on optical image stacks. The results were validated by a virtual phantom and manual measurement. The obtained 3D geometries can be utilized in mathematical models and leads a better understanding of vascular mechanical properties and function.

  19. Gambaran densitas kamar pulpa gigi sulung menggunakan cone beam CT-3D (Description of pulp chamber density in deciduous teeth using cone beam CT-3D)

    OpenAIRE

    Herdiyati Y; Epsilawati L; Oscandar F; Nurianingsih R

    2013-01-01

    Background: Dental caries is the most common chronic diseases. Detection of caries is needed, especially on the deciduous teeth. An examination such as radiological examination is essential. The radiographic figures distinguish radiolucent of the crown. Digital radiography cone beam computed tomography (CBCT) is able to show a more detailed picture. Purpose: This study was aimed to get value of the density of pulp chamber of caries and non caries deciduous teeth using CBCT radiographs. Method...

  20. >Effect of progesterone hormon on cell viability and stem cell activation in dental pulp cells

    OpenAIRE

    Segah Altuntaş; Muhammed Ali Kara; Deniz Selin Aksoy; Zehra Dilşad Çoban; Şefik Güran

    2016-01-01

    Objective: The dental pulp is the part in the center of a tooth made up of living connective tissue and cells called odontoblasts. The vitality of the dentin structure, both during health and after injury, depends on pulp cell activity and the signaling processes that regulate the cell’s behavior. Dental pulp tissue has condensed stem cell activity. Dental pulp stem cells are multipotent stem cells that have the potential to differentiate into a variety of cell types. Several publications hav...

  1. Gambaran densitas kamar pulpa gigi sulung menggunakan cone beam CT-3D (Description of pulp chamber density in deciduous teeth using cone beam CT-3D

    Directory of Open Access Journals (Sweden)

    Herdiyati Y

    2013-06-01

    Full Text Available Background: Dental caries is the most common chronic diseases. Detection of caries is needed, especially on the deciduous teeth. An examination such as radiological examination is essential. The radiographic figures distinguish radiolucent of the crown. Digital radiography cone beam computed tomography (CBCT is able to show a more detailed picture. Purpose: This study was aimed to get value of the density of pulp chamber of caries and non caries deciduous teeth using CBCT radiographs. Methods: The study was conducted by using simple descriptive. The samples were all the data CBCT of pediatric patients aged 7-10 years who visited the Dental Hospital of the Faculty of Dentistry, University of Padjadjaran. The samples were teeth with single and double root. Results: The results showed that the value of the normal pulp density is 422.56 Hu, while the condition of caries decreased becomes -77.89 Hu. Conclusion: The tooth with caries showed a lower density than the non caries/tooth.Latar belakang: Karies gigi merupakan penyakit kronis yang sering terjadi. Deteksi terhadap karies sangat diperlukan terutama pada gigi decidius. Pemeriksaan penunjang berupa pemeriksaan radiologis sangat diperlukan. Secara umum gambaran radiografi dapat membedakan karies berupa gambaran radiolusent pada mahkota. Radiografi digital cone beam computed tomografi (CBCT, merupakan jenis radiografi yang mampu memperlihatkan gambaran yang lebih detail. Tujuan: Penelitian ini bertujuan mendapatkan nilai densitas kamar pulpa gigi sulung yang karies dan non karies menggunakan radiografi CBCT. Metode: Penelitian dilakukan dengan metode simple deskriptif. Sampel penelitian adalah semua data CBCT dari pasien anak berusia 7 - 10 tahun yang berkunjung ke RSGM Fakultas Kedokteran Gigi Universitas Padjadjaran. Gigi yang dianalisa meliputi gigi berakar tunggal dan berakar ganda. Hasil: Hasil penelitian menunjukkan bahwa nilai densitas pulpa normal adalah 422,56 Hu, sedangkan pada kondisi

  2. Differentiation ability of rat postnatal dental pulp cells in vitro.

    NARCIS (Netherlands)

    Zhang, W.; Walboomers, X.F.; Wolke, J.G.C.; Bian, Z.; Fan, M.W.; Jansen, J.A.

    2005-01-01

    The current rapid progression in stem cell research has enhanced our knowledge of dental tissue regeneration. In this study, rat dental pulp cells were isolated and their differentiation ability was evaluated. First, dental pulp cells were obtained from maxillary incisors of male Wistar rats. Immuno

  3. 2D- and 3D-culture of cell

    Directory of Open Access Journals (Sweden)

    Khoruzhenko A. I.

    2011-02-01

    Full Text Available The cultivation of mammalian cells in three-dimensional conditions acquires a priority in a variety of biomedical applications. In the areas of toxicology and anticancer drug development it concerns a significant difference of responses to proapoptotic factors of the cells cultured in 2D versus 3D environment. Besides, the clear-cut differences have been found in cell polarity, cytoskeleton structure, distribution of receptors to wide range of hormones, growth factors, etc. in mammalian cells depending on culture conditions. It is resulted in different response of cultured cells to extracellular stimuli. Multicellular spheroids are regarded presently as the most convenient model of solid tumour growth in vitro. The cultivation of thyroid follicles, mammary acini and other structure units, maintaining initial tissue organization, allows studying the behavior, biochemical features and gene profile of differentiated cells. On the other hand, 3D cultures have some limitations in comparison with a well established monolayer culture. The advantages and disadvantages of each type of cultures and their application in biological and medical researches will be discussed in this review

  4. 3D in vitro cell culture models of tube formation.

    Science.gov (United States)

    Zegers, Mirjam M

    2014-07-01

    Building the complex architecture of tubular organs is a highly dynamic process that involves cell migration, polarization, shape changes, adhesion to neighboring cells and the extracellular matrix, physicochemical characteristics of the extracellular matrix and reciprocal signaling with the mesenchyme. Understanding these processes in vivo has been challenging as they take place over extended time periods deep within the developing organism. Here, I will discuss 3D in vitro models that have been crucial to understand many of the molecular and cellular mechanisms and key concepts underlying branching morphogenesis in vivo. PMID:24613912

  5. A Novel Combinatorial Therapy With Pulp Stem Cells and Granulocyte Colony-Stimulating Factor for Total Pulp Regeneration

    OpenAIRE

    Iohara, Koichiro; Murakami, Masashi; Takeuchi, Norio; Osako, Yohei; Ito, Masataka; Ishizaka, Ryo; Utunomiya, Shinji; Nakamura, Hiroshi; Matsushita, Kenji; Nakashima, Misako

    2013-01-01

    Autologous transplantation of pulp stem cells with granulocyte-colony stimulating factor (G-CSF) in a dog pulpectomized tooth yielded better effects than transplantation of G-CSF or pulp stem cells alone. The combinatorial trophic effects of pulp stem cells and G-CSF are of immediate utility for pulp/dentin regeneration, demonstrating the prerequisites of safety and efficacy critical for clinical applications.

  6. 3D Chromosome Regulatory Landscape of Human Pluripotent Cells.

    Science.gov (United States)

    Ji, Xiong; Dadon, Daniel B; Powell, Benjamin E; Fan, Zi Peng; Borges-Rivera, Diego; Shachar, Sigal; Weintraub, Abraham S; Hnisz, Denes; Pegoraro, Gianluca; Lee, Tong Ihn; Misteli, Tom; Jaenisch, Rudolf; Young, Richard A

    2016-02-01

    In this study, we describe the 3D chromosome regulatory landscape of human naive and primed embryonic stem cells. To devise this map, we identified transcriptional enhancers and insulators in these cells and placed them within the context of cohesin-associated CTCF-CTCF loops using cohesin ChIA-PET data. The CTCF-CTCF loops we identified form a chromosomal framework of insulated neighborhoods, which in turn form topologically associating domains (TADs) that are largely preserved during the transition between the naive and primed states. Regulatory changes in enhancer-promoter interactions occur within insulated neighborhoods during cell state transition. The CTCF anchor regions we identified are conserved across species, influence gene expression, and are a frequent site of mutations in cancer cells, underscoring their functional importance in cellular regulation. These 3D regulatory maps of human pluripotent cells therefore provide a foundation for future interrogation of the relationships between chromosome structure and gene control in development and disease. PMID:26686465

  7. 3D surface topology guides stem cell adhesion and differentiation.

    Science.gov (United States)

    Viswanathan, Priyalakshmi; Ondeck, Matthew G; Chirasatitsin, Somyot; Ngamkham, Kamolchanok; Reilly, Gwendolen C; Engler, Adam J; Battaglia, Giuseppe

    2015-06-01

    Polymerized high internal phase emulsion (polyHIPE) foams are extremely versatile materials for investigating cell-substrate interactions in vitro. Foam morphologies can be controlled by polymerization conditions to result in either open or closed pore structures with different levels of connectivity, consequently enabling the comparison between 2D and 3D matrices using the same substrate with identical surface chemistry conditions. Additionally, here we achieve the control of pore surface topology (i.e. how different ligands are clustered together) using amphiphilic block copolymers as emulsion stabilizers. We demonstrate that adhesion of human mesenchymal progenitor (hES-MP) cells cultured on polyHIPE foams is dependent on foam surface topology and chemistry but is independent of porosity and interconnectivity. We also demonstrate that the interconnectivity, architecture and surface topology of the foams has an effect on the osteogenic differentiation potential of hES-MP cells. Together these data demonstrate that the adhesive heterogeneity of a 3D scaffold could regulate not only mesenchymal stem cell attachment but also cell behavior in the absence of soluble growth factors.

  8. A novel mechanotactic 3D modeling of cell morphology

    International Nuclear Information System (INIS)

    Cell morphology plays a critical role in many biological processes, such as cell migration, tissue development, wound healing and tumor growth. Recent investigations demonstrate that, among other stimuli, cells adapt their shapes according to their substrate stiffness. Until now, the development of this process has not been clear. Therefore, in this work, a new three-dimensional (3D) computational model for cell morphology has been developed. This model is based on a previous cell migration model presented by the same authors. The new model considers that during cell–substrate interaction, cell shape is governed by internal cell deformation, which leads to an accurate prediction of the cell shape according to the mechanical characteristic of its surrounding micro-environment. To study this phenomenon, the model has been applied to different numerical cases. The obtained results, which are qualitatively consistent with well-known related experimental works, indicate that cell morphology not only depends on substrate stiffness but also on the substrate boundary conditions. A cell located within an unconstrained soft substrate (several kPa) with uniform stiffness is unable to adhere to its substrate or to send out pseudopodia. When the substrate stiffness increases to tens of kPa (intermediate and rigid substrates), the cell can adequately adhere to its substrate. Subsequently, as the traction forces exerted by the cell increase, the cell elongates and its shape changes. Within very stiff (hard) substrates, the cell cannot penetrate into its substrate or send out pseudopodia. On the other hand, a cell is found to be more elongated within substrates with a constrained surface. However, this elongation decreases when the cell approaches it. It can be concluded that the higher the net traction force, the greater the cell elongation, the larger the cell membrane area, and the less random the cell alignment. (paper)

  9. [Study progress of dental pulp stem cells in tissue engineering].

    Science.gov (United States)

    Shiyu, Shi; Jiamin, Xie

    2015-12-01

    In recent years, modern tissue engineering is becoming emerging and developing rapidly, and the acquisition, cultivation and differentiation of seed cells is the premise and foundation of the construction of tissue engineering, so more and more scholars pay attention to stem cells as seed cells for tissue engineering construction. Dental pulp stem cells (DPSCs) is a kind of adult stem cells derived from dental pulp, and as a new kind of seed cells of tissue engineering, the study of DPSCs presents important significance in tissue and organ regeneration. In this review, we introduced the progress of studies on dental pulp stem cells and discussed their clinical application prospects. PMID:27051964

  10. Self-Assembled Peptide Gels for 3D Cell Culture

    OpenAIRE

    Tang, Claire

    2010-01-01

    Under specific conditions short peptides modified with an N-terminal fluorenyl-9-methoxycarbonyl (Fmoc) group can self-assemble into hydrogel scaffolds similar in properties to the natural extracellular matrix. Fmoc-diphenylalanine (Fmoc-FF) for instance, has been shown to form hydrogels at physiological pH that have the ability to support 2D and 3D cell culture. The aim of this investigation is to provide further understanding of the self-assembly mechanism of such systems in order to progre...

  11. In Vivo Experiments with Dental Pulp Stem Cells for Pulp-Dentin Complex Regeneration

    OpenAIRE

    Sunil Kim; Su-Jung Shin; Yunjung Song; Euiseong Kim

    2015-01-01

    In recent years, many studies have examined the pulp-dentin complex regeneration with DPSCs. While it is important to perform research on cells, scaffolds, and growth factors, it is also critical to develop animal models for preclinical trials. The development of a reproducible animal model of transplantation is essential for obtaining precise and accurate data in vivo. The efficacy of pulp regeneration should be assessed qualitatively and quantitatively using animal models. This review artic...

  12. Surface modified alginate microcapsules for 3D cell culture

    Science.gov (United States)

    Chen, Yi-Wen; Kuo, Chiung Wen; Chueh, Di-Yen; Chen, Peilin

    2016-06-01

    Culture as three dimensional cell aggregates or spheroids can offer an ideal platform for tissue engineering applications and for pharmaceutical screening. Such 3D culture models, however, may suffer from the problems such as immune response and ineffective and cumbersome culture. This paper describes a simple method for producing microcapsules with alginate cores and a thin shell of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) to encapsulate mouse induced pluripotent stem (miPS) cells, generating a non-fouling surface as an effective immunoisolation barrier. We demonstrated the trapping of the alginate microcapsules in a microwell array for the continuous observation and culture of a large number of encapsulated miPS cells in parallel. miPS cells cultured in the microcapsules survived well and proliferated to form a single cell aggregate. Droplet formation of monodisperse microcapsules with controlled size combined with flow cytometry provided an efficient way to quantitatively analyze the growth of encapsulated cells in a high-throughput manner. The simple and cost-effective coating technique employed to produce the core-shell microcapsules could be used in the emerging field of cell therapy. The microwell array would provide a convenient, user friendly and high-throughput platform for long-term cell culture and monitoring.

  13. Human dental pulp stem cells with highly angiogenic and neurogenic potential for possible use in pulp regeneration.

    Science.gov (United States)

    Nakashima, Misako; Iohara, Koichiro; Sugiyama, Masahiko

    2009-01-01

    Dental caries is a common public health problem, causing early loss of dental pulp and resultant tooth loss. Dental pulp has important functions to sustain teeth providing nutrient and oxygen supply, innervation, reactionary/reparative dentin formation and immune response. Regeneration of pulp is an unmet need in endodontic therapy, and angiogenesis/vasculogenesis and neurogenesis are critical for pulp regeneration. Permanent and deciduous pulp tissue is easily available from teeth after extraction without ethical issues and has potential for clinical use. In this review, we introduce some stem cell subfractions, CD31(-)/CD146(-) SP cells and CD105(+) cells with high angiogenic and neurogenic potential, derived from human adult dental pulp tissue. Potential utility of these cells is addressed as a source of cells for treatment of cerebral and limb ischemia and pulp inflammation complete with angiogenesis and vasculogenesis.

  14. Age estimation on the stricture of pulp cavity of the mandibular first premolar by 3D micro-focus X-CT

    International Nuclear Information System (INIS)

    To know the effect of aging on pulp cavity volumes, 40 mandibular first premolars obtained from 21-75 years old persons and stored in authors' laboratory were subjected to 3D micro-focus X-CT (SMX-130CT-SV, Shimadzu Works) to determine the volume, and ratios to the root canal pulp volume of the crown (C), cervical 1/3 (R1), central (R2) and apical 1/3 (R3) pulp volumes were calculated. In all ages examined, the ratios were in the order of R1>R2>C>R3, and were decreased by aging with exception for R1. Changes in R1 were significant in thirties to fifties and in R2, in twenties to thirties. Multiple regression analysis for age estimation gave the multiple correlation coefficient of 0.82 and coefficient of determination of 0.67, and the item that the partial correlation coefficient was significantly high was R1 (RPVR1) alone. Regression analysis using RPVR1 as a variable gave the multiple correlation coefficient of 0.81 between actual and estimated ages and estimated error (within ±6 years) by the regression was 42.5%. The method was thus found usefully applied for age estimation in forensic judgment. (S.I.)

  15. 3D photospheric velocity field of a Supergranular cell

    CERN Document Server

    Del Moro, Dario; Berrilli, Francesco

    2007-01-01

    We investigate the plasma flow properties inside a Supergranular (SG) cell, in particular its interaction with small scale magnetic field structures. The SG cell has been identified using the magnetic network (CaII wing brightness) as proxy, applying the TST to high spatial, spectral and temporal resolution observations obtained by IBIS. The full 3D velocity vector field for the SG has been reconstructed at two different photospheric heights. In order to strengthen our findings, we also computed the mean radial flow of the SG by means of cork tracing. We also studied the behaviour of the horizontal and Line of Sight plasma flow cospatial with cluster of bright CaII structures of magnetic origin to better understand the interaction between photospheric convection and small scale magnetic features. The SG cell we investigated seems to be organized with an almost radial flow from its centre to the border. The large scale divergence structure is probably created by a compact region of costant up-flow close to the...

  16. In Vivo Experiments with Dental Pulp Stem Cells for Pulp-Dentin Complex Regeneration

    Directory of Open Access Journals (Sweden)

    Sunil Kim

    2015-01-01

    Full Text Available In recent years, many studies have examined the pulp-dentin complex regeneration with DPSCs. While it is important to perform research on cells, scaffolds, and growth factors, it is also critical to develop animal models for preclinical trials. The development of a reproducible animal model of transplantation is essential for obtaining precise and accurate data in vivo. The efficacy of pulp regeneration should be assessed qualitatively and quantitatively using animal models. This review article sought to introduce in vivo experiments that have evaluated the potential of dental pulp stem cells for pulp-dentin complex regeneration. According to a review of various researches about DPSCs, the majority of studies have used subcutaneous mouse and dog teeth for animal models. There is no way to know which animal model will reproduce the clinical environment. If an animal model is developed which is easier to use and is useful in more situations than the currently popular models, it will be a substantial aid to studies examining pulp-dentin complex regeneration.

  17. Identification and Isolation of Human Dental Pulp Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Xue-Chao YANG; Ming-Wen FAN

    2005-01-01

    @@ 1 Introduction Dentinal repair in the postnatal organism occurs through the activity of specialized cells, odontoblasts,that are thought to be maintained by an as yet undefined precursor population associated with pulp tissue.

  18. File list: Pol.ALL.10.Polr3d.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: Pol.ALL.20.Polr3d.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: Pol.ALL.05.Polr3d.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: Pol.ALL.50.Polr3d.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.ALL.50.Polr3d.AllCell mm9 RNA polymerase Polr3d All cell types SRX301459,SRX373...04147 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.ALL.50.Polr3d.AllCell.bed ...

  2. Expression of high mobility group box 1 in inflamed dental pulp and its chemotactic effect on dental pulp cells

    International Nuclear Information System (INIS)

    Highlights: • HMGB1 translocated from nucleus to cytoplasm during dental pulp inflammation. • HMGB1and its receptor RAGE were up-regulated in hDPCs under LPS stimulation. • HMGB1 enhanced hDPCs migration and induces cytoskeleton reorganization. • HMGB1 may play a critical role in dental pulp repair during inflamed state. - Abstract: High mobility group box 1 protein (HMGB1) is a chromatin protein which can be released extracellularly, eliciting a pro-inflammatory response and promoting tissue repair process. This study aimed to examine the expression and distribution of HMGB1 and its receptor RAGE in inflamed dental pulp tissues, and to assess its effects on proliferation, migration and cytoskeleton of cultured human dental pulp cells (DPCs). Our data demonstrated that cytoplasmic expression of HMGB1 was observed in inflamed pulp tissues, while HMGB1 expression was confined in the nuclei in healthy dental pulp. The mRNA expression of HMGB1 and RAGE were significantly increased in inflamed pulps. In in vitro cultured DPCs, expression of HMGB1 in both protein and mRNA level was up-regulated after treated with lipopolysaccharide (LPS). Exogenous HMGB1 enhanced DPCs migration in a dose-dependent manner and induced the reorganization of f-actin in DPCs. Our results suggests that HMGB1 are not only involved in the process of dental pulp inflammation, but also play an important role in the recruitment of dental pulp stem cells, promoting pulp repair and regeneration

  3. Expression of high mobility group box 1 in inflamed dental pulp and its chemotactic effect on dental pulp cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xufang, E-mail: xufang.zhang@student.qut.edu.au [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD 4059 (Australia); Jiang, Hongwei, E-mail: jianghw@163.com [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Gong, Qimei, E-mail: gongqmei@gmail.com [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Fan, Chen, E-mail: c3.fan@student.qut.edu.au [Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD 4059 (Australia); Huang, Yihua, E-mail: enu0701@163.com [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Ling, Junqi, E-mail: lingjq@mail.sysu.edu.cn [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China)

    2014-08-08

    Highlights: • HMGB1 translocated from nucleus to cytoplasm during dental pulp inflammation. • HMGB1and its receptor RAGE were up-regulated in hDPCs under LPS stimulation. • HMGB1 enhanced hDPCs migration and induces cytoskeleton reorganization. • HMGB1 may play a critical role in dental pulp repair during inflamed state. - Abstract: High mobility group box 1 protein (HMGB1) is a chromatin protein which can be released extracellularly, eliciting a pro-inflammatory response and promoting tissue repair process. This study aimed to examine the expression and distribution of HMGB1 and its receptor RAGE in inflamed dental pulp tissues, and to assess its effects on proliferation, migration and cytoskeleton of cultured human dental pulp cells (DPCs). Our data demonstrated that cytoplasmic expression of HMGB1 was observed in inflamed pulp tissues, while HMGB1 expression was confined in the nuclei in healthy dental pulp. The mRNA expression of HMGB1 and RAGE were significantly increased in inflamed pulps. In in vitro cultured DPCs, expression of HMGB1 in both protein and mRNA level was up-regulated after treated with lipopolysaccharide (LPS). Exogenous HMGB1 enhanced DPCs migration in a dose-dependent manner and induced the reorganization of f-actin in DPCs. Our results suggests that HMGB1 are not only involved in the process of dental pulp inflammation, but also play an important role in the recruitment of dental pulp stem cells, promoting pulp repair and regeneration.

  4. Gel de plaquetas: arcabouço 3D para cultura celular Platelet gel: 3D scaffold for cell culture

    Directory of Open Access Journals (Sweden)

    Andrei Moroz

    2009-01-01

    Full Text Available INTRODUÇÃO: O reparo tissular é o objetivo final da cirurgia. A cultura celular requer arcabouço mecânico que dê suporte ao crescimento celular e difusão dos nutrientes. O uso do plasma rico em plaquetas (PRP como um arcabouço 3D possui diversas vantagens: é material biológico, de fácil absorção pós-transplante, rico em fatores de crescimento, em especial PDGF- ββ e TGF-β que estimula síntese de matriz extracelular na cartilagem. OBJETIVO: Desenvolver arcabouço 3D à base de PRP. MATERIAIS E MÉTODOS: Duas formas foram idealizadas: Sphere e Carpet. Condições estéreis foram utilizadas. O gel de plaquetas permaneceu em cultura celular, observado diariamente em microscópio invertido. RESULTADOS: Ambos arcabouços obtiveram sucesso, com aspectos positivos e negativos. DISCUSSÃO: A forma Sphere não aderiu ao plástico. Observou-se retração do gel e investigação ao microscópio dificultada devido às áreas opacas no campo visual. A forma Carpet não aderiu ao plástico e apresentou-se translúcida. O tempo de estudo foi de 20 dias. CONCLUSÕES: A produção de um arcabouço 3D PRP foi um sucesso, e trata-se de uma alternativa que necessita ser mais utilizado e investigado para que se consolide em uma rota eficiente e confiável na tecnologia de engenharia tissular, particularmente em cultura de tecido cartilaginoso.INTRODUCTION: Tissue repair has been the ultimate goal of surgery. Cell culture requires a mechanical scaffold that supports cell growth and nutrient diffusion. Using platelet-rich plasma (PRP as a 3D scaffold presents various advantages: it is a biological material, easily absorbed after transplantation, rich in growth factors, in particular, PDGF-ββ and TGF-β that stimulate extracellular matrix synthesis in cartilage culture. OBJECTIVE: To develop a PRP 3D scaffold. Material and METHODS: Two forms were idealized: Sphere and Carpet. Sterile conditions were used. The platelet gel remained in culture

  5. 3D-Printing Crystallographic Unit Cells for Learning Materials Science and Engineering

    Science.gov (United States)

    Rodenbough, Philip P.; Vanti, William B.; Chan, Siu-Wai

    2015-01-01

    Introductory materials science and engineering courses universally include the study of crystal structure and unit cells, which are by their nature highly visual 3D concepts. Traditionally, such topics are explored with 2D drawings or perhaps a limited set of difficult-to-construct 3D models. The rise of 3D printing, coupled with the wealth of…

  6. Identification and Isolation of Human Dental Pulp Stem Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    1 IntroductionDentinal repair in the postnatal organism occurs through the activity of specialized cells, odontoblasts, that are thought to be maintained by an as yet undefined precursor population associated with pulp tissue. Adult pulp stem cells was found in 2000. Now, it become a critical research in the world and a new stem-cells resourse for potential clinical applications. But, specific marker of DPSCs is not clear. STRO-1 was the marker that used universally. But, the expression rate was very low in...

  7. Allogenic banking of dental pulp stem cells for innovative therapeutics.

    Science.gov (United States)

    Collart-Dutilleul, Pierre-Yves; Chaubron, Franck; De Vos, John; Cuisinier, Frédéric J

    2015-08-26

    Medical research in regenerative medicine and cell-based therapy has brought encouraging perspectives for the use of stem cells in clinical trials. Multiple types of stem cells, from progenitors to pluripotent stem cells, have been investigated. Among these, dental pulp stem cells (DPSCs) are mesenchymal multipotent cells coming from the dental pulp, which is the soft tissue within teeth. They represent an interesting adult stem cell source because they are recovered in large amount in dental pulps with non-invasive techniques compared to other adult stem cell sources. DPSCs can be obtained from discarded teeth, especially wisdom teeth extracted for orthodontic reasons. To shift from promising preclinical results to therapeutic applications to human, DPSCs must be prepared in clinical grade lots and transformed into advanced therapy medicinal products (ATMP). As the production of patient-specific stem cells is costly and time-consuming, allogenic biobanking of clinical grade human leukocyte antigen (HLA)-typed DPSC lines provides efficient innovative therapeutic products. DPSC biobanks represent industrial and therapeutic innovations by using discarded biological tissues (dental pulps) as a source of mesenchymal stem cells to produce and store, in good manufacturing practice (GMP) conditions, DPSC therapeutic batches. In this review, we discuss about the challenges to transfer biological samples from a donor to HLA-typed DPSC therapeutic lots, following regulations, GMP guidelines and ethical principles. We also present some clinical applications, for which there is no efficient therapeutics so far, but that DPSCs-based ATMP could potentially treat.

  8. 3D-printed microfluidic chips with patterned, cell-laden hydrogel constructs.

    Science.gov (United States)

    Knowlton, Stephanie; Yu, Chu Hsiang; Ersoy, Fulya; Emadi, Sharareh; Khademhosseini, Ali; Tasoglu, Savas

    2016-06-01

    Three-dimensional (3D) printing offers potential to fabricate high-throughput and low-cost fabrication of microfluidic devices as a promising alternative to traditional techniques which enables efficient design iterations in the development stage. In this study, we demonstrate a single-step fabrication of a 3D transparent microfluidic chip using two alternative techniques: a stereolithography-based desktop 3D printer and a two-step fabrication using an industrial 3D printer based on polyjet technology. This method, compared to conventional fabrication using relatively expensive materials and labor-intensive processes, presents a low-cost, rapid prototyping technique to print functional 3D microfluidic chips. We enhance the capabilities of 3D-printed microfluidic devices by coupling 3D cell encapsulation and spatial patterning within photocrosslinkable gelatin methacryloyl (GelMA). The platform presented here serves as a 3D culture environment for long-term cell culture and growth. Furthermore, we have demonstrated the ability to print complex 3D microfluidic channels to create predictable and controllable fluid flow regimes. Here, we demonstrate the novel use of 3D-printed microfluidic chips as controllable 3D cell culture environments, advancing the applicability of 3D printing to engineering physiological systems for future applications in bioengineering. PMID:27321481

  9. Allogenic banking of dental pulp stem cells for innovative therapeutics

    Institute of Scientific and Technical Information of China (English)

    Pierre-Yves; Collart-Dutilleul; Franck; Chaubron; John; De; Vos; Frédéric; J; Cuisinier

    2015-01-01

    Medical research in regenerative medicine and cellbased therapy has brought encouraging perspectives for the use of stem cells in clinical trials. Multiple types of stem cells, from progenitors to pluripotent stem cells, have been investigated. Among these, dental pulp stem cells(DPSCs) are mesenchymal multipotent cells coming from the dental pulp, which is the soft tissue within teeth. They represent an interesting adult stem cell source because they are recovered in large amount in dental pulps with non-invasive techniques compared to other adult stem cell sources. DPSCs can be obtained from discarded teeth, especially wisdom teeth extracted for orthodontic reasons. To shift from promising preclinical results to therapeutic applications to human, DPSCs must be prepared in clinical grade lots and transformed into advanced therapy medicinal products(ATMP). As the production of patient-specific stem cells is costly and time-consuming, allogenic biobanking of clinical grade human leukocyte antigen(HLA)-typed DPSC lines provides efficient innovative therapeutic products. DPSC biobanks represent industrial and therapeutic innovations by using discarded biological tissues(dental pulps) as a source of mesenchymal stem cells to produce and store, in good manufacturing practice(GMP) conditions, DPSC therapeutic batches. In this review, we discuss about the challenges to transfer biological samples from a donor to HLA-typed DPSC therapeutic lots, following regulations, GMP guidelines and ethical principles. We also present some clinical applications, for which there is no efficient therapeutics so far, but that DPSCs-based ATMP could potentially treat.

  10. Property Of Human Dental Pulp Stem Cells And Peripheral Blood Hematopoietic Stem Cells That Differentiated Both Group To Cardiac Cells

    OpenAIRE

    Jabari F; Mohammadnejad J; Yavari K

    2013-01-01

    Dental pulp is the soft live tissue inside a tooth. Dental pulp contains stem cells, known as Dental Pulp Stem Cells. The finest Dental Pulp Stem Cells are found in a baby teeth or milk teeth. The stem cells from the milk teeth are ‘mesenchymal’ type of cells. cells that have the ability to generate a wide variety of cell types like chondrocytes, osteoblasts and adipocytes. To isolate high-quality human dental pulp stem cells from accessible resources is an importan...

  11. Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation

    Directory of Open Access Journals (Sweden)

    Hanley Edward N

    2000-10-01

    Full Text Available Abstract Background The relationship between cell shape, proliferation, and extracellular matrix (ECM production, important aspects of cell behavior, is examined in a little-studied cell type, the human annulus cell from the intervertebral disc, during monolayer vs three-dimensional (3D culture. Results Three experimental studies showed that cells respond specifically to culture microenvironments by changes in cell shape, mitosis and ECM production: 1 Cell passages showed extensive immunohistochemical evidence of Type I and II collagens only in 3D culture. Chondroitin sulfate and keratan sulfate were abundant in both monolayer and 3D cultures. 2 Cells showed significantly greater proliferation in monolayer in the presence of platelet-derived growth factor compared to cells in 3D. 3 Cells on Matrigel™-coated monolayer substrates became rounded and formed nodular colonies, a finding absent during monolayer growth. Conclusions The cell's in vivo interactions with the ECM can regulate shape, gene expression and other cell functions. The shape of the annulus cell changes markedly during life: the young, healthy disc contains spindle shaped cells and abundant collagen. With aging and degeneration, many cells assume a strikingly different appearance, become rounded and are surrounded by unusual accumulations of ECM products. In vitro manipulation of disc cells provides an experimental window for testing how disc cells from given individuals respond when they are grown in environments which direct cells to have either spindle- or rounded-shapes. In vitro assessment of the response of such cells to platelet-derived growth factor and to Matrigel™ showed a continued influence of cell shape even in the presence of a growth factor stimulus. These findings contribute new information to the important issue of the influence of cell shape on cell behavior.

  12. Reprogramming mediated radio-resistance of 3D-grown cancer cells

    International Nuclear Information System (INIS)

    In vitro 3D growth of tumors is a new cell culture model that more closely mimics the features of the in vivo environment and is being used increasingly in the field of biological and medical research. It has been demonstrated that cancer cells cultured in 3D matrices are more radio-resistant compared with cells in monolayers. However, the mechanisms causing this difference remain unclear. Here we show that cancer cells cultured in a 3D microenvironment demonstrated an increase in cells with stem cell properties. This was confirmed by the finding that cells in 3D cultures upregulated the gene and protein expression of the stem cell reprogramming factors such as OCT4, SOX2, NANOG, LIN28 and miR-302a, compared with cells in monolayers. Moreover, the expression of β-catenin, a regulating molecule of reprogramming factors, also increased in 3D-grown cancer cells. These findings suggest that cancer cells were reprogrammed to become stem cell-like cancer cells in a 3D growth culture microenvironment. Since cancer stem cell-like cells demonstrate an increased radio-resistance and chemo-resistance, our results offer a new perspective as to why. Our findings shed new light on understanding the features of the 3D growth cell model and its application in basic research into clinical radiotherapy and medicine. (author)

  13. 3D in vitro cell culture models of tube formation

    NARCIS (Netherlands)

    Zegers, M.M.P.

    2014-01-01

    Building the complex architecture of tubular organs is a highly dynamic process that involves cell migration, polarization, shape changes, adhesion to neighboring cells and the extracellular matrix, physicochemical characteristics of the extracellular matrix and reciprocal signaling with the mesench

  14. Pulp tissue from primary teeth: new source of stem cells

    Directory of Open Access Journals (Sweden)

    Paloma Dias Telles

    2011-06-01

    Full Text Available SHED (stem cells from human exfoliated deciduous teeth represent a population of postnatal stem cells capable of extensive proliferation and multipotential differentiation. Primary teeth may be an ideal source of postnatal stem cells to regenerate tooth structures and bone, and possibly to treat neural tissue injury or degenerative diseases. SHED are highly proliferative cells derived from an accessible tissue source, and therefore hold potential for providing enough cells for clinical applications. In this review, we describe the current knowledge about dental pulp stem cells and discuss tissue engineering approaches that use SHED to replace irreversibly inflamed or necrotic pulps with a healthy and functionally competent tissue that is capable of forming new dentin.

  15. Development of 3-D Hydrogel Culture Systems With On-Demand Cell Separation

    OpenAIRE

    Hamilton, Sharon K.; Bloodworth, Nathaniel C.; Massad, Christopher S.; Hammoudi, Taymour M.; Suri, Shalu; Yang, Peter J.; Lu, Hang; Temenoff, Johnna S

    2013-01-01

    Recently there has been an increased interest in the effects of paracrine signaling between groups of cells, particularly in the context of better understanding how stem cells contribute to tissue repair. Most current 3-D co-culture methods lack the ability to effectively separate 2 cell populations after the culture period, which is important for simultaneously analyzing the reciprocal effects of each cell type on the other. Here, we detail the development of a 3-D hydrogel co-culture system...

  16. Screening for Stromal and Matrix Effects in 3D Microenvironments of Breast Cancer Cells

    Science.gov (United States)

    Montanez-Sauri, Sara I.

    Breast cancer progression ensures through the acquisition of genetic mutations, the uncontrollable growth of cells, and their progression to invasion. Studies have shown that the surrounding three-dimensional (3D) microenvironment can also influence breast cancer cell progression by controlling the morphology, differentiation, proliferation, and migration of cells. However, most of the currently available in vitro screening platforms are based on the two-dimensional (2D) culture of cells, and do not provide cells with the complex 3D microenvironment that exists in vivo. Therefore, there is a need for more biologically relevant in vitro platforms to help decipher the complexity of the microenvironment and its influence in breast cancer. In this dissertation we present an automated microfluidic platform that allows to efficiently screen for the effect of multiple matrix and stromal microenvironment in 3D cultures of breast cancer cells. Several extracellular matrix (ECM) compositions and stromal cells are included in the 3D microenvironments to examine their influence on breast cancer cell behavior. The screening results suggest that collagen gels with fibronectin might be influencing paracrine signals between breast cancer cells and stromal cells. The ability of the platform to culture and treat cells in 3D microenvironments offers a powerful screening tool for the identification of compounds and interactions using more in vivo-like 3D microenvironments. The identification of these mechanisms will increase our current understanding of breast cancer, and will aid in the identification of potential therapeutics.

  17. Local 3D matrix confinement determines division axis through cell shape.

    Science.gov (United States)

    He, Lijuan; Chen, Weitong; Wu, Pei-Hsun; Jimenez, Angela; Wong, Bin Sheng; San, Angela; Konstantopoulos, Konstantinos; Wirtz, Denis

    2016-02-01

    How the division axis is determined in mammalian cells embedded in three-dimensional (3D) matrices remains elusive, despite that many types of cells divide in 3D environments. Cells on two-dimensional (2D) substrates typically round up completely to divide. Here, we show that in 3D collagen matrices, mammalian cells such as HT1080 human fibrosarcoma and MDA-MB-231 breast cancer cells exhibit division modes distinct from their Counterparts on 2D substrates, with a markedly higher fraction of cells remaining highly elongated through mitosis in 3D matrices. The long axis of elongated mitotic cells accurately predicts the division axis, independently of matrix density and cell-matrix interactions. This 3D-specific elongated division mode is determined by the local confinement produced by the matrix and the ability of cells to protrude and locally remodel the matrix via β1 integrin. Elongated division is readily recapitulated using collagen-coated microfabricated channels. Cells depleted of β1 integrin still divide in the elongated mode in microchannels, suggesting that 3D confinement is sufficient to induce the elongated cell-division phenotype.

  18. Nonpolarized signaling reveals two distinct modes of 3D cell migration.

    Science.gov (United States)

    Petrie, Ryan J; Gavara, Núria; Chadwick, Richard S; Yamada, Kenneth M

    2012-04-30

    We search in this paper for context-specific modes of three-dimensional (3D) cell migration using imaging for phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and active Rac1 and Cdc42 in primary fibroblasts migrating within different 3D environments. In 3D collagen, PIP3 and active Rac1 and Cdc42 were targeted to the leading edge, consistent with lamellipodia-based migration. In contrast, elongated cells migrating inside dermal explants and the cell-derived matrix (CDM) formed blunt, cylindrical protrusions, termed lobopodia, and Rac1, Cdc42, and PIP3 signaling was nonpolarized. Reducing RhoA, Rho-associated protein kinase (ROCK), or myosin II activity switched the cells to lamellipodia-based 3D migration. These modes of 3D migration were regulated by matrix physical properties. Specifically, experimentally modifying the elasticity of the CDM or collagen gels established that nonlinear elasticity supported lamellipodia-based migration, whereas linear elasticity switched cells to lobopodia-based migration. Thus, the relative polarization of intracellular signaling identifies two distinct modes of 3D cell migration governed intrinsically by RhoA, ROCK, and myosin II and extrinsically by the elastic behavior of the 3D extracellular matrix.

  19. Design of 3D printed insert for hanging culture of Caco-2 cells

    International Nuclear Information System (INIS)

    A Caco-2 cell culture on Transwell, an alternative testing to animal or human testing used in evaluating drug intestinal permeability, incorrectly estimated the absorption of actively transported drugs due to the low expression of membrane transporters. Similarly, three-dimensional (3D) cultures of Caco-2 cells, which have been recommended to be more physiological relevant, were not superior to the Transwell culture in either accuracy or convenience in drug permeability testing. Using rapid 3D printing prototyping techniques, this study proposed a hanging culture of Caco-2 cells that performed with high accuracy in predicting drug permeability in humans. As found, hanging cultured Caco-2 cells formed a confluent monolayer and maintained high cell viability on the 3D printed insert. Compared with the normal culture on Transwell, the Caco-2 cells on the 3D printed insert presented ∼30–100% higher brush border enzyme activity and ∼2–7 folds higher activity of P-glycoprotein/multidrug resistance-associated protein 2 during 21 days of incubation. For the eight membrane transporter substrates, the predictive curve of the 3D printing culture exhibited better linearity (R2 = 0.92) to the human oral adsorption than that of the Transwell culture (R2 = 0.84), indicating better prediction by the 3D printing culture. In this regard, the 3D printed insert for hanging culture could be potentially developed as a convenient and low-cost tool for testing drug oral absorption. (paper)

  20. Cell compatible encapsulation of filaments into 3D hydrogels.

    Science.gov (United States)

    Schirmer, Katharina S U; Gorkin, Robert; Beirne, Stephen; Stewart, Elise; Thompson, Brianna C; Quigley, Anita F; Kapsa, Robert M I; Wallace, Gordon G

    2016-06-01

    Tissue engineering scaffolds for nerve regeneration, or artificial nerve conduits, are particularly challenging due to the high level of complexity the structure of the nerve presents. The list of requirements for artificial nerve conduits is long and includes the ability to physically guide nerve growth using physical and chemical cues as well as electrical stimulation. Combining these characteristics into a conduit, while maintaining biocompatibility and biodegradability, has not been satisfactorily achieved by currently employed fabrication techniques. Here we present a method combining pultrusion and wet-spinning techniques facilitating incorporation of pre-formed filaments into ionically crosslinkable hydrogels. This new biofabrication technique allows the incorporation of conducting or drug-laden filaments, controlled guidance channels and living cells into hydrogels, creating new improved conduit designs. PMID:27213861

  1. Fabrication and optimization of alginate hydrogel constructs for use in 3D neural cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Frampton, J P; Hynd, M R; Shain, W [Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY 12210 (United States); Shuler, M L, E-mail: jf7674@albany.edu [Department of Biomedical Engineering, 270 Olin Hall, Cornell University, Ithaca, NY 14850 (United States)

    2011-02-15

    Two-dimensional (2D) culture systems provide useful information about many biological processes. However, some applications including tissue engineering, drug transport studies, and analysis of cell growth and dynamics are better studied using three-dimensional (3D) culture systems. 3D culture systems can potentially offer higher degrees of organization and control of cell growth environments, more physiologically relevant diffusion characteristics, and permit the formation of more extensive 3D networks of cell-cell interactions. A 3D culture system has been developed using alginate as a cell scaffold, capable of maintaining the viability and function of a variety of neural cell types. Alginate was functionalized by the covalent attachment of a variety of whole proteins and peptide epitopes selected to provide sites for cell attachment. Alginate constructs were used to entrap a variety of neural cell types including astroglioma cells, astrocytes, microglia and neurons. Neural cells displayed process outgrowth over time in culture. Cell-seeded scaffolds were characterized in terms of their biochemical and biomechanical properties, effects on seeded neural cells, and suitability for use as 3D neural cell culture models.

  2. Bioimpedance monitoring of 3D cell culturing--complementary electrode configurations for enhanced spatial sensitivity.

    Science.gov (United States)

    Canali, Chiara; Heiskanen, Arto; Muhammad, Haseena Bashir; Høyum, Per; Pettersen, Fred-Johan; Hemmingsen, Mette; Wolff, Anders; Dufva, Martin; Martinsen, Ørjan Grøttem; Emnéus, Jenny

    2015-01-15

    A bioimpedance platform is presented as a promising tool for non-invasive real-time monitoring of the entire process of three-dimensional (3D) cell culturing in a hydrogel scaffold. In this study, the dynamics involved in the whole process of 3D cell culturing, starting from polymerisation of a bare 3D gelatin scaffold, to human mesenchymal stem cell (MSC) encapsulation and proliferation, was monitored over time. The platform consists of a large rectangular culture chamber with four embedded vertical gold plate electrodes that were exploited in two- and three terminal (2T and 3T) measurement configurations. By switching between the different combinations of electrode couples, it was possible to generate a multiplexing-like approach, which allowed for collecting spatially distributed information within the 3D space. Computational finite element (FE) analysis and electrochemical impedance spectroscopic (EIS) characterisation were used to determine the configurations' sensitivity field localisation. The 2T setup gives insight into the interfacial phenomena at both electrode surfaces and covers the central part of the 3D cell culture volume, while the four 3T modes provide focus on the dynamics at the corners of the 3D culture chamber. By combining a number of electrode configurations, complementary spatially distributed information on a large 3D cell culture can be obtained with maximised sensitivity in the entire 3D space. The experimental results show that cell proliferation can be monitored within the tested biomimetic environment, paving the way to further developments in bioimpedance tracking of 3D cell cultures and tissue engineering. PMID:25058941

  3. In Vitro Guidance of Dental Pulp Cells by Nd:YAG Laser-Irradiated Endothelial Cells

    OpenAIRE

    Masuda, Yoshiko Murakami; Yamada, Yoshishige; Kimura, Yuichi

    2012-01-01

    Objective: After endothelial cells were ablated by neodymium:yttrium-aluminum-garnet (Nd:YAG) laser irradiation, we investigated the response of pulp cells by examining the expression of transforming growth factor beta-1 (TGF-β1). Background data: The reaction of stimulated blood vessels is related to the initiation of dentinogenesis. After artificial injury of endothelial cells, pulp cells migrate to the site of the injured endothelial cells. Materials and methods: Rat aortic endothelial cel...

  4. XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of Telomeres in Tumor Cells.

    Science.gov (United States)

    Taylor-Kashton, Cheryl; Lichtensztejn, Daniel; Baloglu, Erkan; Senapedis, William; Shacham, Sharon; Kauffman, Michael G; Kotb, Rami; Mai, Sabine

    2016-12-01

    Previous work has shown that the three-dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT-185, KPT-330/selinexor, and KPT-8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment-naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non-lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. J. Cell. Physiol. 231: 2711-2719, 2016. © 2016 Wiley Periodicals, Inc. PMID:26991404

  5. >Effect of progesterone hormon on cell viability and stem cell activation in dental pulp cells

    Directory of Open Access Journals (Sweden)

    Segah Altuntaş

    2016-03-01

    Full Text Available Objective: The dental pulp is the part in the center of a tooth made up of living connective tissue and cells called odontoblasts. The vitality of the dentin structure, both during health and after injury, depends on pulp cell activity and the signaling processes that regulate the cell’s behavior. Dental pulp tissue has condensed stem cell activity. Dental pulp stem cells are multipotent stem cells that have the potential to differentiate into a variety of cell types. Several publications have stressed the importance of the expression of pluripotentiality associated markers: the transcription factors Nanog, Sox2, Oct3/4, SSEA4, CD13, Stro1 are indispensable for the stem cells to divide indefinitely without affecting their differentiation potential (self renewal capacity. Progesterone is a steroid hormone leading to menstrual cycle and gestation. There is a widespread rumor among people that pregnancy causes toothy loss. Method: So, progesterone was applied in different concentrations on human dental pulp cells in cell culture. Cell viability assay was applied 24th hour later with trypan blue. RNA isolation, cDNA synthesis and Real Time PCR analysis were applied on selected transcription factors (Nanog and Oct4 (POU5F1 genes which have role on steamness of stem cells. Gene expression analyses results were correlated with the cell viability assay results. Results: Cell viability assay results were 80% viable in control, 82% viable in 7 ml progesterone application, 81% viable in 14 ml progesterone application, 83% viable in 21 ml progesterone application. Due to our findings, progesterone in different concentrations did not chance the cell viability in dental pulpa cells. On gene expression analyses, preliminary results supported that high concentrations of progesterone enhance the gene expressions of steamness genes (Nanog, and Oct4 in dental pulp cells. Conclusions: So, progesterone did not change cell viability in high concentrations. We

  6. Bioimpedance monitoring of 3D cell culturing-Complementary electrode configurations for enhanced spatial sensitivity

    DEFF Research Database (Denmark)

    Canali, Chiara; Heiskanen, Arto; Muhammad, Haseena Bashir;

    2015-01-01

    A bioimpedance platform is presented as a promising tool for non-invasive real-time monitoring of the entire process of three-dimensional (3D) cell culturing in a hydrogel scaffold. In this study, the dynamics involved in the whole process of 3D cell culturing, starting from polymerisation...... of a bare 3D gelatin scaffold, to human mesenchymal stem cell (MSC) encapsulation and proliferation, was monitored over time. The platform consists of a large rectangular culture chamber with four embedded vertical gold plate electrodes that were exploited in two- and three terminal (2T and 3T) measurement...... spectroscopic (EIS) characterisation were used to determine the configurations' sensitivity field localisation. The 2T setup gives insight into the interfacial phenomena at both electrode surfaces and covers the central part of the 3D cell culture volume, while the four 3T modes provide focus on the dynamics...

  7. Impedance Spectroscopic Characterisation of Porosity in 3D Cell Culture Scaffolds with Different Channel Networks

    DEFF Research Database (Denmark)

    Canali, Chiara; Mohanty, Soumyaranjan; Heiskanen, Arto;

    2015-01-01

    We present the application of electrochemical impedance spectroscopy (EIS) as a method for discriminating between different polydimethylsiloxane (PDMS) scaffolds for three-dimensional (3D) cell cultures. The validity of EIS characterisation for scaffolds having different degree of porosity (netwo...

  8. Neural cell 3D microtissue formation is marked by cytokines' up-regulation.

    Directory of Open Access Journals (Sweden)

    Yinzhi Lai

    Full Text Available Cells cultured in three dimensional (3D scaffolds as opposed to traditional two-dimensional (2D substrates have been considered more physiologically relevant based on their superior ability to emulate the in vivo environment. Combined with stem cell technology, 3D cell cultures can provide a promising alternative for use in cell-based assays or biosensors in non-clinical drug discovery studies. To advance 3D culture technology, a case has been made for identifying and validating three-dimensionality biomarkers. With this goal in mind, we conducted a transcriptomic expression comparison among neural progenitor cells cultured on 2D substrates, 3D porous polystyrene scaffolds, and as 3D neurospheres (in vivo surrogate. Up-regulation of cytokines as a group in 3D and neurospheres was observed. A group of 13 cytokines were commonly up-regulated in cells cultured in polystyrene scaffolds and neurospheres, suggesting potential for any or a combination from this list to serve as three-dimensionality biomarkers. These results are supportive of further cytokine identification and validation studies with cells from non-neural tissue.

  9. BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models.

    Directory of Open Access Journals (Sweden)

    Cemal Cagatay Bilgin

    Full Text Available BioSig3D is a computational platform for high-content screening of three-dimensional (3D cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i morphogenesis of a panel of human mammary epithelial cell lines (HMEC, and (ii heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation.

  10. BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models.

    Science.gov (United States)

    Bilgin, Cemal Cagatay; Fontenay, Gerald; Cheng, Qingsu; Chang, Hang; Han, Ju; Parvin, Bahram

    2016-01-01

    BioSig3D is a computational platform for high-content screening of three-dimensional (3D) cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i) morphogenesis of a panel of human mammary epithelial cell lines (HMEC), and (ii) heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation. PMID:26978075

  11. Novel function of complement C3d as an autologous helper T-cell target.

    Science.gov (United States)

    Knopf, Paul M; Rivera, Daniel S; Hai, Si-Han; McMurry, Julie; Martin, William; De Groot, Anne S

    2008-01-01

    The C3d fragment of complement component C3 has been shown to enhance immune responses to antigens that lack T-cell epitopes such as bacterial polysaccharides. C3d binds to the B-cell complement receptor 2 (CR2 or CD21); this binding serves as a co-activation signal to the B cell when the polysaccharide antigen portion binds simultaneously to the B-cell receptor (surface Ig). Bringing together receptor-associated signal transduction molecules CD19 and Igalpha/beta, respectively, results in a lower threshold of activation. Paradoxically, C3d has also been shown to enhance antibody titers in the CD21 knockout (KO) mouse model as well as increase Th1 and Th2 cytokine secretion, suggesting that that an auxiliary CR2-independent pathway of immune activation may exist. We hypothesized that in addition to its molecular adjuvant property that enhances signal 1 during B-cell activation (co-signal 1), C3d also contains T-cell epitopes that are able to stimulate autoreactive C3d peptide-specific helper T cells which we term 'co-signal 2'. Using the EpiMatrix T-cell epitope-mapping algorithm, we identified 11 putative T-cell epitopes in C3d, a very high epitope density for a 302 amino-acid sequence. Eight of these epitope candidates were synthesized and shown to bind a variety of class II HLA-DR molecules of different haplotypes, and to stimulate C3d peptide-specific T cells to secrete pro-inflammatory cytokines in vitro. Further, we demonstrate a C3d-peptide specific increase in CD4(+) intracellular IFN-gamma(+) T cells in peripheral blood mononuclear cells (PBMCs) exposed to C3d peptides in vitro. We believe that the discovery of these autologous T cells autoreactive for C3d provides evidence supporting the 'co-signal 2' hypothesis and may offer a novel explanation of the CD21 KO paradox. PMID:18180801

  12. A novel method for banking dental pulp stem cells.

    Science.gov (United States)

    Gioventù, Silvia; Andriolo, Gabriella; Bonino, Ferruccio; Frasca, Stefania; Lazzari, Lorenza; Montelatici, Elisa; Santoro, Franco; Rebulla, Paolo

    2012-10-01

    Dental pulp stem cells (DPSC), a cell type of mesenchymal origin showing high proliferation and plasticity, are an emerging source of adult stem cells offering interesting features in view of potential applications in regenerative medicine. These features prompted us to develop a new method to cryopreserve DPSC inside a whole tooth, thus avoiding the need to purify the cells before cryopreservation and reducing the initial costs and workload of tooth banking. In this study we cryopreserved 4 human deciduous whole teeth after digging micro-channels into the tooth with an Nd:YAG laser beam (laser piercing) to allow the cryopreservative to reach the dental pulp and preserve the cells at -80°C. Then, we isolated, expanded and characterized in vitro the stem cells after tooth thawing and mechanical fracture. In parallel, we characterized cells extracted from 2 teeth cryopreserved without laser piercing and from 4 non cryopreserved, non laser pierced, freshly fractured teeth. Our data demonstrate that DPSC isolated from laser pierced cryopreserved teeth show mesenchymal stem cells morphology, immunophenotype, viability and proliferation rate similar to those of cells isolated from fresh, non cryopreserved teeth, whereas significant loss of cell viability and proliferation rate was shown by cells isolated from teeth cryopreserved without laser piercing. These data support the use of this method for prospective whole tooth banking.

  13. Cell Proliferation on Macro/Nano Surface Structure and Collagen Immobilization of 3D Polycaprolactone Scaffolds.

    Science.gov (United States)

    Park, Young-Ouk; Myung, Sung-Woon; Kook, Min-Suk; Jung, Sang-Chul; Kim, Byung-Hoon

    2016-02-01

    In this study, 3D polycaprolactone (PCL) scaffolds were fabricated by 3D printing technique. The macro/nano morphology of, 3D PCL scaffolds surface was etched with oxygen plasma. Acrylic acid (AA) plasma-polymerization was performed to functionalize the macro/nano surface with carboxyl groups and then collagen was immobilized with plasma-polymerized 3D PCL scaffolds. After O2 plasma and AA plasma-polymerization, contact angles were decreased. The FE-SEM and AFM results showed that O2 plasma is increased the surface roughness. The MTT assay results showed that proliferation of the M3CT3-E1 cells increased on the oxygen plasma treated and collagen immobilized 3D PCL scaffolds. PMID:27433597

  14. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming

    Science.gov (United States)

    Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

    2016-01-01

    Summary Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated domain (TAD) repositioning and alterations of tissue-restricted genomic neighborhoods and chromatin loops, effectively erasing the somatic-cell-specific genome structures while establishing an embryonic stem-cell-like 3D genome. Yet, early passage iPSCs carry topological hallmarks that enable recognition of their cell of origin. These hallmarks are not remnants of somatic chromosome topologies. Instead, the distinguishing topological features are acquired during reprogramming, as we also find for cell-of-origin-dependent gene expression patterns. PMID:26971819

  15. Micromorph silicon tandem solar cells with fully integrated 3D photonic crystal intermediate reflectors

    Science.gov (United States)

    Üpping, J.; Bielawny, A.; Fahr, S.; Rockstuhl, C.; Lederer, F.; Steidl, L.; Zentel, R.; Beckers, T.; Lambertz, A.; Carius, R.; Wehrspohn, R. B.

    2010-05-01

    A 3D photonic intermediate reflector for textured micromorph silicon tandem solar cells has been investigated. In thin-film silicon tandem solar cells consisting of amorphous and microcrystalline silicon with two junctions of a-Si/c-Si, efficiency enhancements can be achieved by increasing the current density in the a-Si top cell providing an optimized current matching at high current densities. For an ideal photon-management between top and bottom cell, a spectrally-selective intermediate reflective layer (IRL) is necessary. We present the first fully-integrated 3D photonic thin-film IRL device incorporated on a planar substrate. Using a ZnO inverted opal structure the external quantum efficiency of the top cell in the spectral region of interest could be enhanced. As an outlook we present the design and the preparation of a 3D self organized photonic crystal structure in a textured micromorph tandem solar cell.

  16. 3D photonic crystal interlayers for micromorph thin film silicon tandem cell

    Energy Technology Data Exchange (ETDEWEB)

    Uepping, Johannes; Bielawny, Andreas; Otto, Martin; Wehrspohn, Ralf B. [Institute of Physics, University of Halle, Wittenberg (Germany); Steidl, Lorenz; Zentel, Rudolf [Dept. of Chemistry, University of Mainz (Germany); Lee, Seung-Mo; Knez, Mato [Max Planck Institute of Microstructure Physics, Halle (Germany); Beckers, Thomas; Carius, Reinhard [Institute of Energy Research, IEF-5 Photovoltaics, Forschungszentrum Juelich GmbH (Germany)

    2010-07-01

    A 3D photonic intermediate reflector for textured micromorph silicon tandem solar cells has been investigated. In thin-film silicon tandem solar cells consisting of amorphous and microcrystalline silicon with two junctions of a-Si/{mu}c-Si, efficiency enhancements can be achieved by increasing the current density in the a-Si top cell. It is one goal to provide an optimized current matching at high current densities. For an ideal photon-management between top and bottom cell, a spectrally selective intermediate reflective layer (IRL) is necessary. We show results toward the first fully integrated 3D photonic thin-film IRL device incorporated in a state-of-the-art textured tandem solar cell. The design and the preparation of a 3D self organized inverted opal photonic crystal structure in a textured micromorph tandem solar cell is presented.

  17. Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions

    Science.gov (United States)

    Doyle, Andrew D.; Carvajal, Nicole; Jin, Albert; Matsumoto, Kazue; Yamada, Kenneth M.

    2015-11-01

    The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils.

  18. Peptide hydrogels – versatile matrices for 3D cell culture in cancer medicine

    Directory of Open Access Journals (Sweden)

    Peter eWorthington

    2015-04-01

    Full Text Available Traditional two-dimensional (2D cell culture systems have contributed tremendously to our understanding of cancer biology but have significant limitations in mimicking in vivo conditions such as the tumor microenvironment. In vitro, three-dimensional (3D cell culture models represent a more accurate, intermediate platform between simplified 2D culture models and complex and expensive in vivo models. 3D in vitro models can overcome 2D in vitro limitations caused by the oversupply of nutrients, and unphysiological cell-cell and cell-material interactions, and allow for dynamic interactions between cells, stroma, and extracellular matrix. In addition, 3D cultures allow for the development of concentration gradients, including oxygen, metabolites and growth factors, with chemical gradients playing an integral role in many cellular functions ranging from development to signaling in normal epithelia and cancer environments in vivo. Currently, the most common matrices used for 3D culture are biologically derived materials such as matrigel and collagen. However, in recent years, more defined, synthetic materials have become available as scaffolds for 3D culture with the advantage of forming well-defined, designed, tunable materials to control matrix charge, stiffness, porosity, nanostructure, degradability and adhesion properties, in addition to other material and biological properties. One important area of synthetic materials currently available for 3D cell culture are short sequence, self-assembling peptide hydrogels. In addition to the review of recent work towards the control of material, structure, and mechanical properties, we will also discuss the biochemical functionalization of peptide hydrogels and how this functionalization, coupled with desired hydrogel material characteristics, affects tumor cell behavior in 3D culture.

  19. Label-free characterization of white blood cells by measuring 3D refractive index maps

    CERN Document Server

    Yoon, Jonghee; Park, HyunJoo; Choi, Chulhee; Jang, Seongsoo; Park, YongKeun

    2015-01-01

    The characterization of white blood cells (WBCs) is crucial for blood analyses and disease diagnoses. However, current standard techniques rely on cell labeling, a process which imposes significant limitations. Here we present three-dimensional (3D) optical measurements and the label-free characterization of mouse WBCs using optical diffraction tomography. 3D refractive index (RI) tomograms of individual WBCs are constructed from multiple two-dimensional quantitative phase images of samples illuminated at various angles of incidence. Measurements of the 3D RI tomogram of WBCs enable the separation of heterogeneous populations of WBCs using quantitative morphological and biochemical information. Time-lapse tomographic measurements also provide the 3D trajectory of micrometer-sized beads ingested by WBCs. These results demonstrate that optical diffraction tomography can be a useful and versatile tool for the study of WBCs.

  20. FHR3 Blocks C3d-Mediated Coactivation of Human B Cells.

    Science.gov (United States)

    Buhlmann, Denise; Eberhardt, Hannes U; Medyukhina, Anna; Prodinger, Wolfgang M; Figge, Marc Thilo; Zipfel, Peter F; Skerka, Christine

    2016-07-15

    The autoimmune renal disease deficient for complement factor H-related (CFHR) genes and autoantibody-positive form of hemolytic uremic syndrome is characterized by the presence of autoantibodies specific for the central complement regulator, factor H, combined with a homozygous deficiency, mostly in CFHR3 and CFHR1 Because FHR3 and FHR1 bind to C3d and inactivated C3b, which are ligands for complement receptor type 2 (CR2/CD21), the aim of the current study was to examine whether FHR3-C3d or FHR1-C3d complexes modulate B cell activation. Laser-scanning microscopy and automated image-based analysis showed that FHR3, but not FHR1 or factor H, blocked B cell activation by the BCR coreceptor complex (CD19/CD21/CD81). FHR3 bound to C3d, thereby inhibiting the interaction between C3d and CD21 and preventing colocalization of the coreceptor complex with the BCR. FHR3 neutralized the adjuvant effect of C3d on B cells, as shown by inhibited intracellular CD19 and Akt phosphorylation in Raji cells, as well as Ca(2+) release in peripheral B cells. In cases of CFHR3/CFHR1 deficiency, the FHR3 binding sites on C3d are occupied by factor H, which lacks B cell-inhibitory functions. These data provide evidence that FHR3, which is absent in patients with the autoimmune form of hemolytic uremic syndrome, is involved in B cell regulation. PMID:27279373

  1. Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels

    Science.gov (United States)

    Lai, Janice H.; Kajiyama, Glen; Smith, Robert Lane; Maloney, William; Yang, Fan

    2013-12-01

    Cartilage loss is a leading cause of disability among adults and effective therapy remains elusive. Neonatal chondrocytes (NChons) are an attractive allogeneic cell source for cartilage repair, but their clinical translation has been hindered by scarce donor availability. Here we examine the potential for catalyzing cartilage tissue formation using a minimal number of NChons by co-culturing them with adipose-derived stem cells (ADSCs) in 3D hydrogels. Using three different co-culture models, we demonstrated that the effects of co-culture on cartilage tissue formation are dependent on the intercellular distance and cell distribution in 3D. Unexpectedly, increasing ADSC ratio in mixed co-culture led to increased synergy between NChons and ADSCs, and resulted in the formation of large neocartilage nodules. This work raises the potential of utilizing stem cells to catalyze tissue formation by neonatal chondrocytes via paracrine signaling, and highlights the importance of controlling cell distribution in 3D matrices to achieve optimal synergy.

  2. High sensitivity plasmonic biosensor based on nanoimprinted quasi 3D nanosquares for cell detection

    Science.gov (United States)

    Zhu, Shuyan; Li, Hualin; Yang, Mengsu; Pang, Stella W.

    2016-07-01

    Quasi three-dimensional (3D) plasmonic nanostructures consisting of Au nanosquares on top of SU-8 nanopillars and Au nanoholes on the bottom were developed and fabricated using nanoimprint lithography with simultaneous thermal and UV exposure. These 3D plasmonic nanostructures were used to detect cell concentration of lung cancer A549 cells, retinal pigment epithelial (RPE) cells, and breast cancer MCF-7 cells. Nanoimprint technology has the advantage of producing high uniformity plasmonic nanostructures for such biosensors. Multiple resonance modes were observed in these quasi 3D plasmonic nanostructures. The hybrid coupling of localized surface plasmon resonances and Fabry-Perot cavity modes in the quasi 3D nanostructures resulted in high sensitivity of 496 nm/refractive index unit. The plasmonic resonance peak wavelength and sensitivity could be tuned by varying the Au thickness. Resonance peak shifts for different cells at the same concentration were distinct due to their different cell area and confluency. The cell concentration detection limit covered a large range of 5 × 102 to 1 × 107 cells ml-1 with these new plasmonic nanostructures. They also provide a large resonance peak shift of 51 nm for as little as 0.08 cells mm-2 of RPE cells for high sensitivity cell detection.

  3. MULTILEVEL (3D) MICROFLUIDIC TECHNOLOGY FOR AN INNOVATIVE MAGNETIC CELL SEPARATION PLATFORM

    OpenAIRE

    Fouet, Marc; Cargou, Sébastien; Courson, Rémi; Blatché, Charline; Montrose, A.; Reybier, K; Gué, Anne-Marie

    2014-01-01

    We demonstrate a new concept of devices, which by combining 3D fluid engineering and localized mag-netic actuation enables the full integration of a cell tagging and magnetic separation device. We used a low cost, commercially available dry film (EMS Inc, Ohio, USA) that fits microfluidic requirements and gives the possibility to build easily 3D microfluidic structures. The labelling of blood monocytes with su-perparamagnetic particles was performed "up stream" with the aim of a microparticle...

  4. Enhancement of neurite outgrowth in neuron cancer stem cells by growth on 3-D collagen scaffolds

    International Nuclear Information System (INIS)

    Highlights: ► Neuron cancer stem cells (NCSCs) behave high multiply of growth on collagen scaffold. ► Enhancement of NCSCs neurite outgrowth on porous collagen scaffold. ► 3-D collagen culture of NCSCs shows an advance differentiation than 2-D culture. -- Abstract: Collagen is one component of the extracellular matrix that has been widely used for constructive remodeling to facilitate cell growth and differentiation. The 3-D distribution and growth of cells within the porous scaffold suggest a clinical significance for nerve tissue engineering. In the current study, we investigated proliferation and differentiation of neuron cancer stem cells (NCSCs) on a 3-D porous collagen scaffold that mimics the natural extracellular matrix. We first generated green fluorescence protein (GFP) expressing NCSCs using a lentiviral system to instantly monitor the transitions of morphological changes during growth on the 3-D scaffold. We found that proliferation of GFP-NCSCs increased, and a single cell mass rapidly grew with unrestricted expansion between days 3 and 9 in culture. Moreover, immunostaining with neuronal nuclei (NeuN) revealed that NCSCs grown on the 3-D collagen scaffold significantly enhanced neurite outgrowth. Our findings confirmed that the 80 μm porous collagen scaffold could enhance attachment, viability and differentiation of the cancer neural stem cells. This result could provide a new application for nerve tissue engineering and nerve regeneration.

  5. Hypoxia Created Human Mesenchymal Stem Cell Sheet for Prevascularized 3D Tissue Construction.

    Science.gov (United States)

    Zhang, Lijun; Xing, Qi; Qian, Zichen; Tahtinen, Mitchell; Zhang, Zhaoqiang; Shearier, Emily; Qi, Shaohai; Zhao, Feng

    2016-02-01

    3D tissue based on human mesenchymal stem cell (hMSC) sheets offers many interesting opportunities for regenerating multiple types of connective tissues. Prevascularizing hMSC sheets with endothelial cells (ECs) will improve 3D tissue performance by supporting cell survival and accelerating integration with host tissue. It is hypothesized that hypoxia cultured hMSC sheets can promote microvessel network formation and preserve stemness of hMSCs. This study investigates the vascularization of hMSC sheets under different oxygen tensions. It is found that the HN condition, in which hMSC sheets formed under physiological hypoxia (2% O2 ) and then cocultured with ECs under normoxia (20% O2 ), enables longer and more branched microvessel network formation. The observation is corroborated by higher levels of angiogenic factors in coculture medium. Additionally, the hypoxic hMSC sheet is more uniform and less defective, which facilitates fabrication of 3D prevascularized tissue construct by layering the prevascularized hMSC sheets and maturing in rotating wall vessel bioreactor. The hMSCs in the 3D construct still maintain multilineage differentiation ability, which indicates the possible application of the 3D construct for various connective tissues regeneration. These results demonstrate that hypoxia created hMSC sheets benefit the microvessel growth and it is feasible to construct 3D prevascularized tissue construct using the prevascularized hMSC sheets.

  6. Semaphorin 3A Induces Odontoblastic Phenotype in Dental Pulp Stem Cells.

    Science.gov (United States)

    Yoshida, S; Wada, N; Hasegawa, D; Miyaji, H; Mitarai, H; Tomokiyo, A; Hamano, S; Maeda, H

    2016-10-01

    In cases of pulp exposure due to deep dental caries or severe traumatic injuries, existing pulp-capping materials have a limited ability to reconstruct dentin-pulp complexes and can result in pulpectomy because of their low potentials to accelerate dental pulp cell activities, such as migration, proliferation, and differentiation. Therefore, the development of more effective therapeutic agents has been anticipated for direct pulp capping. Dental pulp tissues are enriched with dental pulp stem cells (DPSCs). Here, the authors investigated the effects of semaphorin 3A (Sema3A) on various functions of human DPSCs in vitro and reparative dentin formation in vivo in a rat dental pulp exposure model. Immunofluorescence staining revealed expression of Sema3A and its receptor Nrp1 (neuropilin 1) in rat dental pulp tissue and human DPSC clones. Sema3A induced cell migration, chemotaxis, proliferation, and odontoblastic differentiation of DPSC clones. In addition, Sema3A treatment of DPSC clones increased β-catenin nuclear accumulation, upregulated expression of the FARP2 gene (FERM, RhoGEF, and pleckstrin domain protein 2), and activated Rac1 in DPSC clones. Furthermore, in the rat dental pulp exposure model, Sema3A promoted reparative dentin formation with dentin tubules and a well-aligned odontoblast-like cell layer at the dental pulp exposure site and with novel reparative dentin almost completely covering pulp tissue at 4 wk after direct pulp capping. These findings suggest that Sema3A could play an important role in dentin regeneration via canonical Wnt/β-catenin signaling. Sema3A might be an alternative agent for direct pulp capping, which requires further study. PMID:27302880

  7. Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing

    OpenAIRE

    Akpe Victor; Rydholm Susanna; Liebmann Thomas; Brismar Hjalmar

    2007-01-01

    Abstract Background Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derive...

  8. 3D photonic crystal interlayers for micromorph thin film silicon tandem cell

    Energy Technology Data Exchange (ETDEWEB)

    Bielawny, Andreas; Uepping, Johannes; Miclea, Paul T.; Wehrspohn, Ralf B. [Institute of Physics, University of Halle, Wittenberg (Germany); Rockstuhl, Carsten; Lederer, Falk [Institue of Physics, Solid States Optics, University of Jena (Germany); Peters, Marius [Freiburg Centre for Material Research, University of Freiburg (Germany); Steidl, Lorenz; Zentel, Rudolf [Dept. of Chemistry, Pharmacy and Earth Science, University of Mainz (Germany); Lee, Seung-Mo; Knez, Mato [Max Planck Institute of Microstructure Physics, Halle (Germany); Lambertz, Andreas; Carius, Reinhard [Institute of Energy Research, IEF-5 Photovoltaics, Forschungszentrum Juelich GmbH (Germany)

    2009-07-01

    The concept of 3D photonic intermediate reflectors for micromorph silicon tandem cells has been investigated toward first prototype cells. The reflector enhances the absorption of spectrally selected light in the top cell and decreases the current mismatch between both junctions. Our device is an inverted opal structure made of ZnO and built using self organized nanoparticles and atomic layer deposition coating methods. This 3D photonic crystal intermediate layer is less dependent of the angle of incidence than other state of the art thickness dependent massive interlayers. We present design rules, preparation and characterization of a 3D photonic thin film device. A first prototype is compared to a state of the art reference silicon tandem cell.

  9. Phenomenological modelling and simulation of cell clusters in 3D cultures.

    Science.gov (United States)

    González-Valverde, I; Semino, C; García-Aznar, J M

    2016-10-01

    Cell clustering and aggregation are fundamental processes in the development of several tissues and the progression of many diseases. The formation of these aggregates also has a direct impact on the oxygen concentration in their surroundings due to cellular respiration and poor oxygen diffusion through clusters. In this work, we propose a mathematical model that is capable of simulating cell cluster formation in 3D cultures through combining a particle-based and a finite element approach to recreate complex experimental conditions. Cells are modelled considering cell proliferation, cell death and cell-cell mechanical interactions. Additionally, the oxygen concentration profile is calculated through finite element analysis using a reaction-diffusion model that considers cell oxygen consumption and diffusion through the extracellular matrix and the cell clusters. In our model, the local oxygen concentration in the medium determines both cell proliferation and cell death. Numerical predictions are also compared with experimental data from the literature. The simulation results indicate that our model can predict cell clustering, cluster growth and oxygen distribution in 3D cultures. We conclude that the initial cell distribution, cell death and cell proliferation dynamics determine the size and density of clusters. Moreover, these phenomena are directly affected by the oxygen transport in the 3D culture. PMID:27615191

  10. 3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Machado, C B [Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil); Ventura, J M G [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Lemos, A F [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Ferreira, J M F [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Leite, M F [Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil); Goes, A M [Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil)

    2007-06-01

    A porous 3D scaffold was developed to support and enhance the differentiation process of mesenchymal stem cells (MSC) into osteoblasts in vitro. The 3D scaffold was made with chitosan, gelatin and chondroitin and it was crosslinked by EDAC. The scaffold physicochemical properties were evaluated. SEM revealed the high porosity and interconnection of pores in the scaffold; rheological measurements show that the scaffold exhibits a characteristic behavior of strong gels. The elastic modulus found in compressive tests of the crosslinked scaffold was about 50 times higher than the non-crosslinked one. After 21 days, the 3D matrix submitted to hydrolytic degradation loses above 40% of its weight. MSC were collected from rat bone marrow and seeded in chitosan-gelatin-chondroitin 3D scaffolds and in 2D culture plates as well. MSC were differentiated into osteoblasts for 21 days. Cell proliferation and alkaline phosphatase activity were followed weekly during the osteogenic process. The osteogenic differentiation of MSC was improved in 3D culture as shown by MTT assay and alkaline phosphatase activity. On the 21st day, bone markers, osteopontin and osteocalcin, were detected by the PCR analysis. This study shows that the chitosan-gelatin-chondroitin 3D structure provides a good environment for the osteogenic process and enhances cellular proliferation.

  11. 3D-printed concentrator arrays for external light trapping on thin film solar cells

    NARCIS (Netherlands)

    van Dijk, Lourens; Marcus, E. A. Pepijn; Oostra, A. Jolt; Schropp, Ruud E. I.; Di Vece, Marcel

    2015-01-01

    After our recent demonstration of a 3D-printed external light trap on a small solar cell, we now consider its potential for large solar panels. An external light trap consists of a parabolic concentrator and a spacer that redirects the photons that are reflected by the solar cell back towards the so

  12. 3D Image-Guided Automatic Pipette Positioning for Single Cell Experiments in vivo

    OpenAIRE

    Brian Long; Lu Li; Ulf Knoblich; Hongkui Zeng; Hanchuan Peng

    2015-01-01

    We report a method to facilitate single cell, image-guided experiments including in vivo electrophysiology and electroporation. Our method combines 3D image data acquisition, visualization and on-line image analysis with precise control of physical probes such as electrophysiology microelectrodes in brain tissue in vivo. Adaptive pipette positioning provides a platform for future advances in automated, single cell in vivo experiments.

  13. Cell volume and geometric parameters determination in living cells using confocal microscopy and 3D reconstruction

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: David Hevia, Aida Rodriguez-Garcia, Marta Alonso-Gervós, Isabel Quirós-González, Henar M Cimadevilla, Carmen Gómez-Cordovés, Rosa M Sainz & Juan C Mayo ### Abstract The protocol reported here describes a simple, easy, fast and reproducible method aimed to know the geometric parameters of living cells based on confocal laser scanning microscopy combined with 3D reconstruction software. Briefly, the method is based on intrinsic fluorescence properties of acridine orange (AO...

  14. 3D Reconstitution of the Patterned Neural Tube from Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Andrea Meinhardt

    2014-12-01

    Full Text Available Inducing organogenesis in 3D culture is an important aspect of stem cell research. Anterior neural structures have been produced from large embryonic stem cell (ESC aggregates, but the steps involved in patterning such complex structures have been ill defined, as embryoid bodies typically contained many cell types. Here we show that single mouse ESCs directly embedded in Matrigel or defined synthetic matrices under neural induction conditions can clonally form neuroepithelial cysts containing a single lumen in 3D. Untreated cysts were uniformly dorsal and could be ventralized to floor plate (FP. Retinoic acid posteriorized cysts to cervical levels and induced localize FP formation yielding full patterning along the dorsal/ventral (DV axis. Correct spatial organization of motor neurons, interneurons, and dorsal interneurons along the DV axis was observed. This system serves as a valuable tool for studying morphogen action in 3D and as a source of patterned spinal cord tissue.

  15. 3D reconstitution of the patterned neural tube from embryonic stem cells.

    Science.gov (United States)

    Meinhardt, Andrea; Eberle, Dominic; Tazaki, Akira; Ranga, Adrian; Niesche, Marco; Wilsch-Bräuninger, Michaela; Stec, Agnieszka; Schackert, Gabriele; Lutolf, Matthias; Tanaka, Elly M

    2014-12-01

    Inducing organogenesis in 3D culture is an important aspect of stem cell research. Anterior neural structures have been produced from large embryonic stem cell (ESC) aggregates, but the steps involved in patterning such complex structures have been ill defined, as embryoid bodies typically contained many cell types. Here we show that single mouse ESCs directly embedded in Matrigel or defined synthetic matrices under neural induction conditions can clonally form neuroepithelial cysts containing a single lumen in 3D. Untreated cysts were uniformly dorsal and could be ventralized to floor plate (FP). Retinoic acid posteriorized cysts to cervical levels and induced localize FP formation yielding full patterning along the dorsal/ventral (DV) axis. Correct spatial organization of motor neurons, interneurons, and dorsal interneurons along the DV axis was observed. This system serves as a valuable tool for studying morphogen action in 3D and as a source of patterned spinal cord tissue.

  16. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

    Directory of Open Access Journals (Sweden)

    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  17. CellSegm - a MATLAB toolbox for high-throughput 3D cell segmentation.

    Science.gov (United States)

    Hodneland, Erlend; Kögel, Tanja; Frei, Dominik Michael; Gerdes, Hans-Hermann; Lundervold, Arvid

    2013-08-09

    : The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening.

  18. Heterogeneous Differentiation of Human Mesenchymal Stem Cells in 3D Extracellular Matrix Composites

    Science.gov (United States)

    Jung, Jangwook P.; Bache-Wiig, Meredith K.; Provenzano, Paolo P.; Ogle, Brenda M.

    2016-01-01

    Abstract Extracellular matrix (ECM) proteins are structural elements of tissue and also potent signaling molecules. Previously, our laboratory showed that ECM of 2D coatings can trigger differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into mesodermal lineages in an ECM-specific manner over 14 days, in some cases comparable to chemical induction. To test whether a similar effect was possible in a 3D, tissue-like environment, we designed a synthetic-natural biomaterial composite. The composite can present whole-molecule ECM proteins to cells, even those that do not spontaneously form hydrogels ex vivo, in 3D. To this end, we entrapped collagen type I, laminin-111, or fibronectin in ECM composites with MSCs and directly compared markers of mesodermal differentiation including cardiomyogenic (ACTC1), osteogenic (SPP1), adipogenic (PPARG), and chondrogenic (SOX9) in 2D versus 3D. We found the 3D condition largely mimicked the 2D condition such that the addition of type I collagen was the most potent inducer of differentiation to all lineages tested. One notable difference between 2D and 3D was pronounced adipogenic differentiation in 3D especially in the presence of exogenous collagen type I. In particular, PPARG gene expression was significantly increased ∼16-fold relative to chemical induction, in 3D and not in 2D. Unexpectedly, 3D engagement of ECM proteins also altered immunomodulatory function of MSCs in that expression of IL-6 gene was elevated relative to basal levels in 2D. In fact, levels of IL-6 gene expression in 3D composites containing exogenously supplied collagen type I or fibronectin were statistically similar to levels attained in 2D with tumor necrosis factor-α (TNF-α) stimulation and these levels were sustained over a 2-week period. Thus, this novel biomaterial platform allowed us to compare the biochemical impact of whole-molecule ECM proteins in 2D versus 3D indicating enhanced adipogenic differentiation and IL-6 expression

  19. An Innovative Cell Microincubator for Drug Discovery Based on 3D Silicon Structures

    Directory of Open Access Journals (Sweden)

    Francesca Aredia

    2016-01-01

    Full Text Available We recently employed three-dimensional (3D silicon microstructures (SMSs consisting in arrays of 3 μm-thick silicon walls separated by 50 μm-deep, 5 μm-wide gaps, as microincubators for monitoring the biomechanical properties of tumor cells. They were here applied to investigate the in vitro behavior of HT1080 human fibrosarcoma cells driven to apoptosis by the chemotherapeutic drug Bleomycin. Our results, obtained by fluorescence microscopy, demonstrated that HT1080 cells exhibited a great ability to colonize the narrow gaps. Remarkably, HT1080 cells grown on 3D-SMS, when treated with the DNA damaging agent Bleomycin under conditions leading to apoptosis, tended to shrink, reducing their volume and mimicking the normal behavior of apoptotic cells, and were prone to leave the gaps. Finally, we performed label-free detection of cells adherent to the vertical silicon wall, inside the gap of 3D-SMS, by exploiting optical low coherence reflectometry using infrared, low power radiation. This kind of approach may become a new tool for increasing automation in the drug discovery area. Our results open new perspectives in view of future applications of the 3D-SMS as the core element of a lab-on-a-chip suitable for screening the effect of new molecules potentially able to kill tumor cells.

  20. TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

    Science.gov (United States)

    Gras, Christophe; Smith, Nikaïa; Sengmanivong, Lucie; Gandini, Mariana; Kubelka, Claire Fernandes; Herbeuval, Jean-Philippe

    2013-01-31

    The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images.

  1. TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

    Science.gov (United States)

    Gras, Christophe; Smith, Nikaïa; Sengmanivong, Lucie; Gandini, Mariana; Kubelka, Claire Fernandes; Herbeuval, Jean-Philippe

    2013-01-31

    The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images. PMID:23085529

  2. On-chip clearing of arrays of 3-D cell cultures and micro-tissues.

    Science.gov (United States)

    Grist, S M; Nasseri, S S; Poon, T; Roskelley, C; Cheung, K C

    2016-07-01

    Three-dimensional (3-D) cell cultures are beneficial models for mimicking the complexities of in vivo tissues, especially in tumour studies where transport limitations can complicate response to cancer drugs. 3-D optical microscopy techniques are less involved than traditional embedding and sectioning, but are impeded by optical scattering properties of the tissues. Confocal and even two-photon microscopy limit sample imaging to approximately 100-200 μm depth, which is insufficient to image hypoxic spheroid cores. Optical clearing methods have permitted high-depth imaging of tissues without physical sectioning, but they are difficult to implement for smaller 3-D cultures due to sample loss in solution exchange. In this work, we demonstrate a microfluidic platform for high-throughput on-chip optical clearing of breast cancer spheroids using the SeeDB, Clear(T2), and ScaleSQ clearing methods. Although all three methods are able to effectively clear the spheroids, we find that SeeDB and ScaleSQ more effectively clear the sample than Clear(T2); however, SeeDB induces green autofluorescence while ScaleS causes sample expansion. Our unique on-chip implementation permits clearing arrays of 3-D cultures using perfusion while monitoring the 3-D cultures throughout the process, enabling visualization of the clearing endpoint as well as monitoring of transient changes that could induce image artefacts. Our microfluidic device is compatible with on-chip 3-D cell culture, permitting the use of on-chip clearing at the endpoint after monitoring the same spheroids during their culture. This on-chip method has the potential to improve readout from 3-D cultures, facilitating their use in cell-based assays for high-content drug screening and other applications. PMID:27493703

  3. Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing

    Science.gov (United States)

    Liebmann, Thomas; Rydholm, Susanna; Akpe, Victor; Brismar, Hjalmar

    2007-01-01

    Background Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derived hydrogel for use as a 3D cell culture scaffold at the microscale. Results Phenylalanine derivative hydrogel formation was seen to occur in multiple dispersion media. Cells were immobilized in situ within microchambers designed for cell analysis. Use of the highly biocompatible hydrogel components and simplistic procedures significantly reduced the cytotoxic effects seen with alternate 3D culture materials and microstructure loading methods. Cells were easily immobilized, sustained and removed from microchambers. Differences in growth morphology were seen in the cultured cells, owing to the 3-dimentional character of the gel structure. Degradation improved the removal of hydrogel from the microstructures, permitting reuse of the analysis platforms. Conclusion Self-assembling diphenylalanine derivative hydrogel provided a method to dramatically reduce the typical difficulties of microculture formation. Effective generation of patterned 3D cultures will lead to improved cell study results by better modeling in vivo growth environments and increasing efficiency and specificity of cell studies. Use of simplified growth scaffolds such as peptide-derived hydrogel should be seen as highly advantageous and will likely become more commonplace in cell culture methodology. PMID:18070345

  4. Study of a Microfluidic Chip Integrating Single Cell Trap and 3D Stable Rotation Manipulation

    Directory of Open Access Journals (Sweden)

    Liang Huang

    2016-08-01

    Full Text Available Single cell manipulation technology has been widely applied in biological fields, such as cell injection/enucleation, cell physiological measurement, and cell imaging. Recently, a biochip platform with a novel configuration of electrodes for cell 3D rotation has been successfully developed by generating rotating electric fields. However, the rotation platform still has two major shortcomings that need to be improved. The primary problem is that there is no on-chip module to facilitate the placement of a single cell into the rotation chamber, which causes very low efficiency in experiment to manually pipette single 10-micron-scale cells into rotation position. Secondly, the cell in the chamber may suffer from unstable rotation, which includes gravity-induced sinking down to the chamber bottom or electric-force-induced on-plane movement. To solve the two problems, in this paper we propose a new microfluidic chip with manipulation capabilities of single cell trap and single cell 3D stable rotation, both on one chip. The new microfluidic chip consists of two parts. The top capture part is based on the least flow resistance principle and is used to capture a single cell and to transport it to the rotation chamber. The bottom rotation part is based on dielectrophoresis (DEP and is used to 3D rotate the single cell in the rotation chamber with enhanced stability. The two parts are aligned and bonded together to form closed channels for microfluidic handling. Using COMSOL simulation and preliminary experiments, we have verified, in principle, the concept of on-chip single cell traps and 3D stable rotation, and identified key parameters for chip structures, microfluidic handling, and electrode configurations. The work has laid a solid foundation for on-going chip fabrication and experiment validation.

  5. Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing

    Directory of Open Access Journals (Sweden)

    Akpe Victor

    2007-12-01

    Full Text Available Abstract Background Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derived hydrogel for use as a 3D cell culture scaffold at the microscale. Results Phenylalanine derivative hydrogel formation was seen to occur in multiple dispersion media. Cells were immobilized in situ within microchambers designed for cell analysis. Use of the highly biocompatible hydrogel components and simplistic procedures significantly reduced the cytotoxic effects seen with alternate 3D culture materials and microstructure loading methods. Cells were easily immobilized, sustained and removed from microchambers. Differences in growth morphology were seen in the cultured cells, owing to the 3-dimentional character of the gel structure. Degradation improved the removal of hydrogel from the microstructures, permitting reuse of the analysis platforms. Conclusion Self-assembling diphenylalanine derivative hydrogel provided a method to dramatically reduce the typical difficulties of microculture formation. Effective generation of patterned 3D cultures will lead to improved cell study results by better modeling in vivo growth environments and increasing efficiency and specificity of cell studies. Use of simplified growth scaffolds such as peptide-derived hydrogel should be seen as highly advantageous and will likely become more commonplace in cell culture methodology.

  6. Mesenchymal Stem Cells Derived from Dental Pulp: A Review.

    Science.gov (United States)

    Ledesma-Martínez, Edgar; Mendoza-Núñez, Víctor Manuel; Santiago-Osorio, Edelmiro

    2016-01-01

    The mesenchymal stem cells of dental pulp (DPSCs) were isolated and characterized for the first time more than a decade ago as highly clonogenic cells that were able to generate densely calcified colonies. Now, DPSCs are considered to have potential as stem cell source for orthopedic and oral maxillofacial reconstruction, and it has been suggested that they may have applications beyond the scope of the stomatognathic system. To date, most studies have shown that, regardless of their origin in third molars, incisors, or exfoliated deciduous teeth, DPSCs can generate mineralized tissue, an extracellular matrix and structures type dentin, periodontal ligament, and dental pulp, as well as other structures. Different groups worldwide have designed and evaluated new efficient protocols for the isolation, expansion, and maintenance of clinically safe human DPSCs in sufficient numbers for various therapeutics protocols and have discussed the most appropriate route of administration, the possible contraindications to their clinical use, and the parameters to be considered for monitoring their clinical efficacy and proper biological source. At present, DPSC-based therapy is promising but because most of the available evidence was obtained using nonhuman xenotransplants, it is not a mature technology. PMID:26779263

  7. Mesenchymal Stem Cells Derived from Dental Pulp: A Review

    Directory of Open Access Journals (Sweden)

    Edgar Ledesma-Martínez

    2016-01-01

    Full Text Available The mesenchymal stem cells of dental pulp (DPSCs were isolated and characterized for the first time more than a decade ago as highly clonogenic cells that were able to generate densely calcified colonies. Now, DPSCs are considered to have potential as stem cell source for orthopedic and oral maxillofacial reconstruction, and it has been suggested that they may have applications beyond the scope of the stomatognathic system. To date, most studies have shown that, regardless of their origin in third molars, incisors, or exfoliated deciduous teeth, DPSCs can generate mineralized tissue, an extracellular matrix and structures type dentin, periodontal ligament, and dental pulp, as well as other structures. Different groups worldwide have designed and evaluated new efficient protocols for the isolation, expansion, and maintenance of clinically safe human DPSCs in sufficient numbers for various therapeutics protocols and have discussed the most appropriate route of administration, the possible contraindications to their clinical use, and the parameters to be considered for monitoring their clinical efficacy and proper biological source. At present, DPSC-based therapy is promising but because most of the available evidence was obtained using nonhuman xenotransplants, it is not a mature technology.

  8. Mechano-sensing and cell migration: a 3D model approach

    International Nuclear Information System (INIS)

    Cell migration is essential for tissue development in different physiological and pathological conditions. It is a complex process orchestrated by chemistry, biological factors, microstructure and surrounding mechanical properties. Focusing on the mechanical interactions, cells do not only exert forces on the matrix that surrounds them, but they also sense and react to mechanical cues in a process called mechano-sensing. Here, we hypothesize the involvement of mechano-sensing in the regulation of directional cell migration through a three-dimensional (3D) matrix. For this purpose, we develop a 3D numerical model of individual cell migration, which incorporates the mechano-sensing process of the cell as the main mechanism regulating its movement. Consistent with this hypothesis, we found that factors, such as substrate stiffness, boundary conditions and external forces, regulate specific and distinct cell movements

  9. Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration.

    Science.gov (United States)

    Pati, Falguni; Song, Tae-Ha; Rijal, Girdhari; Jang, Jinah; Kim, Sung Won; Cho, Dong-Woo

    2015-01-01

    3D printing technique is the most sophisticated technique to produce scaffolds with tailorable physical properties. But, these scaffolds often suffer from limited biological functionality as they are typically made from synthetic materials. Cell-laid mineralized ECM was shown to be potential for improving the cellular responses and drive osteogenesis of stem cells. Here, we intend to improve the biological functionality of 3D-printed synthetic scaffolds by ornamenting them with cell-laid mineralized extracellular matrix (ECM) that mimics a bony microenvironment. We developed bone graft substitutes by using 3D printed scaffolds made from a composite of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate (β-TCP) and mineralized ECM laid by human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs). A rotary flask bioreactor was used to culture hTMSCs on the scaffolds to foster formation of mineralized ECM. A freeze/thaw cycle in hypotonic buffer was used to efficiently decellularize (97% DNA reduction) the ECM-ornamented scaffolds while preserving its main organic and inorganic components. The ECM-ornamented 3D printed scaffolds supported osteoblastic differentiation of newly-seeded hTMSCs by upregulating four typical osteoblastic genes (4-fold higher RUNX2; 3-fold higher ALP; 4-fold higher osteocalcin; and 4-fold higher osteopontin) and increasing calcium deposition compared to bare 3D printed scaffolds. In vivo, in ectopic and orthotopic models in rats, ECM-ornamented scaffolds induced greater bone formation than that of bare scaffolds. These results suggest a valuable method to produce ECM-ornamented 3D printed scaffolds as off-the-shelf bone graft substitutes that combine tunable physical properties with physiological presentation of biological signals.

  10. 3D culture broadly regulates tumor cell hypoxia response and angiogenesis via pro-inflammatory pathways.

    Science.gov (United States)

    DelNero, Peter; Lane, Maureen; Verbridge, Scott S; Kwee, Brian; Kermani, Pouneh; Hempstead, Barbara; Stroock, Abraham; Fischbach, Claudia

    2015-07-01

    Oxygen status and tissue dimensionality are critical determinants of tumor angiogenesis, a hallmark of cancer and an enduring target for therapeutic intervention. However, it is unclear how these microenvironmental conditions interact to promote neovascularization, due in part to a lack of comprehensive, unbiased data sets describing tumor cell gene expression as a function of oxygen levels within three-dimensional (3D) culture. Here, we utilized alginate-based, oxygen-controlled 3D tumor models to study the interdependence of culture context and the hypoxia response. Microarray gene expression analysis of tumor cells cultured in 2D versus 3D under ambient or hypoxic conditions revealed striking interdependence between culture dimensionality and hypoxia response, which was mediated in part by pro-inflammatory signaling pathways. In particular, interleukin-8 (IL-8) emerged as a major player in the microenvironmental regulation of the hypoxia program. Notably, this interaction between dimensionality and oxygen status via IL-8 increased angiogenic sprouting in a 3D endothelial invasion assay. Taken together, our data suggest that pro-inflammatory pathways are critical regulators of tumor hypoxia response within 3D environments that ultimately impact tumor angiogenesis, potentially providing important therapeutic targets. Furthermore, these results highlight the importance of pathologically relevant tissue culture models to study the complex physical and chemical processes by which the cancer microenvironment mediates new vessel formation.

  11. Microrheology and ROCK signaling of human endothelial cells embedded in a 3D matrix.

    Science.gov (United States)

    Panorchan, Porntula; Lee, Jerry S H; Kole, Thomas P; Tseng, Yiider; Wirtz, Denis

    2006-11-01

    Cell function is profoundly affected by the geometry of the extracellular environment confining the cell. Whether and how cells plated on a two-dimensional matrix or embedded in a three-dimensional (3D) matrix mechanically sense the dimensionality of their environment is mostly unknown, partly because individual cells in an extended matrix are inaccessible to conventional cell-mechanics probes. Here we develop a functional assay based on multiple particle tracking microrheology coupled with ballistic injection of nanoparticles to measure the local intracellular micromechanical properties of individual cells embedded inside a matrix. With our novel assay, we probe the mechanical properties of the cytoplasm of individual human umbilical vein endothelial cells (HUVECs) embedded in a 3D peptide hydrogel in the presence or absence of vascular endothelial growth factor (VEGF). We found that VEGF treatment, which enhances endothelial migration, increases the compliance and reduces the elasticity of the cytoplasm of HUVECs in a matrix. This VEGF-induced softening response of the cytoplasm is abrogated by specific Rho-kinase (ROCK) inhibition. These results establish combined particle-tracking microrheology and ballistic injection as the first method able to probe the micromechanical properties and mechanical response to agonists and/or drug treatments of individual cells inside a matrix. These results suggest that ROCK plays an essential role in the regulation of the intracellular mechanical response to VEGF of endothelial cells in a 3D matrix.

  12. 3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds

    Science.gov (United States)

    Lee, Wonjae; Park, Jon

    2016-07-01

    Tissue-specific patterned stem cell differentiation serves as the basis for the development, remodeling, and regeneration of the multicellular structure of the native tissues. We herein proposed a cytocompatible 3D casting process to recapitulate this patterned stem cell differentiation for reconstructing multicellular tissues in vitro. We first reconstituted the 2D culture conditions for stem cell fate control within 3D hydrogel by incorporating the sets of the diffusible signal molecules delivered through drug-releasing microparticles. Then, utilizing thermo-responsivity of methylcellulose (MC), we developed a cytocompatible casting process to mold these hydrogels into specific 3D configurations, generating the targeted spatial gradients of diffusible signal molecules. The liquid phase of the MC solution was viscous enough to adopt the shapes of 3D impression patterns, while the gelated MC served as a reliable mold for patterning the hydrogel prepolymers. When these patterned hydrogels were integrated together, the stem cells in each hydrogel distinctly differentiated toward individually defined fates, resulting in the formation of the multicellular tissue structure bearing the very structural integrity and characteristics as seen in vascularized bones and osteochondral tissues.

  13. 3D staggered Lagrangian hydrodynamics scheme with cell-centered Riemann solver-based artificial viscosity

    International Nuclear Information System (INIS)

    The aim of the present work is the 3D extension of a general formalism to derive a staggered discretization for Lagrangian hydrodynamics on unstructured grids. The classical compatible discretization is used; namely, momentum equation is discretized using the fundamental concept of subcell forces. Specific internal energy equation is obtained using total energy conservation. The subcell force is derived by invoking the Galilean invariance and thermodynamic consistency. A general form of the subcell force is provided so that a cell entropy inequality is satisfied. The subcell force consists of a classical pressure term plus a tensorial viscous contribution proportional to the difference between the node velocity and the cell-centered velocity. This cell-centered velocity is an extra degree of freedom solved with a cell-centered approximate Riemann solver. The second law of thermodynamics is satisfied by construction of the local positive definite subcell tensor involved in the viscous term. A particular expression of this tensor is proposed. A more accurate extension of this discretization both in time and space is also provided using a piecewise linear reconstruction of the velocity field and a predictor-corrector time discretization. Numerical tests are presented in order to assess the efficiency of this approach in 3D. Sanity checks show that the 3D extension of the 2D approach reproduces 1D and 2D results. Finally, 3D problems such as Sedov, Noh, and Saltzman are simulated. (authors)

  14. Interfacing polymeric scaffolds with primary pancreatic ductal adenocarcinoma cells to develop 3D cancer models

    NARCIS (Netherlands)

    Ricci, C.; Mota, C.M.; Moscato, S.; Alessandro, D' D.; Ugel, S.; Sartoris, S.; Bronte, V.; Boggi, U.; Campani, D.; Funel, N.; Moroni, L.; Danti, S.

    2014-01-01

    We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol

  15. 3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds

    Science.gov (United States)

    Lee, Wonjae; Park, Jon

    2016-01-01

    Tissue-specific patterned stem cell differentiation serves as the basis for the development, remodeling, and regeneration of the multicellular structure of the native tissues. We herein proposed a cytocompatible 3D casting process to recapitulate this patterned stem cell differentiation for reconstructing multicellular tissues in vitro. We first reconstituted the 2D culture conditions for stem cell fate control within 3D hydrogel by incorporating the sets of the diffusible signal molecules delivered through drug-releasing microparticles. Then, utilizing thermo-responsivity of methylcellulose (MC), we developed a cytocompatible casting process to mold these hydrogels into specific 3D configurations, generating the targeted spatial gradients of diffusible signal molecules. The liquid phase of the MC solution was viscous enough to adopt the shapes of 3D impression patterns, while the gelated MC served as a reliable mold for patterning the hydrogel prepolymers. When these patterned hydrogels were integrated together, the stem cells in each hydrogel distinctly differentiated toward individually defined fates, resulting in the formation of the multicellular tissue structure bearing the very structural integrity and characteristics as seen in vascularized bones and osteochondral tissues. PMID:27381562

  16. A 3D GCL compatible cell-centered Lagrangian scheme for solving gas dynamics equations

    Science.gov (United States)

    Georges, Gabriel; Breil, Jérôme; Maire, Pierre-Henri

    2016-01-01

    Solving the gas dynamics equations under the Lagrangian formalism enables to simulate complex flows with strong shock waves. This formulation is well suited to the simulation of multi-material compressible fluid flows such as those encountered in the domain of High Energy Density Physics (HEDP). These types of flows are characterized by complex 3D structures such as hydrodynamic instabilities (Richtmyer-Meshkov, Rayleigh-Taylor, etc.). Recently, the 3D extension of different Lagrangian schemes has been proposed and appears to be challenging. More precisely, the definition of the cell geometry in the 3D space through the treatment of its non-planar faces and the limiting of a reconstructed field in 3D in the case of a second-order extension are of great interest. This paper proposes two new methods to solve these problems. A systematic and symmetric geometrical decomposition of polyhedral cells is presented. This method enables to define a discrete divergence operator leading to the respect of the Geometric Conservation Law (GCL). Moreover, a multi-dimensional minmod limiter is proposed. This new limiter constructs, from nodal gradients, a cell gradient which enables to ensure the monotonicity of the numerical solution even in presence of strong discontinuity. These new ingredients are employed into a cell-centered Lagrangian scheme. Robustness and accuracy are assessed against various representative test cases.

  17. Static Magnetic Field Attenuates Lipopolysaccharide-Induced Inflammation in Pulp Cells by Affecting Cell Membrane Stability

    Directory of Open Access Journals (Sweden)

    Sung-Chih Hsieh

    2015-01-01

    Full Text Available One of the causes of dental pulpitis is lipopolysaccharide- (LPS- induced inflammatory response. Following pulp tissue inflammation, odontoblasts, dental pulp cells (DPCs, and dental pulp stem cells (DPSCs will activate and repair damaged tissue to maintain homeostasis. However, when LPS infection is too serious, dental repair is impossible and disease may progress to irreversible pulpitis. Therefore, the aim of this study was to examine whether static magnetic field (SMF can attenuate inflammatory response of dental pulp cells challenged with LPS. In methodology, dental pulp cells were isolated from extracted teeth. The population of DPSCs in the cultured DPCs was identified by phenotypes and multilineage differentiation. The effects of 0.4 T SMF on DPCs were observed through MTT assay and fluorescent anisotropy assay. Our results showed that the SMF exposure had no effect on surface markers or multilineage differentiation capability. However, SMF exposure increases cell viability by 15%. In addition, SMF increased cell membrane rigidity which is directly related to higher fluorescent anisotropy. In the LPS-challenged condition, DPCs treated with SMF demonstrated a higher tolerance to LPS-induced inflammatory response when compared to untreated controls. According to these results, we suggest that 0.4 T SMF attenuates LPS-induced inflammatory response to DPCs by changing cell membrane stability.

  18. Fabrication of Dye-Sensitized Solar Cells with a 3D Nanostructured Electrode

    Directory of Open Access Journals (Sweden)

    Guo-Yang Chen

    2010-01-01

    Full Text Available A novel Dye-Sensitized Solar Cell (DSSC scheme for better solar conversion efficiency is proposed. The distinctive characteristic of this novel scheme is that the conventional thin film electrode is replaced by a 3D nanostructured indium tin oxide (ITO electrode, which was fabricated using RF magnetron sputtering with an anodic aluminum oxide (AAO template. The template was prepared by immersing the barrier-layer side of an AAO film into a 30 wt% phosphoric acid solution to produce a contrasting surface. RF magnetron sputtering was then used to deposit a 3D nanostructured ITO thin film on the template. The crystallinity and conductivity of the 3D ITO films were further enhanced by annealing. Titanium dioxide nanoparticles were electrophoretically deposited on the 3D ITO film after which the proposed DSSC was formed by filling vacant spaces in the 3D nanostructured ITO electrode with dye. The measured solar conversion efficiency of the device was 0.125%. It presents a 5-fold improvement over that of conventional spin-coated TiO2 film electrode DSSCs.

  19. Uncovering cancer cell behavioral phenotype in 3-D in vitro metastatic landscapes

    Science.gov (United States)

    Liu, Liyu; Sun, Bo; Duclos, Guillaume; Kam, Yoonseok; Gatenby, Robert; Stone, Howard; Austin, Robert

    2012-02-01

    One well-known fact is that cancer cell genetics determines cell metastatic potentials. However, from a physics point of view, genetics as cell properties cannot directly act on metastasis. An agent is needed to unscramble the genetics first before generating dynamics for metastasis. Exactly this agent is cell behavioral phenotype, which is rarely studied due to the difficulties of real-time cell tracking in in vivo tissue. Here we have successfully constructed a micro in vitro environment with collagen based Extracellular Matrix (ECM) structures for cell 3-D metastasis. With stable nutrition (glucose) gradient inside, breast cancer cell MDA-MB-231 is able to invade inside the collagen from the nutrition poor site towards the nutrition rich site. Continuous confocal microscopy captures images of the cells every 12 hours and tracks their positions in 3-D space. The micro fluorescent beads pre-mixed inside the ECM demonstrate that invasive cells have altered the structures through mechanics. With the observation and the analysis of cell collective behaviors, we argue that game theory may exist between the pioneering cells and their followers in the metastatic cell group. The cell collaboration may explain the high efficiency of metastasis.

  20. 3D-printing of Redox flow batteries for energy storage: a rapid prototype laboratory cell

    OpenAIRE

    Arenas-Martinez, L.F.; Walsh, F.C.; Ponce de Leon, C.

    2015-01-01

    Although interest in redox flow batteries (RFBs) for energy storage has grown over the last few years, implementation of RFB technology has been slow and challenging. Recent developments in 3D-printing of materials enable a transforming technology for fast, reproducible and documented cell manufacture. This technology can give an improved engineering approach to cell design and fabrication, needed to fulfil requirements for lower cost, longer lifetime hardware capable of efficient reliable pe...

  1. Cell counting in human endobronchial biopsies--disagreement of 2D versus 3D morphometry.

    Directory of Open Access Journals (Sweden)

    Vlad A Bratu

    Full Text Available QUESTION: Inflammatory cell numbers are important endpoints in clinical studies relying on endobronchial biopsies. Assumption-based bidimensional (2D counting methods are widely used, although theoretically design-based stereologic three-dimensional (3D methods alone offer an unbiased quantitative tool. We assessed the method agreement between 2D and 3D counting designs in practice when applied to identical samples in parallel. MATERIALS AND METHODS: Biopsies from segmental bronchi were collected from healthy non-smokers (n = 7 and smokers (n = 7, embedded and sectioned exhaustively. Systematic uniform random samples were immunohistochemically stained for macrophages (CD68 and T-lymphocytes (CD3, respectively. In identical fields of view, cell numbers per volume unit (NV were assessed using the physical disector (3D, and profiles per area unit (NA were counted (2D. For CD68+ cells, profiles with and without nucleus were separately recorded. In order to enable a direct comparison of the two methods, the zero-dimensional CD68+/CD3+-ratio was calculated for each approach. Method agreement was tested by Bland-Altmann analysis. RESULTS: In both groups, mean CD68+/CD3+ ratios for NV and NA were significantly different (non-smokers: 0.39 and 0.68, p<0.05; smokers: 0.49 and 1.68, p<0.05. When counting only nucleated CD68+ profiles, mean ratios obtained by 2D and 3D counting were similar, but the regression-based Bland-Altmann analysis indicated a bias of the 2D ratios proportional to their magnitude. This magnitude dependent deviation differed between the two groups. CONCLUSIONS: 2D counts of cell and nuclear profiles introduce a variable size-dependent bias throughout the measurement range. Because the deviation between the 3D and 2D data was different in the two groups, it precludes establishing a 'universal conversion formula'.

  2. A 3D printed nano bone matrix for characterization of breast cancer cell and osteoblast interactions

    Science.gov (United States)

    Zhu, Wei; Castro, Nathan J.; Cui, Haitao; Zhou, Xuan; Boualam, Benchaa; McGrane, Robert; Glazer, Robert I.; Zhang, Lijie Grace

    2016-08-01

    Bone metastasis is one of the most prevalent complications of late-stage breast cancer, in which the native bone matrix components, including osteoblasts, are intimately involved in tumor progression. The development of a successful in vitro model would greatly facilitate understanding the underlying mechanism of breast cancer bone invasion as well as provide a tool for effective discovery of novel therapeutic strategies. In the current study, we fabricated a series of in vitro bone matrices composed of a polyethylene glycol hydrogel and nanocrystalline hydroxyapatite of varying concentrations to mimic the native bone microenvironment for the investigation of breast cancer bone metastasis. A stereolithography-based three-dimensional (3D) printer was used to fabricate the bone matrices with precisely controlled architecture. The interaction between breast cancer cells and osteoblasts was investigated in the optimized bone matrix. Using a Transwell® system to separate the two cell lines, breast cancer cells inhibited osteoblast proliferation, while osteoblasts stimulated breast cancer cell growth, whereas, both cell lines increased IL-8 secretion. Breast cancer cells co-cultured with osteoblasts within the 3D bone matrix formed multi-cellular spheroids in comparison to two-dimensional monolayers. These findings validate the use of our 3D printed bone matrices as an in vitro metastasis model, and highlights their potential for investigating breast cancer bone metastasis.

  3. Defining an optimal surface chemistry for pluripotent stem cell culture in 2D and 3D

    Science.gov (United States)

    Zonca, Michael R., Jr.

    Surface chemistry is critical for growing pluripotent stem cells in an undifferentiated state. There is great potential to engineer the surface chemistry at the nanoscale level to regulate stem cell adhesion. However, the challenge is to identify the optimal surface chemistry of the substrata for ES cell attachment and maintenance. Using a high-throughput polymerization and screening platform, a chemically defined, synthetic polymer grafted coating that supports strong attachment and high expansion capacity of pluripotent stem cells has been discovered using mouse embryonic stem (ES) cells as a model system. This optimal substrate, N-[3-(Dimethylamino)propyl] methacrylamide (DMAPMA) that is grafted on 2D synthetic poly(ether sulfone) (PES) membrane, sustains the self-renewal of ES cells (up to 7 passages). DMAPMA supports cell attachment of ES cells through integrin beta1 in a RGD-independent manner and is similar to another recently reported polymer surface. Next, DMAPMA has been able to be transferred to 3D by grafting to synthetic, polymeric, PES fibrous matrices through both photo-induced and plasma-induced polymerization. These 3D modified fibers exhibited higher cell proliferation and greater expression of pluripotency markers of mouse ES cells than 2D PES membranes. Our results indicated that desirable surfaces in 2D can be scaled to 3D and that both surface chemistry and structural dimension strongly influence the growth and differentiation of pluripotent stem cells. Lastly, the feasibility of incorporating DMAPMA into a widely used natural polymer, alginate, has been tested. Novel adhesive alginate hydrogels have been successfully synthesized by either direct polymerization of DMAPMA and methacrylic acid blended with alginate, or photo-induced DMAPMA polymerization on alginate nanofibrous hydrogels. In particular, DMAPMA-coated alginate hydrogels support strong ES cell attachment, exhibiting a concentration dependency of DMAPMA. This research provides a

  4. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control

    Science.gov (United States)

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L. James; Lu, Wu

    2015-12-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk

  5. Rab3D is critical for secretory granule maturation in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Tanja Kögel

    Full Text Available Neuropeptide- and hormone-containing secretory granules (SGs are synthesized at the trans-Golgi network (TGN as immature secretory granules (ISGs and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs. Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs.

  6. A 3-D Model of a Perennial Ryegrass Primary Cell Wall and Its Enzymatic Degradation

    OpenAIRE

    Indrakumar Vetharaniam; Kelly, William J.; Graeme T. Attwood; Harris, Philip J.

    2014-01-01

    We have developed a novel 3-D, agent-based model of cell-wall digestion to improve our understanding of ruminal cell-wall digestion. It offers a capability to study cell walls and their enzymatic modification, by providing a representation of cellulose microfibrils and non-cellulosic polysaccharides and by simulating their spatial and catalytic interactions with enzymes. One can vary cell-wall composition and the types and numbers of enzyme molecules, allowing the model to be applied to a ran...

  7. Melanin Transfer in Human 3D Skin Equivalents Generated Exclusively from Induced Pluripotent Stem Cells.

    Directory of Open Access Journals (Sweden)

    Karl Gledhill

    Full Text Available The current utility of 3D skin equivalents is limited by the fact that existing models fail to recapitulate the cellular complexity of human skin. They often contain few cell types and no appendages, in part because many cells found in the skin are difficult to isolate from intact tissue and cannot be expanded in culture. Induced pluripotent stem cells (iPSCs present an avenue by which we can overcome this issue due to their ability to be differentiated into multiple cell types in the body and their unlimited growth potential. We previously reported generation of the first human 3D skin equivalents from iPSC-derived fibroblasts and iPSC-derived keratinocytes, demonstrating that iPSCs can provide a foundation for modeling a complex human organ such as skin. Here, we have increased the complexity of this model by including additional iPSC-derived melanocytes. Epidermal melanocytes, which are largely responsible for skin pigmentation, represent the second most numerous cell type found in normal human epidermis and as such represent a logical next addition. We report efficient melanin production from iPSC-derived melanocytes and transfer within an entirely iPSC-derived epidermal-melanin unit and generation of the first functional human 3D skin equivalents made from iPSC-derived fibroblasts, keratinocytes and melanocytes.

  8. 3D Printing of Scaffold for Cells Delivery: Advances in Skin Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Deepti Singh

    2016-01-01

    Full Text Available Injury or damage to tissue and organs is a major health problem, resulting in about half of the world’s annual healthcare expenditure every year. Advances in the fields of stem cells (SCs and biomaterials processing have provided a tremendous leap for researchers to manipulate the dynamics between these two, and obtain a skin substitute that can completely heal the wounded areas. Although wound healing needs a coordinated interplay between cells, extracellular proteins and growth factors, the most important players in this process are the endogenous SCs, which activate the repair cascade by recruiting cells from different sites. Extra cellular matrix (ECM proteins are activated by these SCs, which in turn aid in cellular migrations and finally secretion of growth factors that can seal and heal the wounds. The interaction between ECM proteins and SCs helps the skin to sustain the rigors of everyday activity, and in an attempt to attain this level of functionality in artificial three-dimensional (3D constructs, tissue engineered biomaterials are fabricated using more advanced techniques such as bioprinting and laser assisted printing of the organs. This review provides a concise summary of the most recent advances that have been made in the area of polymer bio-fabrication using 3D bio printing used for encapsulating stem cells for skin regeneration. The focus of this review is to describe, in detail, the role of 3D architecture and arrangement of cells within this system that can heal wounds and aid in skin regeneration.

  9. Slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow.

    Science.gov (United States)

    Jagannadh, Veerendra Kalyan; Mackenzie, Mark D; Pal, Parama; Kar, Ajoy K; Gorthi, Sai Siva

    2016-09-19

    Three-dimensional cellular imaging techniques have become indispensable tools in biological research and medical diagnostics. Conventional 3D imaging approaches employ focal stack collection to image different planes of the cell. In this work, we present the design and fabrication of a slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow. The approach employs slanted microfluidic channels fabricated in glass using ultrafast laser inscription. The slanted nature of the microfluidic channels ensures that samples come into and go out of focus, as they pass through the microscope imaging field of view. This novel approach enables the collection of focal stacks in a straight-forward and automated manner, even with off-the-shelf microscopes that are not equipped with any motorized translation/rotation sample stages. The presented approach not only simplifies conventional focal stack collection, but also enhances the capabilities of a regular widefield fluorescence microscope to match the features of a sophisticated confocal microscope. We demonstrate the retrieval of sectioned slices of microspheres and cells, with the use of computational algorithms to enhance the signal-to-noise ratio (SNR) in the collected raw images. The retrieved sectioned images have been used to visualize fluorescent microspheres and bovine sperm cell nucleus in 3D while using a regular widefield fluorescence microscope. We have been able to achieve sectioning of approximately 200 slices per cell, which corresponds to a spatial translation of ∼ 15 nm per slice along the optical axis of the microscope.

  10. Biomaterials coated by dental pulp cells as substrate for neural stem cell differentiation.

    Science.gov (United States)

    Soria, Jose Miguel; Sancho-Tello, María; Esparza, M Angeles Garcia; Mirabet, Vicente; Bagan, Jose Vicente; Monleón, Manuel; Carda, Carmen

    2011-04-01

    This study is focused on the development of an in vitro hybrid system, consisting in a polymeric biomaterial covered by a dental pulp cellular stroma that acts as a scaffold offering a neurotrophic support for the subsequent survival and differentiation of neural stem cells. In the first place, the behavior of dental pulp stroma on the polymeric biomaterial based on ethyl acrylate and hydroxy ethyl acrylate copolymer was studied. For this purpose, cells from normal human third molars were grown onto 0.5-mm-diameter biomaterial discs. After cell culture, quantification of neurotrophic factors generated by the stromal cells was performed by means of an ELISA assay. In the second place, survival and differentiation of adult murine neural stem cells on the polymeric biomaterials covered by dental pulp stromal cells was studied. The results show the capacity of dental pulp cells to uniformly coat the majority of the material's surface and to secrete neurotrophic factors that become crucial for a subsequent differentiation of neural stem cells. The use of stromal cells cultured on scaffolding biomaterials provides neurotrophic pumps that may suggest new criteria for the design of cell therapy experiments in animal models to assist the repair of lesions in Central Nervous System.

  11. Human dental pulp stem cells express many pluripotency regulators and differentiate into neuronal cells

    Institute of Scientific and Technical Information of China (English)

    Behnam Ebrahimi; Mohammad Mehdi Yaghoobi; Ali Mohammadi Kamal-abadi; Maryam Raoof

    2011-01-01

    Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse transcription-PCR (RT-PCR), and differentiation markers were detected by real-time quantitative RT-PCR and immunocytochemistry. Results showed that dental pulp stem cells actively expressed nanog, oct4, nucleostemin slain-1, jmjd1a, jmjd2c, and cyclin D1. When stem cells were induced to differentiate into neurons, nucleostemin, nanog, and cyclin D1 expres-sion significantly decreased, whereas expression of neuronal markers, such as microtubule asso-ciated protein-2 and neurofilament-heavy, significantly increased. These results suggested that stem cells exited a pluripotent state and entered a neuronal differentiation pathway. In addition, results demonstrated that human dental pulp serves as a reservoir of stem cells that express defined stem cell markers; these cells were easily isolated and were induced to differentiate towards a desired cell lineage.

  12. The influence of printing parameters on cell survival rate and printability in microextrusion-based 3D cell printing technology.

    Science.gov (United States)

    Zhao, Yu; Li, Yang; Mao, Shuangshuang; Sun, Wei; Yao, Rui

    2015-11-02

    Three-dimensional (3D) cell printing technology has provided a versatile methodology to fabricate cell-laden tissue-like constructs and in vitro tissue/pathological models for tissue engineering, drug testing and screening applications. However, it still remains a challenge to print bioinks with high viscoelasticity to achieve long-term stable structure and maintain high cell survival rate after printing at the same time. In this study, we systematically investigated the influence of 3D cell printing parameters, i.e. composition and concentration of bioink, holding temperature and holding time, on the printability and cell survival rate in microextrusion-based 3D cell printing technology. Rheological measurements were utilized to characterize the viscoelasticity of gelatin-based bioinks. Results demonstrated that the bioink viscoelasticity was increased when increasing the bioink concentration, increasing holding time and decreasing holding temperature below gelation temperature. The decline of cell survival rate after 3D cell printing process was observed when increasing the viscoelasticity of the gelatin-based bioinks. However, different process parameter combinations would result in the similar rheological characteristics and thus showed similar cell survival rate after 3D bioprinting process. On the other hand, bioink viscoelasticity should also reach a certain point to ensure good printability and shape fidelity. At last, we proposed a protocol for 3D bioprinting of temperature-sensitive gelatin-based hydrogel bioinks with both high cell survival rate and good printability. This research would be useful for biofabrication researchers to adjust the 3D bioprinting process parameters quickly and as a referable template for designing new bioinks.

  13. Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.

    Science.gov (United States)

    Radtke, Andrea L; Herbst-Kralovetz, Melissa M

    2012-04-03

    Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment. Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues (1-6). The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties

  14. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control.

    Science.gov (United States)

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L James; Lu, Wu

    2016-01-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a "3D nano-channel electroporation (NEP) chip" on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40,000 cells per cm(2) on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells. PMID:26309218

  15. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control.

    Science.gov (United States)

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L James; Lu, Wu

    2016-01-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a "3D nano-channel electroporation (NEP) chip" on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40,000 cells per cm(2) on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.

  16. 3D photonic crystal intermediate reflector for micromorph thin-film tandem solar cell

    Energy Technology Data Exchange (ETDEWEB)

    Uepping, Johannes; Miclea, Paul T.; Wehrspohn, Ralf B. [Institute of Physics, Martin-Luther-University of Halle-Wittenberg, Heinrich-Damerow-Str. 4, 06120 Halle (Germany); Rockstuhl, Carsten; Lederer, Falk [Institute of Condensed Matter Theory and Solid States Optics, Friedrich Schiller University Jena, 07743 Jena (Germany); Peters, Marius [Freiburg Centre for Material Research, University of Freiburg, 79104 Freiburg (Germany); Steidl, Lorenz; Zentel, Rudolf [Dept. of Chemistry, Pharmacy and Earth Science, Johannes Gutenberg University of Mainz, Duesbergweg 10-14 (Germany); Lee, Seung-Mo; Knez, Mato [Max Planck Institute of Microstructure Physics, Weinberg 2, 06120 Halle (Germany); Lambertz, Andreas; Carius, Reinhard [Institute of Energy Research, IEF-5 Photovoltaics, Forschungszentrum Juelich GmbH, 52425 Juelich (Germany); Bielawny, Andreas

    2008-12-15

    The concept of 3D photonic intermediate reflectors for micromorph silicon tandem solar cells has been investigated. In thin-film silicon tandem solar cells consisting of amorphous and microcrystalline silicon with two junctions of a-Si/{mu}c-Si, efficiency enhancements can be achieved by increasing the current density in the a-Si top cell. It is one goal to provide an optimized current matching at high current densities. For an ideal photon-management between top and bottom cell, a spectrally selective intermediate reflective layer (IRL) is necessary, which is less dependent of the angle of incidence than state-of-the-art thickness dependent massive interlayers. The design, preparation and characterization of a 3D photonic thin-film filter device for this purpose has been pursued straight forward in simulation and experimental realization. The inverted opal is capable of providing a suitable optical band stop with high reflectance and the necessary long wavelength transmittance as well and provides further options for improved light trapping. We have determined numerically the relative efficiency enhancement of an a-Si/{mu}c-Si tandem solar cell using a conductive 3D-photonic crystal. We have further fabricated such structures by ZnO-replication of polymeric opals using chemical vapour deposition and atomic layer deposition techniques and present the results of their characterization. Thin film photonic IRL have been prepared at the rear side of a-Si solar cells. Completed with a back contact, this is the first step to integrate this novel technology into an a-Si/{mu}c-Si tandem solar cell process. The spectral response of the cell is presented and compared with reference cells. (copyright 2008 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  17. Total 3D imaging of phase objects using defocusing microscopy: application to red blood cells

    CERN Document Server

    Roma, P M S; Amaral, F T; Agero, U; Mesquita, O N

    2014-01-01

    We present Defocusing Microscopy (DM), a bright-field optical microscopy technique able to perform total 3D imaging of transparent objects. By total 3D imaging we mean the determination of the actual shapes of the upper and lower surfaces of a phase object. We propose a new methodology using DM and apply it to red blood cells subject to different osmolality conditions: hypotonic, isotonic and hypertonic solutions. For each situation the shape of the upper and lower cell surface-membranes (lipid bilayer/cytoskeleton) are completely recovered, displaying the deformation of RBCs surfaces due to adhesion on the glass-substrate. The axial resolution of our technique allowed us to image surface-membranes separated by distances as small as 300 nm. Finally, we determine volume, superficial area, sphericity index and RBCs refractive index for each osmotic condition.

  18. Characterizations of individual mouse red blood cells parasitized by Babesia microti using 3-D holographic microscopy

    CERN Document Server

    Park, HyunJoo; Kim, Kyoohyun; Cho, Shin-Hyeong; Lee, Won-Ja; Kim, Youngchan; Lee, SangEun; Park, YongKeun

    2015-01-01

    Babesia microti causes emergency human babesiosis. However, little is known about the alterations in B. microti invaded red blood cells (Bm-RBCs) at the individual cell level. Through quantitative phase imaging techniques based on laser interferometry, we present the simultaneous measurements of structural, chemical, and mechanical modifications in individual mouse Bm-RBCs. 3-D refractive index maps of individual RBCs and in situ parasite vacuoles are imaged, from which total contents and concentration of dry mass are also precisely quantified. In addition, we examine the dynamic membrane fluctuation of Bm-RBCs, which provide information on cell membrane deformability.

  19. Determination of Drug Toxicity Using 3D Spheroids Constructed From an Immortal Human Hepatocyte Cell Line

    DEFF Research Database (Denmark)

    Fey, S. J.; Wrzesinski, Krzysztof

    2012-01-01

    Numerous publications have documented that the immortal cells grown in three-dimensional (3D) cultures possess physiological behavior, which is more reminiscent of their parental organ than when the same cells are cultivated using classical two-dimensional (2D) culture techniques. The goal...... that a precise dose can be provided in a manner similar to in vivo studies. This avoided correction of the actual dose given based on a protein determination after treatment (when some cells may have lysed). Conversion of published in vitro LC50 data (mM) for six common drugs (acetaminophen, amiodarone...

  20. Digital holography for recovering 3D shape of red blood cells

    Science.gov (United States)

    Memmolo, P.; Miccio, L.; Merola, F.; Gennari, O.; Netti, P.; Ferraro, Pietro

    2015-07-01

    Full morphometric data analysis and 3D rendering of Red Blood Cells (RBCs) is provided by means of Digital Holography (DH) in combination with Optical Tweezers (OT). The proposed method is compared with a geometrical model of RBC in order to evaluate its accuracy and tested for many kinds of RBCs, from healthy ones with double-concavity to that with abnormal shapes. Applications in diagnostics are foreseen.

  1. Non-crimp 3D woven composites unit cell: from geometric modelling to damage simulation

    OpenAIRE

    Bedogni, Enrico

    2013-01-01

    In the last twenty years, the research on composite materials has increased and many progresses have been made. However, there are still unresolved issues concerning the geometric modelling of a material at the meso-level (i.e. on a unit cell) and its damage simulation. In particular, the complexity of the internal geometry of some composite materials, such as 3D textiles, yields to new challenges for the research community. A correct definition of the internal structure in all the important ...

  2. Controlled Positioning of Cells in Biomaterials—Approaches Towards 3D Tissue Printing

    Directory of Open Access Journals (Sweden)

    Sandra Hofmann

    2011-08-01

    Full Text Available Current tissue engineering techniques have various drawbacks: they often incorporate uncontrolled and imprecise scaffold geometries, whereas the current conventional cell seeding techniques result mostly in random cell placement rather than uniform cell distribution. For the successful reconstruction of deficient tissue, new material engineering approaches have to be considered to overcome current limitations. An emerging method to produce complex biological products including cells or extracellular matrices in a controlled manner is a process called bioprinting or biofabrication, which effectively uses principles of rapid prototyping combined with cell-loaded biomaterials, typically hydrogels. 3D tissue printing is an approach to manufacture functional tissue layer-by-layer that could be transplanted in vivo after production. This method is especially advantageous for stem cells since a controlled environment can be created to influence cell growth and differentiation. Using printed tissue for biotechnological and pharmacological needs like in vitro drug-testing may lead to a revolution in the pharmaceutical industry since animal models could be partially replaced by biofabricated tissues mimicking human physiology and pathology. This would not only be a major advancement concerning rising ethical issues but would also have a measureable impact on economical aspects in this industry of today, where animal studies are very labor-intensive and therefore costly. In this review, current controlled material and cell positioning techniques are introduced highlighting approaches towards 3D tissue printing.

  3. Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

    Science.gov (United States)

    Chuang, Han-Ning; Lohaus, Raphaela; Hanisch, Uwe-Karsten; Binder, Claudia

    2013-01-01

    Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7. Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8. For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain

  4. Mesenchymal and embryonic characteristics of stem cells obtained from mouse dental pulp

    DEFF Research Database (Denmark)

    Guimarães, Elisalva Teixeira; Cruz, Gabriela Silva; de Jesus, Alan Araújo;

    2011-01-01

    OBJECTIVE: Several studies have demonstrated that human dental pulp is a source of mesenchymal stem cells. To better understand the biological properties of these cells we isolated and characterized stem cells from the dental pulp of EGFP transgenic mice. METHODS: The pulp tissue was gently...... separated from the roots of teeth extracted from C57BL/6 mice, and cultured under appropriate conditions. Flow cytometry, RT-PCR, light microscopy (staining for alkaline phosphatase) and immunofluorescence were used to investigate the expression of stem cell markers. The presence of chromosomal...... abnormalities was evaluated by G banding. RESULTS: The mouse dental pulp stem cells (mDPSC) were highly proliferative, plastic-adherent, and exhibited a polymorphic morphology predominantly with stellate or fusiform shapes. The presence of cell clusters was observed in cultures of mDPSC. Some cells were...

  5. Effects of microenvironment on growth and differentiation of human dental pulp cells

    Science.gov (United States)

    Datko, Laura Christine

    Dental pulp stem cells (DPSCs) have recently been described as a potential stem cell source for various regenerative medicine and tissue engineering applications. They appear to be multipotent, however more characterization is necessary to determine the true potential of these cells. An important aspect of using DPSCs, or any stem cell type, tissue engineering application is the microenvironment within the construct. The microenvironment could include construct mechanical properties, construct composition, and 3D dynamic conditions in vivo. This work aims to study those specific microenvironment effects on DPSCs. To determine the effects of mechanical properties of the substrate on DPSCs, they were seeded on polyacrylamide (PA) gels of different elastic moduli. These gels ranged from 3 kPa to 75 kPa and a glass coverslip was used as a control. They were also exposed to either standard stem cell media or an osteogenic differentiation media, to determine the potential of the DPSCs for osteogenic/odontogenic differentiation. The cultures were analyzed for morphological changes, osteopontin production, alkaline phosphatase (ALP) production, and mineralization. The results showed that the DPSCs adhered well to the PA gels for the first few days in culture, but by day 7, they were starting to detach from the PA gels and only remain viable in gel defects or along the edges. This selective growth was also reflected in the mineralization, which only occurred in areas of confluence for the cells on the PA gels. Interestingly, all cultures produced osteopontin and ALP, however by the end of the experiment, the cells cultured on glass had the highest ALP production. It appeared that without the addition of growth factors to induce other cell lineages, DPSCs defaulted to an osteogenic/odontogenic lineage. To determine the effect of mineral composition, preliminary studies were done on bone marrow stromal cells (BMSCs) and 7F2 osteoblasts. These two cell types were exposed to

  6. Perfusion Stirred-Tank Bioreactors for 3D Differentiation of Human Neural Stem Cells.

    Science.gov (United States)

    Simão, Daniel; Arez, Francisca; Terasso, Ana P; Pinto, Catarina; Sousa, Marcos F Q; Brito, Catarina; Alves, Paula M

    2016-01-01

    Therapeutic breakthroughs in neurological disorders have been hampered by the lack of accurate central nervous system (CNS) models. The development of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of new therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmental, anatomic, and physiological) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity, etc.). Recapitulation of CNS phenotypic and functional features in vitro requires the implementation of advanced culture strategies, such as 3D culture systems, which enable to mimic the in vivo structural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. The development of robust and scalable processes for the 3D differentiation of hNSC can improve the accuracy of early stage development in preclinical research. In this context, the use of software-controlled stirred-tank bioreactors (STB) provides an efficient technological platform for hNSC aggregation and differentiation. This system enables to monitor and control important physicochemical parameters for hNSC culture, such as dissolved oxygen. Importantly, the adoption of a perfusion operation mode allows a stable flow of nutrients and differentiation/neurotrophic factors, while clearing the toxic by-products. This contributes to a setting closer to the physiological, by mimicking the in vivo microenvironment. In this chapter, we address the technical requirements and procedures for the implementation of 3D differentiation strategies of hNSC, by operating STB under perfusion mode for long-term cultures. This strategy is suitable

  7. Perfusion Stirred-Tank Bioreactors for 3D Differentiation of Human Neural Stem Cells.

    Science.gov (United States)

    Simão, Daniel; Arez, Francisca; Terasso, Ana P; Pinto, Catarina; Sousa, Marcos F Q; Brito, Catarina; Alves, Paula M

    2016-01-01

    Therapeutic breakthroughs in neurological disorders have been hampered by the lack of accurate central nervous system (CNS) models. The development of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of new therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmental, anatomic, and physiological) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity, etc.). Recapitulation of CNS phenotypic and functional features in vitro requires the implementation of advanced culture strategies, such as 3D culture systems, which enable to mimic the in vivo structural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. The development of robust and scalable processes for the 3D differentiation of hNSC can improve the accuracy of early stage development in preclinical research. In this context, the use of software-controlled stirred-tank bioreactors (STB) provides an efficient technological platform for hNSC aggregation and differentiation. This system enables to monitor and control important physicochemical parameters for hNSC culture, such as dissolved oxygen. Importantly, the adoption of a perfusion operation mode allows a stable flow of nutrients and differentiation/neurotrophic factors, while clearing the toxic by-products. This contributes to a setting closer to the physiological, by mimicking the in vivo microenvironment. In this chapter, we address the technical requirements and procedures for the implementation of 3D differentiation strategies of hNSC, by operating STB under perfusion mode for long-term cultures. This strategy is suitable

  8. 3D culture of adult mouse neural stem cells within functionalized self-assembling peptide scaffolds

    Directory of Open Access Journals (Sweden)

    Cunha C

    2011-05-01

    Full Text Available Carla Cunha1,2, Silvia Panseri3,4, Omar Villa1,2, Diego Silva1,2, Fabrizio Gelain1,21Department of Biotechnology and Biosciences, University of Milano-Bicocca; 2Center for Nanomedicine and Tissue Engineering, CNTE – A.O. Ospedale Niguarda Ca' Granda, Milan; 3Laboratory of Biomechanics and Technology Innovation, Rizzoli Orthopaedic Institute, Bologna; 4Laboratory of Nano-Biomagnetism, Institute of Science and Technology for Ceramics, National Research Council, Faenza, ItalyAbstract: Three-dimensional (3D in vitro models of cell culture aim to fill the gap between the standard two-dimensional cell studies and the in vivo environment. Especially for neural tissue regeneration approaches where there is little regenerative capacity, these models are important for mimicking the extracellular matrix in providing support, allowing the natural flow of oxygen, nutrients, and growth factors, and possibly favoring neural cell regrowth. We have previously demonstrated that a new self-assembling nanostructured biomaterial, based on matrigel, was able to support adult neural stem cell (NSC culture. In this study, we developed a new 3D cell culture system that takes advantage of the nano- and microfiber assembling process, under physiologic conditions, of these biomaterials. The assembled scaffold forms an intricate and biologically active matrix that displays specifically designed functional motifs: RGD (Arg-Gly-Asp, BMHP1 (bone marrow homing peptide 1, and BMHP2, for the culture of adult NSCs. These scaffolds were prepared at different concentrations, and microscopic examination of the cell-embedded scaffolds showed that NSCs are viable and they proliferate and differentiate within the nanostructured environment of the scaffold. Such a model has the potential to be tailored to develop ad hoc designed peptides for specific cell lines.Keywords: biomaterials, tissue engineering, 3D in vitro model

  9. Probabilistic Voxel-Fe model for single cell motility in 3D

    Science.gov (United States)

    Borau, Carlos; Polacheck, William J; Kamm, Roger D; García-Aznar, José Manuel

    2015-01-01

    Background Cells respond to a variety of external stimuli regulated by the environment conditions. Mechanical, chemical and biological factors are of great interest and have been deeply studied. Furthermore, mathematical and computational models have been rapidly growing over the past few years, permitting researches to run complex scenarios saving time and resources. Usually these models focus on specific features of cell migration, making them only suitable to study restricted phenomena. Methods Here we present a versatile finite element (FE) cell-scale 3D migration model based on probabilities depending in turn on ECM mechanical properties, chemical, fluid and boundary conditions. Results With this approach we are able to capture important outcomes of cell migration such as: velocities, trajectories, cell shape and aspect ratio, cell stress or ECM displacements. Conclusions The modular form of the model will allow us to constantly update and redefine it as advancements are made in clarifying how cellular events take place. PMID:26290806

  10. Differences in growth properties of endometrial cancer in three dimensional (3D) culture and 2D cell monolayer

    Energy Technology Data Exchange (ETDEWEB)

    Chitcholtan, Kenny, E-mail: kenny.chitcholtan@otago.ac.nz [Department of Obstetrics and Gynaecology, University of Otago, Christchurch, 2 Riccarton Avenue, Christchurch 8011 (New Zealand); Asselin, Eric, E-mail: Eric.Asselin@uqtr.ca [Department of Chemistry and Biology, University of Quebec, at Trois-Rivières, C.P. 500, Trois-Rivières, Quebec, Canada G9A 5H7 (Canada); Parent, Sophie, E-mail: Sophie.Parent@uqtr.ca [Department of Chemistry and Biology, University of Quebec, at Trois-Rivières, C.P. 500, Trois-Rivières, Quebec, Canada G9A 5H7 (Canada); Sykes, Peter H., E-mail: peter.sykes@otago.ac.nz [Department of Obstetrics and Gynaecology, University of Otago, Christchurch, 2 Riccarton Avenue, Christchurch 8011 (New Zealand); Evans, John J., E-mail: john.evans@otago.ac.nz [Department of Obstetrics and Gynaecology, University of Otago, Christchurch, 2 Riccarton Avenue, Christchurch 8011 (New Zealand); Centre of Neuroendocrinology and The MacDiarmid Institute of Advanced Materials and Nanotechnology, University of Otago, Christchurch, 2 Riccarton Avenue, Christchurch 8011 (New Zealand)

    2013-01-01

    Three-dimensional (3D) in vitro models have an invaluable role in understanding the behaviour of tumour cells in a well defined microenvironment. This is because some aspects of tumour characteristics cannot be fully recapitulated in a cell monolayer (2D). In the present study, we compared growth patterns, expression of signalling molecules, and metabolism-associated proteins of endometrial cancer cell lines in 3D and 2D cell cultures. Cancer cells formed spherical structures in 3D reconstituted basement membrane (3D rBM), and the morphological appearance was cell line dependent. Cell differentiation was observed after 8 days in the 3D rBM. There was reduced proliferation, detected by less expression of PCNA in 3D rBM than in 2D cell monolayers. The addition of exogenous epidermal growth factor (EGF) to cancer cells induced phosphorylation of EGFR and Akt in both cell culture conditions. The uptake of glucose was selectively altered in the 3D rBM, but there was a lack of association with Glut-1 expression. The secretion of vascular endothelial growth factor (VEGF) and prostaglandin E{sub 2} (PGE{sub 2}) was selectively altered in 3D rBM, and it was cell line dependent. Our data demonstrated that 3D rBM as an in vitro model can influence proliferation and metabolism of endometrial cancer cell behaviour compared to 2D cell monolayer. Changes are specific to individual cell types. The use of 3D rBM is, therefore, important in the in vitro study of targeted anticancer therapies.

  11. 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

    Directory of Open Access Journals (Sweden)

    Yeonhee Kim

    Full Text Available Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes and non-parenchymal (liver sinusoidal endothelial, LSEC cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs were cultured in a layered three-dimensional (3D configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM, which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1 demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism

  12. Image informatics for studying signal transduction in cells interacting with 3D matrices

    Science.gov (United States)

    Tzeranis, Dimitrios S.; Guo, Jin; Chen, Chengpin; Yannas, Ioannis V.; Wei, Xunbin; So, Peter T. C.

    2014-03-01

    Cells sense and respond to chemical stimuli on their environment via signal transduction pathways, complex networks of proteins whose interactions transmit chemical information. This work describes an implementation of image informatics, imaging-based methodologies for studying signal transduction networks. The methodology developed focuses on studying signal transduction networks in cells that interact with 3D matrices. It utilizes shRNA-based knock down of network components, 3D high-content imaging of cells inside the matrix by spectral multi-photon microscopy, and single-cell quantification using features that describe both cell morphology and cell-matrix adhesion pattern. The methodology is applied in a pilot study of TGFβ signaling via the SMAD pathway in fibroblasts cultured inside porous collagen-GAG scaffolds, biomaterials similar to the ones used clinically to induce skin regeneration. Preliminary results suggest that knocking down all rSMAD components affects fibroblast response to TGFβ1 and TGFβ3 isoforms in different ways, and suggest a potential role for SMAD1 and SMAD5 in regulating TGFβ isoform response. These preliminary results need to be verified with proteomic results that can provide solid evidence about the particular role of individual components of the SMAD pathway.

  13. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry

    Science.gov (United States)

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  14. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

    Science.gov (United States)

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte; Corbeil, Denis; Hoflack, Bernard

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  15. Reconstruction of 3D ion beam micro-tomography data for applications in Cell Biology

    Energy Technology Data Exchange (ETDEWEB)

    Habchi, C. [Universite de Bordeaux, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France)], E-mail: habchi@cenbg.in2p3.fr; Nguyen, D.T.; Barberet, Ph. [Universite de Bordeaux, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France); Incerti, S. [CNRS/IN2P3, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France); Moretto, Ph. [Universite de Bordeaux, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France); Sakellariou, A. [Research School of Physical Sciences and Engineering, Australian National University, Canberra ACT 0200 (Australia); Seznec, H. [CNRS/IN2P3, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France)

    2009-06-15

    The DISRA (Discrete Image Space Reconstruction Algorithm) reconstruction code, created by A. Sakellariou, was conceived for the ideal case of complete three-dimensional (3D) PIXET (Particle Induced X-ray Emission Tomography) data. This implies two major difficulties for biological samples: first, the long duration of such experiments and second, the subsequent damage that occurs on such fragile specimens. For this reason, the DISRA code was extended at CENBG in order to probe isolated PIXET slices, taking into account the sample structure and mass density provided by 3D STIMT (Scanning Transmission Ion Microscopy Tomography) in the volume of interest. This modified version was tested on a phantom sample and first results on human cancer cells are also presented.

  16. Reconstruction of 3D ion beam micro-tomography data for applications in Cell Biology

    Science.gov (United States)

    Habchi, C.; Nguyen, D. T.; Barberet, Ph.; Incerti, S.; Moretto, Ph.; Sakellariou, A.; Seznec, H.

    2009-06-01

    The DISRA (Discrete Image Space Reconstruction Algorithm) reconstruction code, created by A. Sakellariou, was conceived for the ideal case of complete three-dimensional (3D) PIXET (Particle Induced X-ray Emission Tomography) data. This implies two major difficulties for biological samples: first, the long duration of such experiments and second, the subsequent damage that occurs on such fragile specimens. For this reason, the DISRA code was extended at CENBG in order to probe isolated PIXET slices, taking into account the sample structure and mass density provided by 3D STIMT (Scanning Transmission Ion Microscopy Tomography) in the volume of interest. This modified version was tested on a phantom sample and first results on human cancer cells are also presented.

  17. Mechanical Properties of 3-D Printed Cellular Foams with triangular cells

    Science.gov (United States)

    Bunga, Pratap Kumar

    In the present work, poly lactic acid (PLA) is used as a model system to investigate the mechanical behavior of 3-D printed foams with triangular cells. Solid PLA tension and compression specimens and foams made of PLA were fabricated using fused deposition 3-D printing technique. The solid PLA tension specimens were characterized for their densities and found to be about 10% lower in density as compared to their bulk counter parts. The triangular foams had a relative density of about 64%. The relationships between the structure of the foams and its deformation behavior under compression along two in-plane directions were characterized. Furthermore, simple finite element models were developed to understand the observed deformation behavior of triangular foams.

  18. Heritable Genetic Changes in Cells Recovered From Irradiated 3D Tissue Constructs

    Energy Technology Data Exchange (ETDEWEB)

    Michael Cornforth

    2012-03-26

    Combining contemporary cytogenetic methods with DNA CGH microarray technology and chromosome flow-sorting increases substantially the ability to resolve exchange breakpoints associated with interstitial deletions and translocations, allowing the consequences of radiation damage to be directly measured at low doses, while also providing valuable insights into molecular mechanisms of misrepair processes that, in turn, identify appropriate biophysical models of risk at low doses. Specific aims apply to cells recovered from 3D tissue constructs of human skin and, for the purpose of comparison, the same cells irradiated in traditional 2D cultures. The project includes research complementary to NASA/HRP space radiation project.

  19. A method for the evaluation of thousands of automated 3D stem cell segmentations.

    Science.gov (United States)

    Bajcsy, P; Simon, M; Florczyk, S J; Simon, C G; Juba, D; Brady, M C

    2015-12-01

    There is no segmentation method that performs perfectly with any dataset in comparison to human segmentation. Evaluation procedures for segmentation algorithms become critical for their selection. The problems associated with segmentation performance evaluations and visual verification of segmentation results are exaggerated when dealing with thousands of three-dimensional (3D) image volumes because of the amount of computation and manual inputs needed. We address the problem of evaluating 3D segmentation performance when segmentation is applied to thousands of confocal microscopy images (z-stacks). Our approach is to incorporate experimental imaging and geometrical criteria, and map them into computationally efficient segmentation algorithms that can be applied to a very large number of z-stacks. This is an alternative approach to considering existing segmentation methods and evaluating most state-of-the-art algorithms. We designed a methodology for 3D segmentation performance characterization that consists of design, evaluation and verification steps. The characterization integrates manual inputs from projected surrogate 'ground truth' of statistically representative samples and from visual inspection into the evaluation. The novelty of the methodology lies in (1) designing candidate segmentation algorithms by mapping imaging and geometrical criteria into algorithmic steps, and constructing plausible segmentation algorithms with respect to the order of algorithmic steps and their parameters, (2) evaluating segmentation accuracy using samples drawn from probability distribution estimates of candidate segmentations and (3) minimizing human labour needed to create surrogate 'truth' by approximating z-stack segmentations with 2D contours from three orthogonal z-stack projections and by developing visual verification tools. We demonstrate the methodology by applying it to a dataset of 1253 mesenchymal stem cells. The cells reside on 10 different types of biomaterial

  20. Universal lab-on-a-chip platform for complex, perfused 3D cell cultures

    Science.gov (United States)

    Sonntag, F.; Schmieder, F.; Ströbel, J.; Grünzner, S.; Busek, M.; Günther, K.; Steege, T.; Polk, C.; Klotzbach, U.

    2016-03-01

    The miniaturization, rapid prototyping and automation of lab-on-a-chip technology play nowadays a very important role. Lab-on-a-chip technology is successfully implemented not only for environmental analysis and medical diagnostics, but also as replacement of animals used for the testing of substances in the pharmaceutical and cosmetics industries. For that purpose the Fraunhofer IWS and partners developed a lab-on-a-chip platform for perfused cell-based assays in the last years, which includes different micropumps, valves, channels, reservoirs and customized cell culture modules. This technology is already implemented for the characterization of different human cell cultures and organoids, like skin, liver, endothelium, hair follicle and nephron. The advanced universal lab-on-a-chip platform for complex, perfused 3D cell cultures is divided into a multilayer basic chip with integrated micropump and application-specific 3D printed cell culture modules. Moreover a technology for surface modification of the printed cell culture modules by laser micro structuring and a complex and flexibly programmable controlling device based on an embedded Linux system was developed. A universal lab-on-a-chip platform with an optional oxygenator and a cell culture module for cubic scaffolds as well as first cell culture experiments within the cell culture device will be presented. The module is designed for direct interaction with robotic dispenser systems. This offers the opportunity to combine direct organ printing of cells and scaffolds with the microfluidic cell culture module. The characterization of the developed system was done by means of Micro-Particle Image Velocimetry (μPIV) and an optical oxygen measuring system.

  1. Fabrication of 3-D Reconstituted Organoid Arrays by DNA-Programmed Assembly of Cells (DPAC).

    Science.gov (United States)

    Todhunter, Michael E; Weber, Robert J; Farlow, Justin; Jee, Noel Y; Cerchiari, Alec E; Gartner, Zev J

    2016-01-01

    Tissues are the organizational units of function in metazoan organisms. Tissues comprise an assortment of cellular building blocks, soluble factors, and extracellular matrix (ECM) composed into specific three-dimensional (3-D) structures. The capacity to reconstitute tissues in vitro with the structural complexity observed in vivo is key to understanding processes such as morphogenesis, homeostasis, and disease. In this article, we describe DNA-programmed assembly of cells (DPAC), a method to fabricate viable, functional arrays of organoid-like tissues within 3-D ECM gels. In DPAC, dissociated cells are chemically functionalized with degradable oligonucleotide "Velcro," allowing rapid, specific, and reversible cell adhesion to a two-dimensional (2-D) template patterned with complementary DNA. An iterative assembly process builds up organoids, layer-by-layer, from this initial 2-D template and into the third dimension. Cleavage of the DNA releases the completed array of tissues that are captured and fully embedded in ECM gels for culture and observation. DPAC controls the size, shape, composition, and spatial heterogeneity of organoids and permits positioning of constituent cells with single-cell resolution even within cultures several centimeters long. © 2016 by John Wiley & Sons, Inc. PMID:27622567

  2. Current automated 3D cell detection methods are not a suitable replacement for manual stereologic cell counting

    Directory of Open Access Journals (Sweden)

    Christoph eSchmitz

    2014-05-01

    Full Text Available Stereologic cell counting has had a major impact on the field of neuroscience. A major bottleneck in stereologic cell counting is that the user must manually decide whether or not each cell is counted according to three-dimensional (3D stereologic counting rules by visual inspection within hundreds of microscopic fields-of-view per investigated brain or brain region. Reliance on visual inspection forces stereologic cell counting to be very labor-intensive and time-consuming, and is the main reason why biased, non-stereologic two-dimensional (2D cell counting approaches have remained in widespread use. We present an evaluation of the performance of modern automated cell detection and segmentation algorithms as a potential alternative to the manual approach in stereologic cell counting. The image data used in this study were 3D microscopic images of thick brain tissue sections prepared with a variety of commonly used nuclear and cytoplasmic stains. The evaluation compared the numbers and locations of cells identified unambiguously and counted exhaustively by an expert observer with those found by three automated 3D cell detection algorithms: nuclei segmentation from the FARSIGHT toolkit, nuclei segmentation by 3D multiple level set methods, and the 3D object counter plug-in for ImageJ. Of these methods, FARSIGHT performed best, with true-positive detection rates between 38–99% and false-positive rates from 3.6–82%. The results demonstrate that the current automated methods suffer from lower detection rates and higher false-positive rates than are acceptable for obtaining valid estimates of cell numbers. Thus, at present, stereologic cell counting with manual decision for object inclusion according to unbiased stereologic counting rules remains the only adequate method for unbiased cell quantification in histologic tissue sections.

  3. Spontaneous Differentiation of Dental Pulp stem cells on Dental polymers

    Science.gov (United States)

    Bherwani, Aneel; Suarato, Giulia; Qin, Sisi; Chang, Chung-Cheh; Akhavan, Aaron; Spiegel, Joseph; Jurukovski, Vladimir; Rafailovich, Miriam; Simon, Marcia

    2012-02-01

    Dental pulp stem cells were plated on two dentally relevant materials i.e. PMMA commonly used for denture and Titanium used for implants. In both cases, we probed for the role of surface interaction and substrate morphology. Different films of PMMA were spun cast directly onto Si wafers; PMMA fibers of different diameters were electro spun onto some of these substrates. Titanium metal was evaporated onto Si surfaces using an electron beam evaporator. In addition, on some surfaces, P4VP nanofibers were spun cast. DPSC were grown in alpha-MEM supplemented with 10% fetal bovine serum, 0.2mM L-ascorbic acid 2-phosphate, 2mm glutamine and 10mM beta-glycerol phosphate either with or without 10nM dexamethasone. After 21 days samples were examined using confocal microscopy of cells and by scanning electron microscopy (SEM) and Energy dispersive X-ray Analysis (EDAX). In the case of Titanium biomineralization was observed independent of dexamethasone, where the deposits were templated along the fibers. Minimal biomineralization was observed on flat Titanium and PMMA samples. Markers of osteogenesis and specific signaling pathways are being evaluated by RT-PCR, which are up regulated on each surface, to understand the fundamental manner in which surfaces interact with cell differentiation.

  4. 3D measurements of live cells via digital holographic microscopy and terahertz spectroscopy

    Science.gov (United States)

    Park, Jun Yong; Oser, Dorian; Iapozzuto, Peter; Norbury, Sean; Mahajan, Supriya; Khmaladze, Alexander; Sharikova, Anna

    2016-03-01

    This is a study of the central nervous system (CNS) cells, including brain micro vascular endothelial cells (BMV) that constitute the blood brain barrier, and C6 glial cells that are the predominant cell in the brain. The cells are exposed to various chemicals by non-invasive, label-free methods. Digital holographic microscopy (DHM) is a technique that records an interference pattern between an object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information, and 3D images are obtained. The measurement of cell cultures by digital holographic microscopy yields information about cell death mechanisms, since these processes are correlated with individual cell volume. Our in-house DHM combines a visible (red) laser source with a conventional microscope base, and LabVIEW-run data processing. Terahertz spectral signatures are associated with structural changes in molecules and provide complementary information about cells. Both CNS cells BMV and C6 cells are treated with the drug "Methamphetamine" (METH), which induces apoptosis in neuronal cells and exhibits decrease in cell volume, a characteristic of cells undergoing apoptosis (induced cell death). METH can cause CNS cell death by cross-talk between mitochondria-, endoplasmic reticulum-, and receptor-mediated apoptotic events, all of which results in drug induced changes in neuroplasticity and significant neuropathology. Doxorubicin (DOX), a popular anticancer drug, is used as a control. We observe that METH treatment resulted in more pronounced cell volume shrinkage in both the BMV and C6 cells, as compared to DOX-induced cell apoptosis.

  5. Low-level laser therapy in 3D cell culture model using gingival fibroblasts.

    Science.gov (United States)

    Basso, Fernanda G; Soares, Diana G; de Souza Costa, Carlos Alberto; Hebling, Josimeri

    2016-07-01

    Besides extensive data about the effects of low-level laser therapy (LLLT) on different cell types, so far, these results were obtained from monolayer cell culture models, which have limitations in terms of cell morphology and phenotype expression. Therefore, for better in vitro evaluation of the effects of LLLT, this study was performed with a 3D cell culture model, where gingival fibroblasts were seeded in collagen matrix. Cells isolated from a healthy patient were seeded in wells of 24-well plates with culture medium (DMEM) supplemented with 10 % fetal bovine serum and collagen type I solution. After 5 days, a serum-free DMEM was added to the matrices with cells that were subjected or not to three consecutive irradiations of LLLT by means of the LaserTABLE diode device (780 nm, 25 mW) at 0.5, 1.5, and 3 J/cm(2). Twenty-four hours after the last irradiation, cell viability and morphology as well as gene expression of growth factors were assessed. Histological evaluation of matrices demonstrated uniform distribution and morphology of gingival fibroblasts within the collagen matrix. LLLT at 3 J/cm(2) increased gingival fibroblast viability. Enhanced gene expression of hCOL-I and hEGF was observed for 0.5 J/cm(2), while no significant changes were detected for the other irradiation densities tested. In conclusion, LLLT promoted biostimulation of gingival fibroblasts seeded in a 3D cell culture model, demonstrating that this model can be applied for phototherapy studies and that LLLT could penetrate the collagen matrix to increase cell functions related to tissue repair. PMID:27126408

  6. Direct 3D-printing of cell-laden constructs in microfluidic architectures.

    Science.gov (United States)

    Liu, Justin; Hwang, Henry H; Wang, Pengrui; Whang, Grace; Chen, Shaochen

    2016-04-21

    Microfluidic platforms have greatly benefited the biological and medical fields, however standard practices require a high cost of entry in terms of time and energy. The utilization of three-dimensional (3D) printing technologies has greatly enhanced the ability to iterate and build functional devices with unique functions. However, their inability to fabricate within microfluidic devices greatly increases the cost of producing several different devices to examine different scientific questions. In this work, a variable height micromixer (VHM) is fabricated using projection 3D-printing combined with soft lithography. Theoretical and flow experiments demonstrate that altering the local z-heights of VHM improved mixing at lower flow rates than simple geometries. Mixing of two fluids occurs as low as 320 μL min(-1) in VHM whereas the planar zigzag region requires a flow rate of 2.4 mL min(-1) before full mixing occurred. Following device printing, to further demonstrate the ability of this projection-based method, complex, user-defined cell-laden scaffolds are directly printed inside the VHM. The utilization of this unique ability to produce 3D tissue models within a microfluidic system could offer a unique platform for medical diagnostics and disease modeling. PMID:26980159

  7. 3D cut-cell modelling for high-resolution atmospheric simulations

    CERN Document Server

    Yamazaki, H; Nikiforakis, N

    2015-01-01

    With the recent, rapid development of computer technology, the resolution of atmospheric numerical models has increased substantially. As a result, steep gradients in mountainous terrain are now being resolved in high-resolution models. This results in large truncation errors in those models using terrain-following coordinates. In this study, a new 3D Cartesian coordinate non-hydrostatic atmospheric model is developed. A cut-cell representation of topography based on finite-volume discretization is combined with a cell-merging approach, in which small cut-cells are merged with neighboring cells either vertically or horizontally. In addition, a block-structured mesh-refinement technique achieves a variable resolution on the model grid with the finest resolution occurring close to the terrain surface. The model successfully reproduces a flow over a 3D bell-shaped hill that shows a good agreement with the flow predicted by the linear theory. The ability of the model to simulate flows over steep terrain is demons...

  8. Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells.

    Science.gov (United States)

    Rimann, Markus; Bono, Epifania; Annaheim, Helene; Bleisch, Matthias; Graf-Hausner, Ursula

    2016-08-01

    Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application. PMID:25609254

  9. Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells.

    Science.gov (United States)

    Rimann, Markus; Bono, Epifania; Annaheim, Helene; Bleisch, Matthias; Graf-Hausner, Ursula

    2016-08-01

    Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application.

  10. Regulation of mesenchymal stem cell 3D microenvironment: From macro to microfluidic bioreactors.

    Science.gov (United States)

    Sart, Sébastien; Agathos, Spiros N; Li, Yan; Ma, Teng

    2016-01-01

    Human mesenchymal stem cells (hMSCs) have emerged as an important cell type in cell therapy and tissue engineering. In these applications, maintaining the therapeutic properties of hMSCs requires tight control of the culture environments and the structural cell organizations. Bioreactor systems are essential tools to achieve these goals in the clinical-scale expansion and tissue engineering applications. This review summarizes how different bioreactors provide cues to regulate the structure and the chemico-mechanical microenvironment of hMSCs with a focus on 3D organization. In addition to conventional bioreactors, recent advances in microfluidic bioreactors as a novel approach to better control the hMSC microenvironment are also discussed. These advancements highlight the key role of bioreactor systems in preserving hMSC's functional properties by providing dynamic and temporal regulation of in vitro cellular microenvironment.

  11. Assessing Drug Efficacy in a Miniaturized Pancreatic Cancer In Vitro 3D Cell Culture Model.

    Science.gov (United States)

    Shelper, Todd B; Lovitt, Carrie J; Avery, Vicky M

    2016-09-01

    Pancreatic cancer continues to have one of the poorest prognoses among all cancers. The drug discovery efforts for this disease have largely failed, with no significant improvement in survival outcomes for advanced pancreatic cancer patients over the past 20 years. Traditional in vitro cell culture techniques have been used extensively in both basic and early drug discovery; however, these systems offer poor models to assess emerging therapeutics. More predictive cell-based models, which better capture the cellular heterogeneity and complexities of solid pancreatic tumors, are urgently needed not only to improve drug discovery success but also to provide insight into the tumor biology. Pancreatic tumors are characterized by a unique micro-environment that is surrounded by a dense stroma. A complex network of interactions between extracellular matrix (ECM) components and the effects of cell-to-cell contacts may enhance survival pathways within in vivo tumors. This biological and physical complexity is lost in traditional cell monolayer models. To explore the predictive potential of a more complex cellular system, a three-dimensional (3D) micro-tumor assay was evaluated. Efficacy of six current chemotherapeutics was determined against a panel of primary and metastatic pancreatic tumor cell lines in a miniaturized ECM-based 3D cell culture system. Suitability for potential use in high-throughput screening applications was assessed, including ascertaining the effects that miniaturization and automation had on assay robustness. Cellular health was determined by utilizing an indirect population-based metabolic activity assay and a direct imaging-based cell viability assay. PMID:27552143

  12. Biomimetic Extracellular Matrix Mediated Somatic Stem Cell Differentiation: Applications in Dental Pulp Tissue Regeneration.

    Directory of Open Access Journals (Sweden)

    Sriram eRavindran

    2015-04-01

    Full Text Available Dental caries is one of the most widely prevalent infectious diseases in the world. It affects more than half of the world’s population. The current treatment for necrotic dental pulp tissue arising from dental caries is root canal therapy. This treatment results in loss of tooth sensitivity and vitality making it prone for secondary infections. Over the past decade, several tissue-engineering approaches have attempted regeneration of the dental pulp tissue. Although several studies have highlighted the potential of dental stem cells, none have transitioned into a clinical setting owing to limited availability of dental stem cells and the need for growth factor delivery systems. Our strategy is to utilize the intact ECM of pulp cells to drive lineage specific differentiation of bone marrow derived mesenchymal stem cells. From a clinical perspective, pulp ECM scaffolds can be generated using cell lines and patient specific somatic stem cells can be used for regeneration. Our published results have shown the feasibility of using pulp ECM scaffolds for odontogenic differentiation of non-dental mesenchymal cells. This focused review discusses the issues surrounding dental pulp tissue regeneration and the potential of our strategy to overcome these issues.

  13. Biomimetic extracellular matrix mediated somatic stem cell differentiation: applications in dental pulp tissue regeneration.

    Science.gov (United States)

    Ravindran, Sriram; George, Anne

    2015-01-01

    Dental caries is one of the most widely prevalent infectious diseases in the world. It affects more than half of the world's population. The current treatment for necrotic dental pulp tissue arising from dental caries is root canal therapy. This treatment results in loss of tooth sensitivity and vitality making it prone for secondary infections. Over the past decade, several tissue-engineering approaches have attempted regeneration of the dental pulp tissue. Although several studies have highlighted the potential of dental stem cells, none have transitioned into a clinical setting owing to limited availability of dental stem cells and the need for growth factor delivery systems. Our strategy is to utilize the intact ECM of pulp cells to drive lineage specific differentiation of bone marrow derived mesenchymal stem cells. From a clinical perspective, pulp ECM scaffolds can be generated using cell lines and patient specific somatic stem cells can be used for regeneration. Our published results have shown the feasibility of using pulp ECM scaffolds for odontogenic differentiation of non-dental mesenchymal cells. This focused review discusses the issues surrounding dental pulp tissue regeneration and the potential of our strategy to overcome these issues.

  14. Regeneration of Dental-Pulp-like Tissue by Chemotaxis-Induced Cell Homing

    OpenAIRE

    Kim, Jin Y.; Xin, Xuejun; Moioli, Eduardo K.; Chung, Jenny; Lee, Chang Hun; Chen, Mo; Fu, Susan Y.; Koch, Peter D.; Mao, Jeremy J.

    2010-01-01

    Tooth infections or injuries involving dental pulp are treated routinely by root canal therapy. Endodontically treated teeth are devitalized, susceptible to re-infections, fractures, and subsequent tooth loss. Here, we report regeneration of dental-pulp-like tissue by cell homing and without cell transplantation. Upon in vivo implantation of endodontically treated real-size, native human teeth in mouse dorsum for the tested 3 weeks, delivery of basic fibroblast growth factor and/or vascular e...

  15. Enabling Flexible Polymer Tandem Solar Cells by 3D Ptychographic Imaging

    DEFF Research Database (Denmark)

    Dam, Henrik Friis; Andersen, Thomas Rieks; Pedersen, Emil Bøje Lind;

    2015-01-01

    one after the other by wet processing leaves plenty of room for error and the process development calls for an analytical technique that enables 3D reconstruction of the layer stack with the possibility to probe thickness, density, and chemistry of the individual layers in the stack. The use......The realization of a complete tandem polymer solar cell under ambient conditions using only printing and coating methods on a flexible substrate results in a fully scalable process but also requires accurate control during layer formation to succeed. The serial process where the layers are added...

  16. Tuning 3D Collagen Matrix Stiffness Independently of Collagen Concentration Modulates Endothelial Cell Behavior

    Science.gov (United States)

    Mason, Brooke N.; Starchenko, Alina; Williams, Rebecca M.; Bonassar, Lawrence J.; Reinhart-King, Cynthia A.

    2012-01-01

    Numerous studies have described the effects of matrix stiffening on cell behavior using two dimensional (2D) synthetic surfaces; however less is known about the effects of matrix stiffening on cells embedded in three dimensional (3D) in vivo-like matrices. A primary limitation in investigating the effects of matrix stiffness in 3D is the lack of materials that can be tuned to control stiffness independently of matrix density. Here, we use collagen-based scaffolds where the mechanical properties are tuned using non-enzymatic glycation of the collagen in solution, prior to polymerization. Collagen solutions glycated prior to polymerization result in collagen gels with a 3-fold increase in compressive modulus without significant changes to the collagen architecture. Using these scaffolds, we show that endothelial cell spreading increases with matrix stiffness, as does the number and length of angiogenic sprouts and the overall spheroid outgrowth. Differences in sprout length are maintained even when the receptor for advanced glycation endproducts is inhibited. Our results demonstrate the ability to de-couple matrix stiffness from matrix density and structure in collagen gels, and that increased matrix stiffness results in increased sprouting and outgrowth. PMID:22902816

  17. Brownian nanoimaging of interface dynamics and ligand-receptor binding at cell surfaces in 3-D.

    Science.gov (United States)

    Kuznetsov, Igor R; Evans, Evan A

    2013-04-01

    We describe a method for nanoimaging interfacial dynamics and ligand-receptor binding at surfaces of live cells in 3-D. The imaging probe is a 1-μm diameter glass bead confined by a soft laser trap to create a "cloud" of fluctuating states. Using a facile on-line method of video image analysis, the probe displacements are reported at ~10 ms intervals with bare precisions (±SD) of 4-6 nm along the optical axis (elevation) and 2 nm in the transverse directions. We demonstrate how the Brownian distributions are analyzed to characterize the free energy potential of each small probe in 3-D taking into account the blur effect of its motions during CCD image capture. Then, using the approach to image interactions of a labeled probe with lamellae of leukocytic cells spreading on cover-glass substrates, we show that deformations of the soft distribution in probe elevations provide both a sensitive long-range sensor for defining the steric topography of a cell lamella and a fast telemetry for reporting rare events of probe binding with its surface receptors. Invoking established principles of Brownian physics and statistical thermodynamics, we describe an off-line method of super resolution that improves precision of probe separations from a non-reactive steric boundary to ~1 nm.

  18. A harmonic polynomial cell (HPC) method for 3D Laplace equation with application in marine hydrodynamics

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Yan-Lin, E-mail: yanlin.shao@dnvgl.com; Faltinsen, Odd M.

    2014-10-01

    We propose a new efficient and accurate numerical method based on harmonic polynomials to solve boundary value problems governed by 3D Laplace equation. The computational domain is discretized by overlapping cells. Within each cell, the velocity potential is represented by the linear superposition of a complete set of harmonic polynomials, which are the elementary solutions of Laplace equation. By its definition, the method is named as Harmonic Polynomial Cell (HPC) method. The characteristics of the accuracy and efficiency of the HPC method are demonstrated by studying analytical cases. Comparisons will be made with some other existing boundary element based methods, e.g. Quadratic Boundary Element Method (QBEM) and the Fast Multipole Accelerated QBEM (FMA-QBEM) and a fourth order Finite Difference Method (FDM). To demonstrate the applications of the method, it is applied to some studies relevant for marine hydrodynamics. Sloshing in 3D rectangular tanks, a fully-nonlinear numerical wave tank, fully-nonlinear wave focusing on a semi-circular shoal, and the nonlinear wave diffraction of a bottom-mounted cylinder in regular waves are studied. The comparisons with the experimental results and other numerical results are all in satisfactory agreement, indicating that the present HPC method is a promising method in solving potential-flow problems. The underlying procedure of the HPC method could also be useful in other fields than marine hydrodynamics involved with solving Laplace equation.

  19. Fabrication and evaluation of electrohydrodynamic jet 3D printed polycaprolactone/chitosan cell carriers using human embryonic stem cell-derived fibroblasts.

    Science.gov (United States)

    Wu, Yang; Sriram, Gopu; Fawzy, Amr S; Fuh, Jerry Yh; Rosa, Vinicius; Cao, Tong; Wong, Yoke San

    2016-08-01

    Biological function of adherent cells depends on the cell-cell and cell-matrix interactions in three-dimensional space. To understand the behavior of cells in 3D environment and their interactions with neighboring cells and matrix requires 3D culture systems. Here, we present a novel 3D cell carrier scaffold that provides an environment for routine 3D cell growth in vitro We have developed thin, mechanically stable electrohydrodynamic jet (E-jet) 3D printed polycaprolactone and polycaprolactone/Chitosan macroporous scaffolds with precise fiber orientation for basic 3D cell culture application. We have evaluated the application of this technology by growing human embryonic stem cell-derived fibroblasts within these 3D scaffolds. Assessment of cell viability and proliferation of cells seeded on polycaprolactone and polycaprolactone/Chitosan 3D-scaffolds show that the human embryonic stem cell-derived fibroblasts could adhere and proliferate on the scaffolds over time. Further, using confocal microscopy we demonstrate the ability to use fluorescence-labelled cells that could be microscopically monitored in real-time. Hence, these 3D printed polycaprolactone and polycaprolactone/Chitosan scaffolds could be used as a cell carrier for in vitro 3D cell culture-, bioreactor- and tissue engineering-related applications in the future. PMID:27252227

  20. AC electric field induced dipole-based on-chip 3D cell rotation.

    Science.gov (United States)

    Benhal, Prateek; Chase, J Geoffrey; Gaynor, Paul; Oback, Björn; Wang, Wenhui

    2014-08-01

    The precise rotation of suspended cells is one of the many fundamental manipulations used in a wide range of biotechnological applications such as cell injection and enucleation in nuclear transfer (NT) cloning. Noticeably scarce among the existing rotation techniques is the three-dimensional (3D) rotation of cells on a single chip. Here we present an alternating current (ac) induced electric field-based biochip platform, which has an open-top sub-mm square chamber enclosed by four sidewall electrodes and two bottom electrodes, to achieve rotation about the two axes, thus 3D cell rotation. By applying an ac potential to the four sidewall electrodes, an in-plane (yaw) rotating electric field is generated and in-plane rotation is achieved. Similarly, by applying an ac potential to two opposite sidewall electrodes and the two bottom electrodes, an out-of-plane (pitch) rotating electric field is generated and rolling rotation is achieved. As a prompt proof-of-concept, bottom electrodes were constructed with transparent indium tin oxide (ITO) using the standard lift-off process and the sidewall electrodes were constructed using a low-cost micro-milling process and then assembled to form the chip. Through experiments, we demonstrate rotation of bovine oocytes of ~120 μm diameter about two axes, with the capability of controlling the rotation direction and the rate for each axis through control of the ac potential amplitude, frequency, and phase shift, and cell medium conductivity. The maximum observed rotation rate reached nearly 140° s⁻¹, while a consistent rotation rate reached up to 40° s⁻¹. Rotation rate spectra for zona pellucida-intact and zona pellucida-free oocytes were further compared and found to have no effective difference. This simple, transparent, cheap-to-manufacture, and open-top platform allows additional functional modules to be integrated to become a more powerful cell manipulation system.

  1. Engineering a perfusable 3D human liver platform from iPS cells.

    Science.gov (United States)

    Schepers, Arnout; Li, Cheri; Chhabra, Arnav; Seney, Benjamin Tschudy; Bhatia, Sangeeta

    2016-07-01

    In vitro models of human tissue are crucial to our ability to study human disease as well as develop safe and effective drug therapies. Models of single organs in static and microfluidic culture have been established and shown utility for modeling some aspects of health and disease; however, these systems lack multi-organ interactions that are critical to some aspects of drug metabolism and toxicity. Thus, as part of a consortium of researchers, we have developed a liver chip that meets the following criteria: (1) employs human iPS cells from a patient of interest, (2) cultures cells in perfusable 3D organoids, and (3) is robust to variations in perfusion rate so as to be compatible in series with other specialized tissue chips (e.g. heart, lung). In order to achieve this, we describe methods to form hepatocyte aggregates from primary and iPS-derived cells, alone and in co-culture with support cells. This necessitated a novel culture protocol for the interrupted differentiation of iPS cells that permits their removal from a plated surface and aggregation while maintaining phenotypic hepatic functions. In order to incorporate these 3D aggregates in a perfusable platform, we next encapsulated the cells in a PEG hydrogel to prevent aggregation and overgrowth once on chip. We adapted a C-trap chip architecture from the literature that enabled robust loading with encapsulated organoids and culture over a range of flow rates. Finally, we characterize the liver functions of this iHep organoid chip under perfusion and demonstrate a lifetime of at least 28 days. We envision that such this strategy can be generalized to other microfluidic tissue models and provides an opportunity to query patient-specific liver responses in vitro. PMID:27296616

  2. One-step fabrication of 3D silver paste electrodes into microfluidic devices for enhanced droplet-based cell sorting

    Directory of Open Access Journals (Sweden)

    Lang Rao

    2015-05-01

    Full Text Available 3D microelectrodes are one-step fabricated into a microfluidic droplet separator by filling conductive silver paste into PDMS microchambers. The advantages of 3D silver paste electrodes in promoting droplet sorting accuracy are systematically demonstrated by theoretical calculation, numerical simulation and experimental validation. The employment of 3D electrodes also helps to decrease the droplet sorting voltage, guaranteeing that cells encapsulated in droplets undergo chip-based sorting processes are at better metabolic status for further potential cellular assays. At last, target droplet containing single cell are selectively sorted out from others by an appropriate electric pulse. This method provides a simple and inexpensive alternative to fabricate 3D electrodes, and it is expected our 3D electrode-integrated microfluidic droplet separator platform can be widely used in single cell operation and analysis.

  3. Accessible bioprinting: adaptation of a low-cost 3D-printer for precise cell placement and stem cell differentiation.

    Science.gov (United States)

    Reid, John A; Mollica, Peter A; Johnson, Garett D; Ogle, Roy C; Bruno, Robert D; Sachs, Patrick C

    2016-06-01

    The precision and repeatability offered by computer-aided design and computer-numerically controlled techniques in biofabrication processes is quickly becoming an industry standard. However, many hurdles still exist before these techniques can be used in research laboratories for cellular and molecular biology applications. Extrusion-based bioprinting systems have been characterized by high development costs, injector clogging, difficulty achieving small cell number deposits, decreased cell viability, and altered cell function post-printing. To circumvent the high-price barrier to entry of conventional bioprinters, we designed and 3D printed components for the adaptation of an inexpensive 'off-the-shelf' commercially available 3D printer. We also demonstrate via goal based computer simulations that the needle geometries of conventional commercially standardized, 'luer-lock' syringe-needle systems cause many of the issues plaguing conventional bioprinters. To address these performance limitations we optimized flow within several microneedle geometries, which revealed a short tapered injector design with minimal cylindrical needle length was ideal to minimize cell strain and accretion. We then experimentally quantified these geometries using pulled glass microcapillary pipettes and our modified, low-cost 3D printer. This systems performance validated our models exhibiting: reduced clogging, single cell print resolution, and maintenance of cell viability without the use of a sacrificial vehicle. Using this system we show the successful printing of human induced pluripotent stem cells (hiPSCs) into Geltrex and note their retention of a pluripotent state 7 d post printing. We also show embryoid body differentiation of hiPSC by injection into differentiation conducive environments, wherein we observed continuous growth, emergence of various evaginations, and post-printing gene expression indicative of the presence of all three germ layers. These data demonstrate an

  4. High power density microbial fuel cell with flexible 3D graphene-nickel foam as anode

    Science.gov (United States)

    Wang, Hanyu; Wang, Gongming; Ling, Yichuan; Qian, Fang; Song, Yang; Lu, Xihong; Chen, Shaowei; Tong, Yexiang; Li, Yat

    2013-10-01

    The structure and electrical conductivity of anode play a significant role in the power generation of microbial fuel cells (MFCs). In this study, we developed a three-dimensional (3D) reduced graphene oxide-nickel (denoted as rGO-Ni) foam as an anode for MFC through controlled deposition of rGO sheets onto the nickel foam substrate. The loading amount of rGO sheets and electrode surface area can be controlled by the number of rGO loading cycles. 3D rGO-Ni foam anode provides not only a large accessible surface area for microbial colonization and electron mediators, but also a uniform macro-porous scaffold for effective mass diffusion of the culture medium. Significantly, at a steady state of the power generation, the MFC device with flexible rGO-Ni electrodes produced an optimal volumetric power density of 661 W m-3 calculated based on the volume of anode material, or 27 W m-3 based on the volume of the anode chamber. These values are substantially higher than that of plain nickel foam, and other conventional carbon based electrodes (e.g., carbon cloth, carbon felt, and carbon paper) measured in the same conditions. To our knowledge, this is the highest volumetric power density reported for mL-scale MFC device with a pure strain of Shewanella oneidensis MR-1. We also demonstrated that the MFC device can be operated effectively in a batch-mode at least for a week. These new 3D rGO-Ni electrodes show great promise for improving the power generation of MFC devices.The structure and electrical conductivity of anode play a significant role in the power generation of microbial fuel cells (MFCs). In this study, we developed a three-dimensional (3D) reduced graphene oxide-nickel (denoted as rGO-Ni) foam as an anode for MFC through controlled deposition of rGO sheets onto the nickel foam substrate. The loading amount of rGO sheets and electrode surface area can be controlled by the number of rGO loading cycles. 3D rGO-Ni foam anode provides not only a large accessible

  5. Enzyme separation techniques for the study of growth of cells from layers of bovine dental pulp.

    Science.gov (United States)

    Miller, W A; Everett, M M; Freedman, J T; Feagans, W C; Cramer, J F

    1976-08-01

    Effects of the enzymes trypsin, papain, bromelains and ficin on bovine dental pulp tissue were studied. Minced or whole pulps were subjected to each enzyme at 17 degrees, 20 degrees and 37 degrees C for set time intervals, after which aliquots of supernatant fluid were removed for cell counts and viability tests. Pooled samples were subsequently cultured as monolayers in Eagle's MEM plus 10% calf serum. The dissociation characteristics were quite distinct for each enzyme, although quite similar between minced and whole pulp. A parallel histological study was made of the residual pulp tissue. Ficin was found to be the most suitable enzyme for future studies on the growth of isolated pulp cells from various layers of the bovine pulp, due to its even rate of cell removal, and the good initial viability and subsequent growth of the separated cells in monolayer culture. Further studies on ficin may show that it is more suitable for enzymatic separation of tissues generally than the more commonly used trypsin, a major advantage being its use in media containing Ca2+ and Mg2+.

  6. Temperature distributions in the laser-heated diamond anvil cell from 3-D numerical modeling

    International Nuclear Information System (INIS)

    We present TempDAC, a 3-D numerical model for calculating the steady-state temperature distribution for continuous wave laser-heated experiments in the diamond anvil cell. TempDAC solves the steady heat conduction equation in three dimensions over the sample chamber, gasket, and diamond anvils and includes material-, temperature-, and direction-dependent thermal conductivity, while allowing for flexible sample geometries, laser beam intensity profile, and laser absorption properties. The model has been validated against an axisymmetric analytic solution for the temperature distribution within a laser-heated sample. Example calculations illustrate the importance of considering heat flow in three dimensions for the laser-heated diamond anvil cell. In particular, we show that a “flat top” input laser beam profile does not lead to a more uniform temperature distribution or flatter temperature gradients than a wide Gaussian laser beam

  7. Additive manufactured polymeric 3D scaffolds with tailored surface topography influence mesenchymal stromal cells activity.

    Science.gov (United States)

    Neves, Sara C; Mota, Carlos; Longoni, Alessia; Barrias, Cristina C; Granja, Pedro L; Moroni, Lorenzo

    2016-06-01

    Additive manufactured three-dimensional (3D) scaffolds with tailored surface topography constitute a clear advantage in tissue regeneration strategies to steer cell behavior. 3D fibrous scaffolds of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) block copolymer presenting different fiber surface features were successfully fabricated by additive manufacturing combined with wet-spinning, in a single step, without any post-processing. The optimization of the processing parameters, mainly driven by different solvent/non-solvent combinations, led to four distinct scaffold types, with average surface roughness values ranging from 0.071 ± 0.012 μm to 1.950 ± 0.553 μm, average pore sizes in the x- and y-axis between 351.1 ± 33.6 μm and 396.1 ± 32.3 μm, in the z-axis between 36.5 ± 5.3 μm and 70.7 ± 8.8 μm, average fiber diameters between 69.4 ± 6.1 μm and 99.0 ± 9.4 μm, and porosity values ranging from 60.2 ± 0.8% to 71.7 ± 2.6%. Human mesenchymal stromal cells (hMSCs) cultured on these scaffolds adhered, proliferated, and produced endogenous extracellular matrix. The effect of surface roughness and topography on hMSCs differentiation was more evident for cells seeded at lower density, where the percentage of cells in direct contact with the surface was higher compared to more densely seeded scaffolds. Under osteogenic conditions, lower surface roughness values (0.227 ± 0.035 μm) had a synergistic effect on hMSCs behavior, while chondrogenesis was favored on rougher surfaces (1.950 ± 0.553 μm). PMID:27219645

  8. Dynamic 3D cell rearrangements guided by a fibronectin matrix underlie somitogenesis.

    Directory of Open Access Journals (Sweden)

    Gabriel G Martins

    Full Text Available Somites are transient segments formed in a rostro-caudal progression during vertebrate development. In chick embryos, segmentation of a new pair of somites occurs every 90 minutes and involves a mesenchyme-to-epithelium transition of cells from the presomitic mesoderm. Little is known about the cellular rearrangements involved, and, although it is known that the fibronectin extracellular matrix is required, its actual role remains elusive. Using 3D and 4D imaging of somite formation we discovered that somitogenesis consists of a complex choreography of individual cell movements. Epithelialization starts medially with the formation of a transient epithelium of cuboidal cells, followed by cell elongation and reorganization into a pseudostratified epithelium of spindle-shaped epitheloid cells. Mesenchymal cells are then recruited to this medial epithelium through accretion, a phenomenon that spreads to all sides, except the lateral side of the forming somite, which epithelializes by cell elongation and intercalation. Surprisingly, an important contribution to the somite epithelium also comes from the continuous egression of mesenchymal cells from the core into the epithelium via its apical side. Inhibition of fibronectin matrix assembly first slows down the rate, and then halts somite formation, without affecting pseudopodial activity or cell body movements. Rather, cell elongation, centripetal alignment, N-cadherin polarization and egression are impaired, showing that the fibronectin matrix plays a role in polarizing and guiding the exploratory behavior of somitic cells. To our knowledge, this is the first 4D in vivo recording of a full mesenchyme-to-epithelium transition. This approach brought new insights into this event and highlighted the importance of the extracellular matrix as a guiding cue during morphogenesis.

  9. Complete factorial design experiment for 3D load cell instrumented crank validation.

    Science.gov (United States)

    Omar, Valle-Casas; Rafael, Dalazen; Vinicius, Cene; Alexandre, Balbinot

    2015-08-01

    Developing of instrumentation systems for sport medicine is a promising area, that's why this research evaluates the design of a new instrumented crank arm prototype for a race bicycle projecting an experiment for indoor - outdoor comparison. This study investigated the viability of an instrumentation 3D load cell for force measurement crank, implementing a design of experiment. A Complete factorial design experiment was developed for data validation, with an Analysis of Variance (ANOVA) throwing significant results for controlled factors with response variables rms, mean and variance. A software routine allowed to obtained system variables metrics for Symmetry and Cadence analysis, which came out from Effective force bilateral comparing and speed computation. Characterization allowed achieving calibration curves that were used for data conversion in force projection channels with a linearity error of 0.29% (perpendicular), 0.55% (parallel) and 0.10% (lateral). Interactions of factors resulted significant mainly for indoor tests in symmetry and cadence was significant in interactions generally for outdoor tests. Implemented system was able to generate Effective Force graph for 3D plot symmetry analysis, torque and power symmetry for specialist's analysis. PMID:26737085

  10. A MULTISCALE APPROACH TO THE REPRESENTATION OF 3D IMAGES, WITH APPLICATION TO POLYMER SOLAR CELLS

    Directory of Open Access Journals (Sweden)

    Ralf Thiedmann

    2011-03-01

    Full Text Available A multiscale approach to the description of geometrically complex 3D image data is proposed which distinguishes between morphological features on a ‘macro-scale’ and a ‘micro-scale’. Since our method is mainly tailored to nanostructures observed in composite materials consisting of two different phases, an appropriate binarization of grayscale images is required first. Then, a morphological smoothing is applied to extract the structural information from binarized image data on the ‘macro-scale’. A stochastic algorithm is developed for the morphologically smoothed images whose goal is to find a suitable representation of the macro-scale structure by unions of overlapping spheres. Such representations can be interpreted as marked point patterns. They lead to an enormous reduction of data and allow the application of well-known tools from point-process theory for their analysis and structural modeling. All those voxels which have been ‘misspecified’ by the morphological smoothing and subsequent representation by unions of overlapping spheres are interpreted as ‘micro-scale’ structure. The exemplary data sets considered in this paper are 3D grayscale images of photoactive layers in hybrid solar cells gained by electron tomography. These composite materials consist of two phases: a polymer phase and a zinc oxide phase. The macro-scale structure of the latter is represented by unions of overlapping spheres.

  11. Challenges of stem cell-based pulp and dentin regeneration: a clinical perspective

    OpenAIRE

    Huang, George T.-J.; Al-Habib, Mey; Gauthier, Philippe

    2013-01-01

    There are two types of approaches to regenerate tissues: cell-based and cell-free. The former approach is to introduce exogenous cells into the host to regenerate tissues, and the latter is to use materials other than cells in an attempt to regenerate tissues. There has been a significant advancement in stem cell-based pulp and dentin regeneration research in the past few years. Studies in small and large animals have demonstrated that pulp/dentin-like tissues can be regenerated partially or ...

  12. 3D Printing Bioceramic Porous Scaffolds with Good Mechanical Property and Cell Affinity.

    Directory of Open Access Journals (Sweden)

    Chih-Hao Chang

    Full Text Available Artificial bone grafting is widely used in current orthopedic surgery for bone defect problems. Unfortunately, surgeons remain unsatisfied with the current commercially available products. One of the major complaints is that these products cannot provide sufficient mechanical strength to support the human skeletal structure. In this study, we aimed to develop a bone scaffold with better mechanical property and good cell affinity by 3D printing (3DP techniques. A self-developed 3D printer with laser-aided gelling (LAG process was used to fabricate bioceramic scaffolds with inter-porous structures. To improve the mechanical property of the bioceramic parts after heating, CaCO3 was added to the silica ceramic slurry. CaCO3 was blended into a homogenous SiO2-sol dispersion at weight ratios varying from 0/100 to 5/95 to 9/91 (w/w. Bi-component CaCO3/SiO2-sol was prepared as a biocomposite for the 3DP scaffold. The well-mixed biocomposite was used to fabricate the bioceramic green part using the LAG method. The varied scaffolds were sintered at different temperatures ranging from 900 to 1500°C, and the mechanical property was subsequently analyzed. The scaffolds showed good property with the composite ratio of 5:95 CaCO3:SiO2 at a sintering temperature of 1300°C. The compressive strength was 47 MPa, and the porosity was 34%. The topography of the sintered 3DP bioceramic scaffold was examined by SEM, EDS and XRD. The silica bioceramic presented no cytotoxicity and good MG-63 osteoblast-like cell affinity, demonstrating good biocompatibility. Therefore, the new silica biocomposite is viable for fabricating 3DP bone bioceramics with improved mechanical property and good cell affinity.

  13. 3D Printing Bioceramic Porous Scaffolds with Good Mechanical Property and Cell Affinity.

    Science.gov (United States)

    Chang, Chih-Hao; Lin, Chih-Yang; Liu, Fwu-Hsing; Chen, Mark Hung-Chih; Lin, Chun-Pin; Ho, Hong-Nerng; Liao, Yunn-Shiuan

    2015-01-01

    Artificial bone grafting is widely used in current orthopedic surgery for bone defect problems. Unfortunately, surgeons remain unsatisfied with the current commercially available products. One of the major complaints is that these products cannot provide sufficient mechanical strength to support the human skeletal structure. In this study, we aimed to develop a bone scaffold with better mechanical property and good cell affinity by 3D printing (3DP) techniques. A self-developed 3D printer with laser-aided gelling (LAG) process was used to fabricate bioceramic scaffolds with inter-porous structures. To improve the mechanical property of the bioceramic parts after heating, CaCO3 was added to the silica ceramic slurry. CaCO3 was blended into a homogenous SiO2-sol dispersion at weight ratios varying from 0/100 to 5/95 to 9/91 (w/w). Bi-component CaCO3/SiO2-sol was prepared as a biocomposite for the 3DP scaffold. The well-mixed biocomposite was used to fabricate the bioceramic green part using the LAG method. The varied scaffolds were sintered at different temperatures ranging from 900 to 1500°C, and the mechanical property was subsequently analyzed. The scaffolds showed good property with the composite ratio of 5:95 CaCO3:SiO2 at a sintering temperature of 1300°C. The compressive strength was 47 MPa, and the porosity was 34%. The topography of the sintered 3DP bioceramic scaffold was examined by SEM, EDS and XRD. The silica bioceramic presented no cytotoxicity and good MG-63 osteoblast-like cell affinity, demonstrating good biocompatibility. Therefore, the new silica biocomposite is viable for fabricating 3DP bone bioceramics with improved mechanical property and good cell affinity.

  14. Effects of hTERT immortalization on osteogenic and adipogenic differentiation of dental pulp stem cells

    Directory of Open Access Journals (Sweden)

    Ikbale El-Ayachi

    2016-03-01

    Full Text Available These data relate to the differentiation of human dental pulp stem cells (DPSC and DPSC immortalized by constitutively expressing human telomerase reverse transcriptase (hTERT through both osteogenic and adipogenic lineages (i.e. to make bone producing and fat producing cells from these dental pulp stem cells. The data augment another study to characterize immortalized DPSC for the study of neurogenetic “Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders” [1]. Two copies of one typical control cell line (technical replicates were used in this study. The data represent the differentiation of primary DPSC into osteoblast cells approximately 60% more effectively than hTERT immortalized DPSC. Conversely, both primary and immortalized DPSC are poorly differentiated into adipocytes. The mRNA expression levels for both early and late adipogenic and osteogenic gene markers are shown.

  15. Metabolic response of lung cancer cells to radiation in a paper-based 3D cell culture system.

    Science.gov (United States)

    Simon, Karen A; Mosadegh, Bobak; Minn, Kyaw Thu; Lockett, Matthew R; Mohammady, Marym R; Boucher, Diane M; Hall, Amy B; Hillier, Shawn M; Udagawa, Taturo; Eustace, Brenda K; Whitesides, George M

    2016-07-01

    This work demonstrates the application of a 3D culture system-Cells-in-Gels-in-Paper (CiGiP)-in evaluating the metabolic response of lung cancer cells to ionizing radiation. The 3D tissue-like construct-prepared by stacking multiple sheets of paper containing cell-embedded hydrogels-generates a gradient of oxygen and nutrients that decreases monotonically in the stack. Separating the layers of the stack after exposure enabled analysis of the cellular response to radiation as a function of oxygen and nutrient availability; this availability is dictated by the distance between the cells and the source of oxygenated medium. As the distance between the cells and source of oxygenated media increased, cells show increased levels of hypoxia-inducible factor 1-alpha, decreased proliferation, and reduced sensitivity to ionizing radiation. Each of these cellular responses are characteristic of cancer cells observed in solid tumors. With this setup we were able to differentiate three isogenic variants of A549 cells based on their metabolic radiosensitivity; these three variants have known differences in their metastatic behavior in vivo. This system can, therefore, capture some aspects of radiosensitivity of populations of cancer cells related to mass-transport phenomenon, carry out systematic studies of radiation response in vitro that decouple effects from migration and proliferation of cells, and regulate the exposure of oxygen to subpopulations of cells in a tissue-like construct either before or after irradiation. PMID:27116031

  16. SURVIVAL OF LIVER CELLS, IMMOBILIZED ON 3D-MATRIXES, IN LIVER FAILURE MODEL

    Directory of Open Access Journals (Sweden)

    M. Y. Shagidulin

    2011-01-01

    Full Text Available It was examined a new method for correction of hepatic failure by transplantation of liver support biounit (liver cells, immobilized on biocompatible and biodegradable 3D-matrixes ElastoPOB® into small intestine mesentery. It was determined that after modeling of acute hepatic failure on dogs by 65–70% liver resection and transplantation liver support biounit the restoration of disturbed biochemical indecies (such as total protein, lactate, cytolytic ensymes-ALT, AST, ALP, LDH, fibrinogen, protrombine index and others took place more rapidly on 9–14th day instead of 18th day in control. It was made a preposition about efficiency of the suggested method for correction both acute hepatic failure because even 90 days after transplantation of liver support biounit alive hepatocytes and neogenic plethoric vessels, growing through matrix were revealed. 

  17. Novel carbocyclic curcumin analog CUR3d modulates genes involved in multiple apoptosis pathways in human hepatocellular carcinoma cells.

    Science.gov (United States)

    Bhullar, Khushwant S; Jha, Amitabh; Rupasinghe, H P Vasantha

    2015-12-01

    Anticancer activity of a novel curcumin analog (E)-2-(4-hydroxy-3-methoxybenzylidene)-5-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)cyclopentanone (CUR3d) was studied using a human hepatocellular carcinoma cell line (HepG2). The results showed that CUR3d completely inhibits the tumor cell proliferation in a dose- and time-dependent manner. CUR3d at 100 μmol/L activated the pro-apoptotic caspase-3 along with downregulation of anti-apoptotic BIRC5 and Bcl2. CUR3d treatment controlled the cancer cell growth by downregulating the expression of PI3K/Akt (Akt1, Akt2) pathway along with NF-κB. CUR3d down-regulated the members of epidermal growth receptor family (EGFR, ERBB3, ERBB2) and insulin like growth receptors (IGF1, IGF-1R, IGF2). This correlated with the downregulation of G-protein (RHOA, RHOB) and RAS (ATF2, HRAS, KRAS, NRAS) pathway signaling. CUR3d also arrested cell cycle via inhibition of CDK2, CDK4, CDK5, CDK9, MDM2, MDM4 and TERT genes. Cell cycle essential aurora kinases (AURKα, AURKβ) and polo-like kinases (PLK1, PLK2, PLK3) were also modulated by CUR3d. Topoisomerases (TOP2α, TOP2β), important factors in cancer cell immortality, as well as HIF-1α were downregulated following CUR3d treatment. The expression of protein kinase-C family (PRKC-A, PRKC-D, PRKC-E) was also attenuated by CUR3d. The downregulation of histone deacetylases (Class I, II, IV) and PARP I further strengthened the anticancer efficacy of CUR3d. Downregulation of carcinogenic cathepsins (CTSB, CTSD) and heat shock proteins exhibited CUR3d's potency as a potential immunological adjuvant. Finally, the non-toxic manifestation of CUR3d in healthy liver and lung cells along with downregulation of drug resistant gene ABCC1 further warrant need for advance investigations. PMID:26409325

  18. Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells.

    Science.gov (United States)

    Ouyang, Liliang; Yao, Rui; Zhao, Yu; Sun, Wei

    2016-01-01

    3D cell printing is an emerging technology for fabricating complex cell-laden constructs with precise and pre-designed geometry, structure and composition to overcome the limitations of 2D cell culture and conventional tissue engineering scaffold technology. This technology enables spatial manipulation of cells and biomaterials, also referred to as 'bioink', and thus allows study of cellular interactions in a 3D microenvironment and/or in the formation of functional tissues and organs. Recently, many efforts have been made to develop new bioinks and to apply more cell sources for better biocompatibility and biofunctionality. However, the influences of printing parameters on the shape fidelity of 3D constructs as well as on cell viability after the cell printing process have been poorly characterized. Furthermore, parameter optimization based on a specific cell type might not be suitable for other types of cells, especially cells with high sensibility. In this study, we systematically studied the influence of bioink properties and printing parameters on bioink printability and embryonic stem cell (ESC) viability in the process of extrusion-based cell printing, also known as bioplotting. A novel method was established to determine suitable conditions for bioplotting ESCs to achieve both good printability and high cell viability. The rheological properties of gelatin/alginate bioinks were evaluated to determine the gelation properties under different bioink compositions, printing temperatures and holding times. The bioink printability was characterized by a newly developed semi-quantitative method. The results demonstrated that bioinks with longer gelation times would result in poorer printability. The live/dead assay showed that ESC viability increased with higher printing temperatures and lower gelatin concentrations. Furthermore, an exponential relationship was obtained between ESC viability and induced shear stress. By defining the proper printability and

  19. Using Polymer Confinement for Stem Cell Differentiation: 3D Printed vs Molded Scaffolds

    Science.gov (United States)

    Rafailovich, Miriam

    Additive manufacturing technologies are increasingly being used to replace standard extrusion or molding methods in engineering polymeric biomedical implants, which can be further seeded with cells for tissue regeneration. The principal advantage of this new technology is the ability to print directly from a scan and hence produce parts which are an ideal fit for an individual, eliminating much of the sizing and fitting associated with standard manufacturing methods. The question though arises whether devices which may be macroscopically similar, serve identical functions and are produced from the same material, interact in the same manner with cells and living tissue. Here we show that fundamental differences can exist between 3-D printed and extruded scaffolds which can impact stem cell differentiation and lineage selection. We will show how polymer confinement inherent in these methods affect the printed features on multiple length scales. We will also and how the differentiation of stem cells is affected by substrate heterogeneity in both morphological and mechanical features. NSF-Inspire award # 1344267.

  20. Improved Human Bone Marrow Mesenchymal Stem Cell Osteogenesis in 3D Bioprinted Tissue Scaffolds with Low Intensity Pulsed Ultrasound Stimulation.

    Science.gov (United States)

    Zhou, Xuan; Castro, Nathan J; Zhu, Wei; Cui, Haitao; Aliabouzar, Mitra; Sarkar, Kausik; Zhang, Lijie Grace

    2016-09-06

    3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm(2) intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application.

  1. Improved Human Bone Marrow Mesenchymal Stem Cell Osteogenesis in 3D Bioprinted Tissue Scaffolds with Low Intensity Pulsed Ultrasound Stimulation.

    Science.gov (United States)

    Zhou, Xuan; Castro, Nathan J; Zhu, Wei; Cui, Haitao; Aliabouzar, Mitra; Sarkar, Kausik; Zhang, Lijie Grace

    2016-01-01

    3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm(2) intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application. PMID:27597635

  2. Improved Human Bone Marrow Mesenchymal Stem Cell Osteogenesis in 3D Bioprinted Tissue Scaffolds with Low Intensity Pulsed Ultrasound Stimulation

    Science.gov (United States)

    Zhou, Xuan; Castro, Nathan J.; Zhu, Wei; Cui, Haitao; Aliabouzar, Mitra; Sarkar, Kausik; Zhang, Lijie Grace

    2016-01-01

    3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm2 intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application. PMID:27597635

  3. Development of bioartificial myocardium by electrostimulation of 3D collagen scaffolds seeded with stem cells

    Directory of Open Access Journals (Sweden)

    Alain Carpentier

    2012-06-01

    Full Text Available Electrostimulation (ES can be defined as a safe physical method to induce stem cell differentiation. The aim of this study is to evaluate the effectiveness of ES on bone marrow mesenchymal stem cells (BMSCs seeded in collagen scaffolds in terms of proliferation and differentiation into cardiomyocytes. BMSCs were isolated from Wistar rats and seeded into 3D collagen type 1 templates measuring 25 x 25 x 6 mm. Bipolar in vitro ES was performed during 21 days. Electrical impedance and cell proliferation were measured. Expression of cardiac markers was assessed by immunocytochemistry. Viscoelasticity of collagen matrix was evaluated. Electrical impedance assessments showed a low resistance of 234±41 Ohms which indicates good electrical conductivity of collagen matrix. Cell proliferation at 570 nm as significantly increased in ES groups after seven day (ES 0.129±0.03 vs non-stimulated control matrix 0.06±0.01, P=0.002 and after 21 days, (ES 0.22±0.04 vs control 0.13±0.01, P=0.01. Immunocytochemistry of BMSCs after 21 days ES showed positive staining of cardiac markers, troponin I, connexin 43, sarcomeric alpha-actinin, slow myosin, fast myosin and desmin. Staining for BMSCs marker CD29 after 21 days was negative. Electrostimulation of cell-seeded collagen matrix changed stem cell morphology and bio- chemical characteristics, increasing the expression of cardiac markers. Thus, MSC-derived differentiated cells by electrostimulation grafted in biological scaffolds might result in a convenient tissue engineering source for myocardial diseases.

  4. Development of bioartificial myocardium by electrostimulation of 3D collagen scaffolds seeded with stem cells.

    Science.gov (United States)

    Haneef, Kanwal; Lila, Nermine; Benadda, Samira; Legrand, Fabien; Carpentier, Alain; Chachques, Juan C

    2012-06-01

    Electrostimulation (ES) can be defined as a safe physical method to induce stem cell differentiation. The aim of this study is to evaluate the effectiveness of ES on bone marrow mesenchymal stem cells (BMSCs) seeded in collagen scaffolds in terms of proliferation and differentiation into cardiomyocytes. BMSCs were isolated from Wistar rats and seeded into 3D collagen type 1 templates measuring 25 × 25 × 6 mm. Bipolar in vitro ES was performed during 21 days. Electrical impedance and cell proliferation were measured. Expression of cardiac markers was assessed by immunocytochemistry. Viscoelasticity of collagen matrix was evaluated. Electrical impedance assessments showed a low resistance of 234±41 Ohms which indicates good electrical conductivity of collagen matrix. Cell proliferation at 570 nm as significantly increased in ES groups after seven day (ES 0.129±0.03 vs non-stimulated control matrix 0.06±0.01, P=0.002) and after 21 days, (ES 0.22±0.04 vs control 0.13±0.01, P=0.01). Immunocytoche mistry of BMSCs after 21 days ES showed positive staining of cardiac markers, troponin I, connexin 43, sarcomeric alpha-actinin, slow myosin, fast myosin and desmin. Staining for BMSCs marker CD29 after 21 days was negative. Electrostimulation of cell-seeded collagen matrix changed stem cell morphology and biochemical characteristics, increasing the expression of cardiac markers. Thus, MSC-derived differentiated cells by electrostimulation grafted in biological scaffolds might result in a convenient tissue engineering source for myocardial diseases.

  5. A Hyaluronan-Based Scaffold for the in Vitro Construction of Dental Pulp-Like Tissue

    OpenAIRE

    Letizia Ferroni; Chiara Gardin; Stefano Sivolella; Giulia Brunello; Mario Berengo; Adriano Piattelli; Eriberto Bressan; Barbara Zavan

    2015-01-01

    Dental pulp tissue supports the vitality of the tooth, but it is particularly vulnerable to external insults, such as mechanical trauma, chemical irritation or microbial invasion, which can lead to tissue necrosis. In the present work, we present an endodontic regeneration method based on the use of a tridimensional (3D) hyaluronan scaffold and human dental pulp stem cells (DPSCs) to produce a functional dental pulp-like tissue in vitro. An enriched population of DPSCs was seeded onto hyalur...

  6. Histone deacetylase inhibitors induced differentiation and accelerated mineralization of pulp-derived cells.

    LENUS (Irish Health Repository)

    Duncan, Henry F

    2012-03-01

    Histone deacetylase inhibitors (HDACis) alter the homeostatic balance between 2 groups of cellular enzymes, histone deacetylases (HDACs) and histone acetyltransferases (HATs), increasing transcription and influencing cell behavior. This study investigated the potential of 2 HDACis, valproic acid (VPA) and trichostatin A (TSA), to promote reparative processes in pulp cells as assayed by viability, cell cycle, and mineralization analyses.

  7. THE COMPARISON OF STRENGTH PROPERTY BETWWEEN KRAFT PULP AND ALKALINE SULFITE-ANTHRAQUINONE PULP FOR THICKER CELL WALL FIBER MATERIALS

    Institute of Scientific and Technical Information of China (English)

    LiLi; FeifeiWang; YunzhanZhang

    2004-01-01

    The comparison of strength property between kraftpulp ( KP ) and Alkaline Sulfite-Anthraquinone(AS-AQ) pulp for thicker cell wall fiber materialsLarch and Quercus as examples was studied. Theaverage coefficient of flexibility of Larch andQuercus are 0.6-0.7 and 0.45-0.50, respectively. Theresults showed that the strength property of thickercell wall pulp is some what different from thosereported earlier. The strengths of AS-AQ are allhigher than those of KP for Larch and Quercus. ForLarch, under same beating degree the breaking lengthof AS-AQ is 8-16% higher than that of KP, burstindex 3-14% higher, folding endurance 30% higher,tear index slightly higher. For Quercus, the breakinglength of AS-AQ is 5-10% higher then that of KP,burst index 10-15% higher, folding endurance30-50% higher, tear index 5-15% higher. Under thesame breaking length the tear index of AS-AQ pulpis significantly higher than that of KP for both Larchand Quercus.

  8. Interfacing polymeric scaffolds with primary pancreatic ductal adenocarcinoma cells to develop 3D cancer models.

    Science.gov (United States)

    Ricci, Claudio; Mota, Carlos; Moscato, Stefania; D'Alessandro, Delfo; Ugel, Stefano; Sartoris, Silvia; Bronte, Vincenzo; Boggi, Ugo; Campani, Daniela; Funel, Niccola; Moroni, Lorenzo; Danti, Serena

    2014-01-01

    We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol)/gelatin (PVA/G) mixture and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer, were obtained via different techniques, namely, emulsion and freeze-drying, compression molding followed by salt leaching, and electrospinning. In this way, primary PDAC cells interfaced with different pore topographies, such as sponge-like pores of different shape and size or nanofiber interspaces. The aim of this study was to investigate the influence played by the scaffold architecture over cancerous cell growth and function. In all scaffolds, primary PDAC cells showed good viability and synthesized tumor-specific metalloproteinases (MMPs) such as MMP-2, and MMP-9. However, only sponge-like pores, obtained via emulsion-based and salt leaching-based techniques allowed for an organized cellular aggregation very similar to the native PDAC morphological structure. Differently, these cell clusters were not observed on PEOT/PBT electrospun scaffolds. MMP-2 and MMP-9, as active enzymes, resulted to be increased in PVA/G and PEOT/PBT sponges, respectively. These findings suggested that spongy scaffolds supported the generation of pancreatic tumor models with enhanced aggressiveness. In conclusion, primary PDAC cells showed diverse behaviors while interacting with different scaffold types that can be potentially exploited to create stage-specific pancreatic cancer models likely to provide new knowledge on the modulation and drug susceptibility of MMPs.

  9. A Novel Flow-Perfusion Bioreactor Supports 3D Dynamic Cell Culture

    Directory of Open Access Journals (Sweden)

    Alexander M. Sailon

    2009-01-01

    Full Text Available Background. Bone engineering requires thicker three-dimensional constructs than the maximum thickness supported by standard cell-culture techniques (2 mm. A flow-perfusion bioreactor was developed to provide chemotransportation to thick (6 mm scaffolds. Methods. Polyurethane scaffolds, seeded with murine preosteoblasts, were loaded into a novel bioreactor. Control scaffolds remained in static culture. Samples were harvested at days 2, 4, 6, and 8 and analyzed for cellular distribution, viability, metabolic activity, and density at the periphery and core. Results. By day 8, static scaffolds had a periphery cell density of 67%±5.0%, while in the core it was 0.3%±0.3%. Flow-perfused scaffolds demonstrated peripheral cell density of 94%±8.3% and core density of 76%±3.1% at day 8. Conclusions. Flow perfusion provides chemotransportation to thick scaffolds. This system may permit high throughput study of 3D tissues in vitro and enable prefabrication of biological constructs large enough to solve clinical problems.

  10. Improving organic tandem solar cells based on water-processed nanoparticles by quantitative 3D nanoimaging.

    Science.gov (United States)

    Pedersen, E B L; Angmo, D; Dam, H F; Thydén, K T S; Andersen, T R; Skjønsfjell, E T B; Krebs, F C; Holler, M; Diaz, A; Guizar-Sicairos, M; Breiby, D W; Andreasen, J W

    2015-08-28

    Organic solar cells have great potential for upscaling due to roll-to-roll processing and a low energy payback time, making them an attractive sustainable energy source for the future. Active layers coated with water-dispersible Landfester particles enable greater control of the layer formation and easier access to the printing industry, which has reduced the use of organic solvents since the 1980s. Through ptychographic X-ray computed tomography (PXCT), we image quantitatively a roll-to-roll coated photovoltaic tandem stack consisting of one bulk heterojunction active layer and one Landfester particle active layer. We extract the layered morphology with structural and density information including the porosity present in the various layers and the silver electrode with high resolution in 3D. The Landfester particle layer is found to have an undesired morphology with negatively correlated top- and bottom interfaces, wide thickness distribution and only partial surface coverage causing electric short circuits through the layer. By top coating a polymer material onto the Landfester nanoparticles we eliminate the structural defects of the layer such as porosity and roughness, and achieve the increased performance larger than 1 V expected for a tandem cell. This study highlights that quantitative imaging of weakly scattering stacked layers of organic materials has become feasible by PXCT, and that this information cannot be obtained by other methods. In the present study, this technique specifically reveals the need to improve the coatability and layer formation of Landfester nanoparticles, thus allowing improved solar cells to be produced. PMID:26220159

  11. X-ray tomography: Biological cells in 3-D at better than 50 nm resolution

    International Nuclear Information System (INIS)

    Full text: X-ray microscopy can be used to image whole, hydrated, specimens with a spatial resolution 5-10 times better than that obtained using visible light microscopy. X-ray imaging at photon energies below the K- absorption edge of oxygen, referred to as the water window, exploits the strong natural contrast for organic material embedded in a mostly water matrix. With a transmission X-ray microscope using Fresnel zone plate optics, specimens up to 10 microns thick can be examined. The highest X-ray transmission in hydrated samples is obtained at a wavelength of 2.4 nm but, due to the low numerical aperture of zone plate lenses operated in st order diffraction mode the structures resolved are much larger than the X-ray wavelength. Because of the low NA of X-ray lenses (NA=0.05), combined with the effect of polychromatic illumination and a wavelength dependant focal length, the effective depth of ld is large (6-10 microns). The experiments presented here were performed at the Advanced Light Source using the full ld transmission X-ray microscope, XM-1. This microscope employs a bend magnet X-ray source and zone plate condenser and objective lenses. The condenser zone plate acts as a monochromator and the X-ray images are recorded directly on a cooled, back-thinned 1024x1024 pixel CCD camera. The sample holder was a rotationally symmetric glass tube; the region containing the sample was 10 microns in diameter with a wall thickness of 200 nm. Live yeast cells were loaded into the tube, rapidly frozen by a blast of liquid nitrogen-cooled helium gas, and maintained at 140 deg C by a steady flow of cold helium. The image sequence spanned 180 deg and consisted of 45 images spaced by 4 deg. The images were aligned to a common axis and computed tomographic reconstruction was used to obtain the 3-D X-ray linear absorption coefficient. Volume rendering and animation of reconstructed data was performed using the 3-D program, Amira. Acquisition time for 90 images was 3 min

  12. Effect of sodium hypochlorite on human pulp cells: an in vitro study

    Science.gov (United States)

    Essner, Mark D.; Javed, Amjad; Eleazer, Paul D.

    2014-01-01

    Background The purpose of this study was to determine the effect of sodium hypochlorite (NaOCl) on human pulp cells to provide an aid in determining its optimum concentration in maintaining the viability of remaining pulp cells in the revascularization of immature permanent teeth with apical periodontitis. Study design Human pulp tissue cells taken from extracted third molars were plated, incubated, and subjected to various concentrations of NaOCl (0.33%, 0.16%, 0.08%, and 0.04%) for 5-, 10-, and 15-minute time intervals to simulate possible contact times in vivo. The Cell Titer–Glo Luminescent Cell Viability Assay was used to determine the number of viable cells present in culture following treatment. Results The results showed an increase in cell viability with the lowering of NaOCl concentration. The use of 0.04% NaOCl was similar to the control, indicating nearly complete preservation of cell viability at all time intervals tested. As sodium hypochlorite concentration increased from 0.04% to 0.33%, cell viability decreased correspondingly. Conclusions The results indicate that the lowest concentration of NaOCl tested did not affect the viability of cells. This may prove beneficial in developing a new treatment protocol to help preserve existing vital pulp cells in revascularization cases. PMID:21821446

  13. Heritable Genetic Changes in Cells Recovered From Irradiated 3D Tissue Contracts. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Cornforth, Michael N. [The University of Texas Medical Branch at Galveston, TX (United States)

    2013-05-03

    Combining contemporary cytogenetic methods with DNA CGH microarray technology and chromosome flow-sorting increases substantially the ability to resolve exchange breakpoints associated with interstitial deletions and translocations, allowing the consequences of radiation damage to be directly measured at low doses, while also providing valuable insights into molecular mechanisms of misrepair processes that, in turn, identify appropriate biophysical models of risk at low doses. The aims of this work apply to cells recovered from 3D tissue constructs of human skin and, for the purpose of comparison, the same cells irradiated in traditional 2D cultures. These aims are: to analyze by multi-flour fluorescence in situ hybridization (mFISH) the chromosomes in clonal descendents of individual human fibroblasts that were previously irradiated; to examine irradiated clones from Aim 1 for submicroscopic deletions by subjecting their DNA to comparative genomic hybridization (CGH) microarray analysis; and to flow-sort aberrant chromosomes from clones containing stable radiation-induced translocations and map the breakpoints to within an average resolution of 100 kb using the technique of 'array painting'.

  14. Tuning Cell Differentiation into a 3D Scaffold Presenting a Pore Shape Gradient for Osteochondral Regeneration.

    Science.gov (United States)

    Di Luca, Andrea; Lorenzo-Moldero, Ivan; Mota, Carlos; Lepedda, Antonio; Auhl, Dietmar; Van Blitterswijk, Clemens; Moroni, Lorenzo

    2016-07-01

    Osteochondral regeneration remains nowadays a major problem since the outcome of current techniques is not satisfactory in terms of functional tissue formation and development. A possible solution is the combination of human mesenchymal stem cells (hMSCs) with additive manufacturing technologies to fabricate scaffolds with instructive properties. In this study, the differentiation of hMSCs within a scaffold presenting a gradient in pore shape is presented. The variation in pore shape is determined by varying the angle formed by the fibers of two consequent layers. The fiber deposition patterns are 0-90, which generate squared pores, 0-45, 0-30, and 0-15, that generate rhomboidal pores with an increasing major axis as the deposition angle decreases. Within the gradient construct, squared pores support a better chondrogenic differentiation whereas cells residing in the rhomboidal pores display a better osteogenic differentiation. When cultured under osteochondral conditions the trend in both osteogenic and chondrogenic markers is maintained. Engineering the pore shape, thus creating axial gradients in structural properties, seems to be an instructive strategy to fabricate functional 3D scaffolds that are able to influence hMSCs differentiation for osteochondral tissue regeneration. PMID:27109461

  15. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming

    NARCIS (Netherlands)

    Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

    2016-01-01

    Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated doma

  16. Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture.

    Directory of Open Access Journals (Sweden)

    Michael S Weiss

    Full Text Available BACKGROUND: Extracellular activation of signal transduction pathways and their downstream target transcription factors (TFs are critical regulators of cellular processes and tissue development. The intracellular signaling network is complex, and techniques that quantify the activities of numerous pathways and connect their activities to the resulting phenotype would identify the signals and mechanisms regulating tissue development. The ability to investigate tissue development should capture the dynamic pathway activity and requires an environment that supports cellular organization into structures that mimic in vivo phenotypes. Taken together, our objective was to develop cellular arrays for dynamic, large-scale quantification of TF activity as cells organized into spherical structures within 3D culture. METHODOLOGY/PRINCIPAL FINDINGS: TF-specific and normalization reporter constructs were delivered in parallel to a cellular array containing a well-established breast cancer cell line cultured in Matrigel. Bioluminescence imaging provided a rapid, non-invasive, and sensitive method to quantify luciferase levels, and was applied repeatedly on each sample to monitor dynamic activity. Arrays measuring 28 TFs identified up to 19 active, with 13 factors changing significantly over time. Stimulation of cells with β-estradiol or activin A resulted in differential TF activity profiles evolving from initial stimulation of the ligand. Many TFs changed as expected based on previous reports, yet arrays were able to replicate these results in a single experiment. Additionally, arrays identified TFs that had not previously been linked with activin A. CONCLUSIONS/SIGNIFICANCE: This system provides a method for large-scale, non-invasive, and dynamic quantification of signaling pathway activity as cells organize into structures. The arrays may find utility for investigating mechanisms regulating normal and abnormal tissue growth, biomaterial design, or as a

  17. Osteogenic differentiation of dental pulp stem cells under the influence of three different materials

    DEFF Research Database (Denmark)

    Ajlan, S. A.; Ashri, N. Y.; Aldahmash, Abdullah M.;

    2015-01-01

    Background: Regeneration of periodontal tissues is a major goal of periodontal therapy. Dental pulp stem cells (DPSCs) show mesenchymal cell properties with the potential for dental tissue engineering. Enamel matrix derivative (EMD) and platelet-derived growth factor (PDGF) are examples...

  18. Mechanical Changes in Human Dental Pulp Stem Cells during Early Odontogenic Differentiation

    OpenAIRE

    Jones, Taneka D.; Naimipour, Hamed; Sun, Shan; Cho, Michael; Alapati, Satish B.

    2014-01-01

    Cell adhesion and migration in bioactive scaffolds require actin cytoskeleton remodeling and focal adhesion formation. Additionally, human dental pulp stem cells (hDPSCs) undergo several changes in their mechanical properties during odontogenic differentiation. The effect of factors essential for odontogenesis on the actin stress fiber elasticity and focal adhesion formation is not known.

  19. Chitosan-collagen biomembrane embedded with calcium-aluminate enhances dentinogenic potential of pulp cells.

    Science.gov (United States)

    Soares, Diana Gabriela; Rosseto, Hebert Luís; Basso, Fernanda Gonçalves; Scheffel, Débora Salles; Hebling, Josimeri; Costa, Carlos Alberto de Souza

    2016-01-01

    The development of biomaterials capable of driving dental pulp stem cell differentiation into odontoblast-like cells able to secrete reparative dentin is the goal of current conservative dentistry. In the present investigation, a biomembrane (BM) composed of a chitosan/collagen matrix embedded with calcium-aluminate microparticles was tested. The BM was produced by mixing collagen gel with a chitosan solution (2:1), and then adding bioactive calcium-aluminate cement as the mineral phase. An inert material (polystyrene) was used as the negative control. Human dental pulp cells were seeded onto the surface of certain materials, and the cytocompatibility was evaluated by cell proliferation and cell morphology, assessed after 1, 7, 14 and 28 days in culture. The odontoblastic differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, total protein production, gene expression of DMP-1/DSPP and mineralized nodule deposition. The pulp cells were able to attach onto the BM surface and spread, displaying a faster proliferative rate at initial periods than that of the control cells. The BM also acted on the cells to induce more intense ALP activity, protein production at 14 days, and higher gene expression of DSPP and DMP-1 at 28 days, leading to the deposition of about five times more mineralized matrix than the cells in the control group. Therefore, the experimental biomembrane induced the differentiation of pulp cells into odontoblast-like cells featuring a highly secretory phenotype. This innovative bioactive material can drive other protocols for dental pulp exposure treatment by inducing the regeneration of dentin tissue mediated by resident cells. PMID:27119587

  20. Chitosan-collagen biomembrane embedded with calcium-aluminate enhances dentinogenic potential of pulp cells

    Directory of Open Access Journals (Sweden)

    Diana Gabriela SOARES

    2016-01-01

    Full Text Available Abstract The development of biomaterials capable of driving dental pulp stem cell differentiation into odontoblast-like cells able to secrete reparative dentin is the goal of current conservative dentistry. In the present investigation, a biomembrane (BM composed of a chitosan/collagen matrix embedded with calcium-aluminate microparticles was tested. The BM was produced by mixing collagen gel with a chitosan solution (2:1, and then adding bioactive calcium-aluminate cement as the mineral phase. An inert material (polystyrene was used as the negative control. Human dental pulp cells were seeded onto the surface of certain materials, and the cytocompatibility was evaluated by cell proliferation and cell morphology, assessed after 1, 7, 14 and 28 days in culture. The odontoblastic differentiation was evaluated by measuring alkaline phosphatase (ALP activity, total protein production, gene expression of DMP-1/DSPP and mineralized nodule deposition. The pulp cells were able to attach onto the BM surface and spread, displaying a faster proliferative rate at initial periods than that of the control cells. The BM also acted on the cells to induce more intense ALP activity, protein production at 14 days, and higher gene expression of DSPP and DMP-1 at 28 days, leading to the deposition of about five times more mineralized matrix than the cells in the control group. Therefore, the experimental biomembrane induced the differentiation of pulp cells into odontoblast-like cells featuring a highly secretory phenotype. This innovative bioactive material can drive other protocols for dental pulp exposure treatment by inducing the regeneration of dentin tissue mediated by resident cells.

  1. A Bio-Acoustic Levitational (BAL) Assembly Method for Engineering of Multilayered, 3D Brain-Like Constructs, Using Human Embryonic Stem Cell Derived Neuro-Progenitors.

    Science.gov (United States)

    Bouyer, Charlène; Chen, Pu; Güven, Sinan; Demirtaş, Tuğrul Tolga; Nieland, Thomas J F; Padilla, Frédéric; Demirci, Utkan

    2016-01-01

    A bio-acoustic levitational assembly method for engineering of multilayered, 3D brainlike constructs is presented. Acoustic radiation forces are used to levitate neuroprogenitors derived from human embryonic stem cells in 3D multilayered fibrin tissue constructs. The neuro-progenitor cells are subsequently differentiated in neural cells, resulting in a 3D neuronal construct with inter and intralayer neurite elongations.

  2. Functional 3D Neural Mini-Tissues from Printed Gel-Based Bioink and Human Neural Stem Cells.

    Science.gov (United States)

    Gu, Qi; Tomaskovic-Crook, Eva; Lozano, Rodrigo; Chen, Yu; Kapsa, Robert M; Zhou, Qi; Wallace, Gordon G; Crook, Jeremy M

    2016-06-01

    Direct-write printing of stem cells within biomaterials presents an opportunity to engineer tissue for in vitro modeling and regenerative medicine. Here, a first example of constructing neural tissue by printing human neural stem cells that are differentiated in situ to functional neurons and supporting neuroglia is reported. The supporting biomaterial incorporates a novel clinically relevant polysaccharide-based bioink comprising alginate, carboxymethyl-chitosan, and agarose. The printed bioink rapidly gels by stable cross-linking to form a porous 3D scaffold encapsulating stem cells for in situ expansion and differentiation. Differentiated neurons form synaptic contacts, establish networks, are spontaneously active, show a bicuculline-induced increased calcium response, and are predominantly gamma-aminobutyric acid expressing. The 3D tissues will facilitate investigation of human neural development, function, and disease, and may be adaptable for engineering other 3D tissues from different stem cell types. PMID:27028356

  3. Biocompatibility and bioactivity of calcium silicate-based endodontic sealers in human dental pulp cells

    OpenAIRE

    Leticia Boldrin MESTIERI; GOMES-CORNÉLIO, Ana Lívia; RODRIGUES, Elisandra Márcia; SALLES, Loise Pedrosa; BOSSO-MARTELO, Roberta; Juliane Maria GUERREIRO-TANOMARU; TANOMARU-FILHO, Mário

    2015-01-01

    Mineral Trioxide Aggregate (MTA) is a calcium silicate-based material. New sealers have been developed based on calcium silicate as MTA Fillapex and MTA Plus. Objective The aim of this study was to evaluate biocompatibility and bioactivity of these two calcium silicate-based sealers in culture of human dental pulp cells (hDPCs). Material and Methods The cells were isolated from third molars extracted from a 16-year-old patient. Pulp tissue was sectioned into fragments with approximately 1 mm3...

  4. Effect of an Experimental Direct Pulp-capping Material on the Properties and Osteogenic Differentiation of Human Dental Pulp Stem Cells

    Science.gov (United States)

    Yu, Fan; Dong, Yan; Yang, Yan-wei; Lin, Ping-ting; Yu, Hao-han; Sun, Xiang; Sun, Xue-fei; Zhou, Huan; Huang, Li; Chen, Ji-hua

    2016-01-01

    Effective pulp-capping materials must have antibacterial properties and induce dentin bridge formation; however, many current materials do not satisfy clinical requirements. Accordingly, the effects of an experiment pulp-capping material (Exp) composed of an antibacterial resin monomer (MAE-DB) and Portland cement (PC) on the viability, adhesion, migration, and differentiation of human dental pulp stem cells (hDPSCs) were examined. Based on a Cell Counting Kit-8 assay, hDPSCs exposed to Exp extracts showed limited viability at 24 and 48 h, but displayed comparable viability to the control at 72 h. hDPSC treatment with Exp extracts enhanced cellular adhesion and migration according to in vitro scratch wound healing and Transwell migration assays. Exp significantly upregulated the expression of osteogenesis-related genes. The hDPSCs cultured with Exp exhibited higher ALP activity and calcium deposition in vitro compared with the control group. The novel material showed comparable cytocompatibility to control cells and promoted the adhesion, migration, and osteogenic differentiation of hDPSCs, indicating excellent biocompatibility. This new direct pulp-capping material containing MAE-DB and PC shows promise as a potential alternative to conventional materials for direct pulp capping. PMID:27698421

  5. Dental pulp stem cells as a multifaceted tool for bioengineering and the regeneration of craniomaxillofacial tissues

    OpenAIRE

    Aurrekoetxea, Maitane; Garcia-Gallastegui, Patricia; Irastorza, Igor; Luzuriaga, Jon; Uribe-Etxebarria, Verónica; Unda, Fernando; Ibarretxe, Gaskon

    2015-01-01

    Dental pulp stem cells, or DPSC, are neural crest-derived cells with an outstanding capacity to differentiate along multiple cell lineages of interest for cell therapy. In particular, highly efficient osteo/dentinogenic differentiation of DPSC can be achieved using simple in vitro protocols, making these cells a very attractive and promising tool for the future treatment of dental and periodontal diseases. Among craniomaxillofacial organs, the tooth and salivary gland are two such cases in wh...

  6. Dental Pulp Stem Cells as a multifaceted tool for bioengineering and the regeneration of craniomaxillofacial tissues

    OpenAIRE

    Maitane eAurrekoetxea; Patricia eGarcía-Gallastegui; Igor eIrastorza; Jon eLuzuriaga; Verónica eUribe-Etxebarria; Fernando eUnda; Gaskon eIbarretxe

    2015-01-01

    Dental pulp stem cells, or DPSC, are neural crest-derived cells with an outstanding capacity to differentiate along multiple cell lineages of interest for cell therapy. In particular, highly efficient osteo/dentinogenic differentiation of DPSC can be achieved using simple in vitro protocols, making these cells a very attractive and promising tool for the future treatment of dental and periodontal diseases. Among craniomaxillofacial organs, the tooth and salivary gland are two such cases in wh...

  7. Human Serum Promotes Osteogenic Differentiation of Human Dental Pulp Stem Cells In Vitro and In Vivo

    OpenAIRE

    Alessandra Pisciotta; Massimo Riccio; Gianluca Carnevale; Francesca Beretti; Lara Gibellini; Tullia Maraldi; Gian Maria Cavallini; Adriano Ferrari; Giacomo Bruzzesi; Anto De Pol

    2012-01-01

    Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS). FCS is known to contain a great quantity of growth factors, and thus to promote cell attachmen...

  8. Magnetic Nanocomposite Scaffold-Induced Stimulation of Migration and Odontogenesis of Human Dental Pulp Cells through Integrin Signaling Pathways.

    Directory of Open Access Journals (Sweden)

    Hyung-Mun Yun

    Full Text Available Magnetism is an intriguing physical cue that can alter the behaviors of a broad range of cells. Nanocomposite scaffolds that exhibit magnetic properties are thus considered useful 3D matrix for culture of cells and their fate control in repair and regeneration processes. Here we produced magnetic nanocomposite scaffolds made of magnetite nanoparticles (MNPs and polycaprolactone (PCL, and the effects of the scaffolds on the adhesion, growth, migration and odontogenic differentiation of human dental pulp cells (HDPCs were investigated. Furthermore, the associated signaling pathways were examined in order to elucidate the molecular mechanisms in the cellular events. The magnetic scaffolds incorporated with MNPs at varying concentrations (up to 10%wt supported cellular adhesion and multiplication over 2 weeks, showing good viability. The cellular constructs in the nanocomposite scaffolds played significant roles in the stimulation of adhesion, migration and odontogenesis of HDPCs. Cells were shown to adhere to substantially higher number when affected by the magnetic scaffolds. Cell migration tested by in vitro wound closure model was significantly enhanced by the magnetic scaffolds. Furthermore, odontogenic differentiation of HDPCs, as assessed by the alkaline phosphatase activity, mRNA expressions of odontogenic markers (DMP-1, DSPP,osteocalcin, and ostepontin, and alizarin red staining, was significantly stimulated by the magnetic scaffolds. Signal transduction was analyzed by RT-PCR, Western blotting, and confocal microscopy. The magnetic scaffolds upregulated the integrin subunits (α1, α2, β1 and β3 and activated downstream pathways, such as FAK, paxillin, p38, ERK MAPK, and NF-κB. The current study reports for the first time the significant impact of magnetic scaffolds in stimulating HDPC behaviors, including cell migration and odontogenesis, implying the potential usefulness of the magnetic scaffolds for dentin-pulp tissue engineering.

  9. Magnetic Nanocomposite Scaffold-Induced Stimulation of Migration and Odontogenesis of Human Dental Pulp Cells through Integrin Signaling Pathways

    Science.gov (United States)

    Kim, Mi-joo; Kim, Jung-Ju; Lee, Jung-Hwan; Lee, Hae-Hyoung; Park, Kyung-Ran; Yi, Jin-Kyu; Kim, Hae-Won; Kim, Eun-cheol

    2015-01-01

    Magnetism is an intriguing physical cue that can alter the behaviors of a broad range of cells. Nanocomposite scaffolds that exhibit magnetic properties are thus considered useful 3D matrix for culture of cells and their fate control in repair and regeneration processes. Here we produced magnetic nanocomposite scaffolds made of magnetite nanoparticles (MNPs) and polycaprolactone (PCL), and the effects of the scaffolds on the adhesion, growth, migration and odontogenic differentiation of human dental pulp cells (HDPCs) were investigated. Furthermore, the associated signaling pathways were examined in order to elucidate the molecular mechanisms in the cellular events. The magnetic scaffolds incorporated with MNPs at varying concentrations (up to 10%wt) supported cellular adhesion and multiplication over 2 weeks, showing good viability. The cellular constructs in the nanocomposite scaffolds played significant roles in the stimulation of adhesion, migration and odontogenesis of HDPCs. Cells were shown to adhere to substantially higher number when affected by the magnetic scaffolds. Cell migration tested by in vitro wound closure model was significantly enhanced by the magnetic scaffolds. Furthermore, odontogenic differentiation of HDPCs, as assessed by the alkaline phosphatase activity, mRNA expressions of odontogenic markers (DMP-1, DSPP,osteocalcin, and ostepontin), and alizarin red staining, was significantly stimulated by the magnetic scaffolds. Signal transduction was analyzed by RT-PCR, Western blotting, and confocal microscopy. The magnetic scaffolds upregulated the integrin subunits (α1, α2, β1 and β3) and activated downstream pathways, such as FAK, paxillin, p38, ERK MAPK, and NF-κB. The current study reports for the first time the significant impact of magnetic scaffolds in stimulating HDPC behaviors, including cell migration and odontogenesis, implying the potential usefulness of the magnetic scaffolds for dentin-pulp tissue engineering. PMID:26382272

  10. Highly ordered large-scale neuronal networks of individual cells - toward single cell to 3D nanowire intracellular interfaces.

    Science.gov (United States)

    Kwiat, Moria; Elnathan, Roey; Pevzner, Alexander; Peretz, Asher; Barak, Boaz; Peretz, Hagit; Ducobni, Tamir; Stein, Daniel; Mittelman, Leonid; Ashery, Uri; Patolsky, Fernando

    2012-07-25

    The use of artificial, prepatterned neuronal networks in vitro is a promising approach for studying the development and dynamics of small neural systems in order to understand the basic functionality of neurons and later on of the brain. The present work presents a high fidelity and robust procedure for controlling neuronal growth on substrates such as silicon wafers and glass, enabling us to obtain mature and durable neural networks of individual cells at designed geometries. It offers several advantages compared to other related techniques that have been reported in recent years mainly because of its high yield and reproducibility. The procedure is based on surface chemistry that allows the formation of functional, tailormade neural architectures with a micrometer high-resolution partition, that has the ability to promote or repel cells attachment. The main achievements of this work are deemed to be the creation of a large scale neuronal network at low density down to individual cells, that develop intact typical neurites and synapses without any glia-supportive cells straight from the plating stage and with a relatively long term survival rate, up to 4 weeks. An important application of this method is its use on 3D nanopillars and 3D nanowire-device arrays, enabling not only the cell bodies, but also their neurites to be positioned directly on electrical devices and grow with registration to the recording elements underneath.

  11. Mesenchymal stem cells enhance ovarian cancer cell infiltration through IL6 secretion in an amniochorionic membrane based 3D model

    Directory of Open Access Journals (Sweden)

    Touboul Cyril

    2013-01-01

    Full Text Available Abstract Background The early peritoneal invasion of epithelial ovarian cancer (EOC by tumoral aggregates presents in ascites is a major concern. The role of the microenvironment seems to be important in this process but the lack of adequate models to study cellular interactions between cancer cells and stromal cells does not allow to uncover the molecular pathways involved. Our goal was to study the interactions between ovarian cancer cells (OCC and mesenchymal stem cells (MSC using a 3D model. Methods We used millimetric pieces of amniochorionic membrane - referred to as amniotic membrane scaffold (AMS - to create 3D peritoneal nodules mimicking EOC early invasion. We were able to measure the distribution and the depth of infiltration using confocal microsopy. We extracted MSC from the amniochorionic membrane using the markers CD34-, CD45-, CD73+, CD90+, CD105+ and CD29+ at the Fluorescence Activated Cell Sorting (FACS analysis. We used transwell and wound healing tests to test OCC migration and invasion in vitro. Results Here we show that OCC tumors were located in regions rich in MSC (70%. The tumors infiltrated deeper within AMS in regions rich in MSC (p Conclusions The use of tridimensional models using AMS could be a useful tool to decipher early molecular events in ovarian cancer metastasis. Cytokine inhibitors interrupting the cross-talk between OCCs and MSCs such as IL6 should be investigated as a new therapeutic approach in ovarian cancer.

  12. Prospective use of the 3D printing technology for the microstructural engineering of Solid Oxide Fuel Cell components

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez-Rodriguez, E. M.; Acosta-Mora, P.; Mendez-Ramos, J.; Borges Chinea, E.; Esparza Ferrera, P.; Canales-Vazquez, J.; Nunez, P.; Ruiz-Morales, J.

    2014-07-01

    A cost-effective micro-manufacturing process to accurately build 3D microstructures for their prospective use in the fabrication of Solid Oxide Fuel Cells components has been tested. The 3D printing method, based on the stereo lithography, allows solidifying layer by layer a dispersion of ceramic material in a liquid photosensitive organic monomer. A simple projector, a computer-controlled z-stage and a few PowerPoint slides may be used for the fabrication of a wide range of complex 3D microstructures in few minutes. In this work, 3D ceramic microstructures based on the yttria-stabilized zirconia (YSZ) were successfully fabricated. The micro structured ceramic components produced were stable after sintering at 1400 degree centigrade for 4 h. Impedance measurements show that the fabrication process does not have any detrimental effect on the electrical properties of the structured material. (Author)

  13. Prospective use of the 3D printing technology for the microstructural engineering of Solid Oxide Fuel Cell components

    International Nuclear Information System (INIS)

    A cost-effective micro-manufacturing process to accurately build 3D microstructures for their prospective use in the fabrication of Solid Oxide Fuel Cells components has been tested. The 3D printing method, based on the stereo lithography, allows solidifying layer by layer a dispersion of ceramic material in a liquid photosensitive organic monomer. A simple projector, a computer-controlled z-stage and a few PowerPoint slides may be used for the fabrication of a wide range of complex 3D microstructures in few minutes. In this work, 3D ceramic microstructures based on the yttria-stabilized zirconia (YSZ) were successfully fabricated. The micro structured ceramic components produced were stable after sintering at 1400 degree centigrade for 4 h. Impedance measurements show that the fabrication process does not have any detrimental effect on the electrical properties of the structured material. (Author)

  14. Surface Acoustic Waves (SAW-Based Biosensing for Quantification of Cell Growth in 2D and 3D Cultures

    Directory of Open Access Journals (Sweden)

    Tao Wang

    2015-12-01

    Full Text Available Detection and quantification of cell viability and growth in two-dimensional (2D and three-dimensional (3D cell cultures commonly involve harvesting of cells and therefore requires a parallel set-up of several replicates for time-lapse or dose–response studies. Thus, developing a non-invasive and touch-free detection of cell growth in longitudinal studies of 3D tumor spheroid cultures or of stem cell regeneration remains a major unmet need. Since surface acoustic waves (SAWs permit mass loading-based biosensing and have been touted due to their many advantages including low cost, small size and ease of assembly, we examined the potential of SAW-biosensing to detect and quantify cell growth. Herein, we demonstrate that a shear horizontal-surface acoustic waves (SH-SAW device comprising two pairs of resonators consisting of interdigital transducers and reflecting fingers can be used to quantify mass loading by the cells in suspension as well as within a 3D cell culture platform. A 3D COMSOL model was built to simulate the mass loading response of increasing concentrations of cells in suspension in the polydimethylsiloxane (PDMS well in order to predict the characteristics and optimize the design of the SH-SAW biosensor. The simulated relative frequency shift from the two oscillatory circuit systems (one of which functions as control were found to be concordant to experimental data generated with RAW264.7 macrophage and A549 cancer cells. In addition, results showed that SAW measurements per se did not affect viability of cells. Further, SH-SAW biosensing was applied to A549 cells cultured on a 3D electrospun nanofiber scaffold that generate tumor spheroids (tumoroids and the results showed the device's ability to detect changes in tumor spheroid growth over the course of eight days. Taken together, these results demonstrate the use of SH-SAW device for detection and quantification of cell growth changes over time in 2D suspension cultures and in

  15. An impedance method for spatial sensing of 3D cell constructs – towards applications in tissue engineering

    DEFF Research Database (Denmark)

    Canali, Chiara; Mazzoni, Chiara; Larsen, Layla Bashir;

    2015-01-01

    ) cells were encapsulated in gelatin to form artificial 3D cell constructs and detected when placed in different positions inside large gelatin scaffolds. Taken together, these results open new perspectives for impedance-based sensing technologies for non-invasive monitoring in tissue engineering...

  16. Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening.

    Science.gov (United States)

    Wollrab, Viktoria; Caballero, David; Thiagarajan, Raghavan; Riveline, Daniel

    2016-01-01

    Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 10(5) cavities/cm(2)), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, 'eggcups', onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this

  17. Immunogenicity and T cell recognition in swine of foot-and-mouth disease virus polymerase 3D

    International Nuclear Information System (INIS)

    Immunization of domestic pigs with a vaccinia virus (VV) recombinant expressing foot-and-mouth disease virus (FMDV) 3D protein conferred partial protection against challenge with infectious virus. The severity reduction of the clinical symptoms developed by the challenged animals occurred in the absence of significant levels of anti-3D circulating antibodies. This observation suggested that the partial protection observed was mediated by the induction of a 3D-specific cellular immune response. To gain information on the T cell recognition of FMDV 3D protein, we conducted in vitro proliferative assays using lymphocytes from outbred pigs experimentally infected with FMDV and 90 overlapping peptides spanning the complete 3D sequence. The use of pools of two to three peptides allowed the identification of T cell epitopes that were efficiently recognized by lymphocytes from at least four of the five animals analyzed. This recognition was heterotypic because anti-peptide responses increased upon reinfection of animals with a FMDV isolate from a different serotype. The results obtained with individual peptides confirmed the antigenicity observed with peptide pools. Detection of cytokine mRNAs by RT-PCR in lymphocytes stimulated in vitro by individual 3D peptides revealed that IFN-γ mRNA was the most consistently induced, suggesting that the activated T cells belong to the Th 1 subset. These results indicate that 3D protein contains epitopes that can be efficiently recognized by porcine T lymphocytes from different infected animals, both upon primary and secondary (heterotypic) FMDV infection. These epitopes can extend the repertoire of viral T cell epitopes to be included in subunit and synthetic FMD vaccines

  18. Human dental pulp stem cells derived from cryopreserved dental pulp tissues of vital extracted teeth with disease demonstrate hepatic-like differentiation.

    Science.gov (United States)

    Chen, Y K; Huang, Anderson H C; Chan, Anthony W S; Lin, L M

    2016-06-01

    Reviewing the literature, hepatic differentiation of human dental pulp stem cells (hDPSCs) from cryopreserved dental pulp tissues of vital extracted teeth with disease has not been studied. This study is aimed to evaluate the hypothesis that hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease could possess potential hepatic differentiation. Forty vital extracted teeth with disease recruited for hDPSCs isolation, stem cell characterization and hepatic differentiation were randomly and equally divided into group A (liquid nitrogen-stored dental pulp tissues) and group B (freshly derived dental pulp tissues). Samples of hDPSCs isolated from groups A and B but without hepatic growth factors formed negative controls. A well-differentiated hepatocellular carcinoma cell line was employed as a positive control. All the isolated hDPSCs from groups A and B showed hepatic-like differentiation with morphological change from a spindle-shaped to a polygonal shape and normal karyotype. Differentiated hDPSCs and the positive control expressed hepatic metabolic function genes and liver-specific genes. Glycogen storage of differentiated hDPSCs was noted from day 7 of differentiation-medium culture. Positive immunofluorescence staining of low-density lipoprotein and albumin was observed from day 14 of differentiation-medium culture; urea production in the medium was noted from week 6. No hepatic differentiation was observed for any of the samples of the negative controls. We not only demonstrated the feasibility of hepatic-like differentiation of hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease but also indicated that the differentiated cells possessed normal karyotype and were functionally close to normal hepatic-like cells. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23950016

  19. A 3D culture system enhances the ability of human bone marrow stromal cells to support the growth of limbal stem/progenitor cells

    Directory of Open Access Journals (Sweden)

    Sheyla González

    2016-03-01

    Full Text Available The standard method of cultivating limbal epithelial progenitor/stem cells (LSCs on a monolayer of mouse 3T3 feeder cells possesses the risk of cross-contamination in clinical applications. Human feeder cells have been used to eliminate this risk; however, efficiency from xenobiotic-free cultures on a monolayer appears to be lower than in the standard method using 3T3 cells. We investigated whether bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells, could serve as feeder cells for the expansion of LSCs in the 3-dimensional (3D system. Primary single human LSCs on a monolayer of 3T3s served as the control. Very poor growth was observed when single LSCs were cultured on BMSCs. When LSC clusters were cultured on a BMSC monolayer (CC-BM, 3D culture system (3D CC-BM and fibrin 3D system (fibrin 3D CC-BM, the 3D CC-BM method supported a greater LSC expansion. The 3D CC-BM system produced a 2.5-fold higher cell growth rate than the control (p  0.05, whereas the proportion of K12+ cells was lower (p < 0.05. These results indicate that BMSCs can efficiently support the expansion of the LSC population in the 3D culture.

  20. Quantitative data analysis methods for 3D microstructure characterization of Solid Oxide Cells

    DEFF Research Database (Denmark)

    Jørgensen, Peter Stanley

    the microstructure. The methods are exemplied by the analysis of Ni-YSZ and LSC-CGO electrode samples. Automatic methods for preprocessing the raw 3D image data are developed. The preprocessing steps correct for errors introduced by the image acquisition by the focused ion beam serial sectioning. Alignment...... for gaining further fundamental understanding of how microstructure affects performance. In this work, methods for automatic 3D characterization of microstructure are studied: from the acquisition of 3D image data by focused ion beam tomography to the extraction of quantitative measures that characterize...... of the individual image slices is performed by automatic detection of ducial marks. Uneven illumination is corrected by tting hypersurfaces to the spatial intensity variation in the 3D image data. Routine use of quantitative three dimensional analysis of microstructure is generally restricted by the time consuming...

  1. Calcium Electroporation: Evidence for Differential Effects in Normal and Malignant Cell Lines, Evaluated in a 3D Spheroid Model

    OpenAIRE

    Frandsen, Stine Krog; Gibot, Laure; Madi, Moinecha; Gehl, Julie; Rols, Marie-Pierre

    2015-01-01

    Background Calcium electroporation describes the use of high voltage electric pulses to introduce supraphysiological calcium concentrations into cells. This promising method is currently in clinical trial as an anti-cancer treatment. One very important issue is the relation between tumor cell kill efficacy–and normal cell sensitivity. Methods Using a 3D spheroid cell culture model we have tested the effect of calcium electroporation and electrochemotherapy using bleomycin on three different h...

  2. Pulmonary surfactant expression analysis--role of cell-cell interactions and 3-D tissue-like architecture.

    Science.gov (United States)

    Nandkumar, Maya A; Ashna, U; Thomas, Lynda V; Nair, Prabha D

    2015-03-01

    Surfactant production is important in maintaining alveolar function both in vivo and in vitro, but surfactant expression is the primary property lost by alveolar Type II Pneumocytes in culture and its maintenance is a functional requirement. To develop a functional tissue-like model, the in vivo cell-cell interactions and three dimensional architecture has to be reproduced. To this end, 3D button-shaped synthetic gelatin vinyl acetate (GeVAc) co-polymer scaffold was seeded with different types of lung cells. Functionality of the construct was studied under both static and dynamic conditions. The construct was characterized by Environmental Scanning Electron and fluorescent microscopy, and functionality of the system was analyzed by studying mRNA modulations of all four surfactant genes A, B, C, and D by real time-PCR and varying culture conditions. The scaffold supports alveolar cell adhesion and maintenance of cuboidal morphology, and the alveolar-specific property of surfactant synthesis, which would otherwise be rapidly lost in culture. This is a novel 3D system that expresses all 4 surfactants for a culture duration of 3 weeks.

  3. High-Throughput Cancer Cell Sphere Formation for Characterizing the Efficacy of Photo Dynamic Therapy in 3D Cell Cultures

    Science.gov (United States)

    Chen, Yu-Chih; Lou, Xia; Zhang, Zhixiong; Ingram, Patrick; Yoon, Euisik

    2015-07-01

    Photodynamic therapy (PDT), wherein light sensitive non-toxic agents are locally and selectively activated using light, has emerged as an appealing alternative to traditional cancer chemotherapy. Yet to date, PDT efficacy has been mostly characterized using 2D cultures. Compared to 2D cultures, 3D sphere culture generates unique spatial distributions of nutrients and oxygen for the cells that better mimics the in-vivo conditions. Using a novel polyHEMA (non-adherent polymer) fabrication process, we developed a microfluidic sphere formation platform that can (1) generate 1,024 uniform (size variation cancer spheres within a 2 cm by 2 cm core area, (2) culture spheres for more than 2 weeks, and (3) allow the retrieval of spheres. Using the presented platform, we have successfully characterized the different responses in 2D and 3D cell culture to PDT. Furthermore, we investigated the treatment resistance effect in cancer cells induced by tumor associated fibroblasts (CAF). Although the CAFs can enhance the resistance to traditional chemotherapy agents, no significant difference in PDT was observed. The preliminary results suggest that the PDT can be an attractive alternative cancer therapy, which is less affected by the therapeutic resistance induced by cancer associated cells.

  4. Pep-3D-Search: a method for B-cell epitope prediction based on mimotope analysis

    Directory of Open Access Journals (Sweden)

    Wang Yan

    2008-12-01

    Full Text Available Abstract Background The prediction of conformational B-cell epitopes is one of the most important goals in immunoinformatics. The solution to this problem, even if approximate, would help in designing experiments to precisely map the residues of interaction between an antigen and an antibody. Consequently, this area of research has received considerable attention from immunologists, structural biologists and computational biologists. Phage-displayed random peptide libraries are powerful tools used to obtain mimotopes that are selected by binding to a given monoclonal antibody (mAb in a similar way to the native epitope. These mimotopes can be considered as functional epitope mimics. Mimotope analysis based methods can predict not only linear but also conformational epitopes and this has been the focus of much research in recent years. Though some algorithms based on mimotope analysis have been proposed, the precise localization of the interaction site mimicked by the mimotopes is still a challenging task. Results In this study, we propose a method for B-cell epitope prediction based on mimotope analysis called Pep-3D-Search. Given the 3D structure of an antigen and a set of mimotopes (or a motif sequence derived from the set of mimotopes, Pep-3D-Search can be used in two modes: mimotope or motif. To evaluate the performance of Pep-3D-Search to predict epitopes from a set of mimotopes, 10 epitopes defined by crystallography were compared with the predicted results from a Pep-3D-Search: the average Matthews correlation oefficient (MCC, sensitivity and precision were 0.1758, 0.3642 and 0.6948. Compared with other available prediction algorithms, Pep-3D-Search showed comparable MCC, specificity and precision, and could provide novel, rational results. To verify the capability of Pep-3D-Search to align a motif sequence to a 3D structure for predicting epitopes, 6 test cases were used. The predictive performance of Pep-3D-Search was demonstrated to be

  5. 1-integrin and MT1-MMP promote tumor cell migration in 2D but not in 3D fibronectin microenvironments

    Science.gov (United States)

    Corall, Silke; Haraszti, Tamas; Bartoschik, Tanja; Spatz, Joachim Pius; Ludwig, Thomas; Cavalcanti-Adam, Elisabetta Ada

    2014-03-01

    Cell migration is a crucial event for physiological processes, such as embryonic development and wound healing, as well as for pathological processes, such as cancer dissemination and metastasis formation. Cancer cell migration is a result of the concerted action of matrix metalloproteinases (MMPs), expressed by cancer cells to degrade the surrounding matrix, and integrins, the transmembrane receptors responsible for cell binding to matrix proteins. While it is known that cell-microenvironment interactions are essential for migration, the role of the physical state of such interactions remains still unclear. In this study we investigated human fibrosarcoma cell migration in two-dimensional (2D) and three-dimensional (3D) fibronectin (FN) microenvironments. By using antibody blocking approach and cell-binding site mutation, we determined that -integrin is the main mediator of fibrosarcoma cell migration in 2D FN, whereas in 3D fibrillar FN, the binding of - and -integrins is not necessary for cell movement in the fibrillar network. Furthermore, while the general inhibition of MMPs with GM6001 has no effect on cell migration in both 2D and 3D FN matrices, we observed opposing effect after targeted silencing of a membrane-bound MMP, namely MT1-MMP. In 2D fibronectin, silencing of MT1-MMP results in decreased migration speed and loss of directionality, whereas in 3D FN matrices, cell migration speed is increased and integrin-mediated signaling for actin dynamics is promoted. Our results suggest that the fibrillar nature of the matrix governs the migratory behavior of fibrosarcoma cells. Therefore, to hinder migration and dissemination of diseased cells, matrix molecules should be directly targeted, rather than specific subtypes of receptors at the cell membrane.

  6. Successful isolation, in vitro expansion and characterization of stem cells from Human Dental Pulp

    Directory of Open Access Journals (Sweden)

    Preethy SP

    2010-01-01

    Full Text Available BACKGROUND: Recent studies have shown that mesenchymal stem cells isolated from post natal human dental pulp, (Dental pulp stem cells-DPSCs which is from permanent teeth and SHED (stem cells from human exfoliated deciduous teeth,the Periodontal ligament stem cells (PDLSC and Stem cells from root Apical papilla(SCAPhave the potential to differentiate into cells of a variety of tissues including heart, muscle, cartilage, bone, nerve, salivary glands, teeth etc(1,2,3,4.This multipotential ability of DPSCs is being researched for clinical application for treating a variety of diseases like myocardial infarction, muscular dystrophy, neuro-degenerative disorders, cartilage replacement, tooth regeneration and for repair of bone defects to mention a few. Moreover, the isolation of stem cells from teeth is minimally invasive, readily accessible and the non immunogenic characteristic of dental stem cells has paved the way for efforts to store the exfoliated deciduous teeth or milk teeth which is usually discarded, for use in the future. In this study we have isolated and expanded in vitro, the cells obtained from human dental pulp. MATERIALS AND METHODS: After obtaining written informed consent, 24 teeth that were extracted for therapeutic or cosmetic reasons from 16 patients were used in this study. The specimens were transported from the clinic to NCRM lab taking 6 to 48 Hrs. For removal of the pulp tissue, the teeth were split obliquely at the Cementoenamel junction and the pulp tissue was isolated using brooches. The extracted pulp tissues were subjected to digestion using Collagenase type-I and type II at 37˚C for 15- 30 minutes. The digested cells were filtered with 70µm filter and centrifuged at 1800 rpm for 10 minutes. The pellet was then suspended in Dulbecco’s modified Eagle’s medium (DMEM/Ham’s F12 supplemented with 15% fetal bovine serum , 100 U/ml penicillin, 100 µg/ml streptomycin,2 m M L -glutamine, and 2 m M nonessential amino

  7. Lamellipodin promotes invasive 3D cancer cell migration via regulated interactions with Ena/VASP and SCAR/WAVE

    Science.gov (United States)

    Carmona, Guillaume; Perera, Upamali; Gillett, Cheryl; Naba, Alexandra; Law, Ah-Lai; Sharma, Ved P.; Wang, Jian; Wyckoff, Jeffrey; Balsamo, Michele; Mosis, Fuad; De Piano, Mario; Monypenny, James; Woodman, Natalie; McConnell, Russell E.; Mouneimne, Ghassan; Van Hemelrijck, Mieke; Cao, Yihai; Condeelis, John; Hynes, Richard O.; Gertler, Frank B.; Krause, Matthias

    2016-01-01

    Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlates with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation, and matrix degradation were impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not Ena/VASP is required for random 2D cell migration. We identify a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, while Src-dependent phosphorylation enhances binding to Scar/WAVE but not Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of EGF gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis. PMID:26996666

  8. The microenvironment determines the breast cancer cells' phenotype: organization of MCF7 cells in 3D cultures

    Directory of Open Access Journals (Sweden)

    Soto Ana M

    2010-06-01

    Full Text Available Abstract Background Stromal-epithelial interactions mediate breast development, and the initiation and progression of breast cancer. In the present study, we developed 3-dimensional (3D in vitro models to study breast cancer tissue organization and the role of the microenvironment in phenotypic determination. Methods The human breast cancer MCF7 cells were grown alone or co-cultured with primary human breast fibroblasts. Cells were embedded in matrices containing either type I collagen or a combination of reconstituted basement membrane proteins and type I collagen. The cultures were carried out for up to 6 weeks. For every time point (1-6 weeks, the gels were fixed and processed for histology, and whole-mounted for confocal microscopy evaluation. The epithelial structures were characterized utilizing immunohistochemical techniques; their area and proliferation index were measured using computerized morphometric analysis. Statistical differences between groups were analyzed by ANOVA, Dunnett's T3 post-hoc test and chi-square. Results Most of the MCF7 cells grown alone within a collagen matrix died during the first two weeks; those that survived organized into large, round and solid clusters. The presence of fibroblasts in collagen gels reduced MCF7 cell death, induced cell polarity, and the formation of round and elongated epithelial structures containing a lumen. The addition of reconstituted basement membrane to collagen gels by itself had also survival and organizational effects on the MCF7 cells. Regardless of the presence of fibroblasts, the MCF7 cells both polarized and formed a lumen. The addition of fibroblasts to the gel containing reconstituted basement membrane and collagen induced the formation of elongated structures. Conclusions Our results indicate that a matrix containing both type I collagen and reconstituted basement membrane, and the presence of normal breast fibroblasts constitute the minimal permissive microenvironment to

  9. The microenvironment determines the breast cancer cells' phenotype: organization of MCF7 cells in 3D cultures

    International Nuclear Information System (INIS)

    Stromal-epithelial interactions mediate breast development, and the initiation and progression of breast cancer. In the present study, we developed 3-dimensional (3D) in vitro models to study breast cancer tissue organization and the role of the microenvironment in phenotypic determination. The human breast cancer MCF7 cells were grown alone or co-cultured with primary human breast fibroblasts. Cells were embedded in matrices containing either type I collagen or a combination of reconstituted basement membrane proteins and type I collagen. The cultures were carried out for up to 6 weeks. For every time point (1-6 weeks), the gels were fixed and processed for histology, and whole-mounted for confocal microscopy evaluation. The epithelial structures were characterized utilizing immunohistochemical techniques; their area and proliferation index were measured using computerized morphometric analysis. Statistical differences between groups were analyzed by ANOVA, Dunnett's T3 post-hoc test and chi-square. Most of the MCF7 cells grown alone within a collagen matrix died during the first two weeks; those that survived organized into large, round and solid clusters. The presence of fibroblasts in collagen gels reduced MCF7 cell death, induced cell polarity, and the formation of round and elongated epithelial structures containing a lumen. The addition of reconstituted basement membrane to collagen gels by itself had also survival and organizational effects on the MCF7 cells. Regardless of the presence of fibroblasts, the MCF7 cells both polarized and formed a lumen. The addition of fibroblasts to the gel containing reconstituted basement membrane and collagen induced the formation of elongated structures. Our results indicate that a matrix containing both type I collagen and reconstituted basement membrane, and the presence of normal breast fibroblasts constitute the minimal permissive microenvironment to induce near-complete tumor phenotype reversion. These human

  10. Development of 3D in vitro platform technology to engineer mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Hosseinkhani H

    2012-06-01

    Full Text Available Hossein Hosseinkhani,1 Po-Da Hong,1 Dah-Shyong Yu,2 Yi-Ru Chen,3 Diana Ickowicz,4 Ira-Yudovin Farber,4 Abraham J Domb41Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology (TAIWANTECH, 2Nanomedicine Research Center, National Defense Medical Center, Taipei, Taiwan, 3Department of Biomedical Engineering, National Yang-Ming University, Taipei, Taiwan, 4Institute of Drug Research, The Center for Nanoscience and Nanotechnology, School of Pharmacy-Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, IsraelAbstract: This study aims to develop a three-dimensional in vitro culture system to genetically engineer mesenchymal stem cells (MSC to express bone morphogenic protein-2. We employed nanofabrication technologies borrowed from the spinning industry, such as electrospinning, to mass-produce identical building blocks in a variety of shapes and sizes to fabricate electrospun nanofiber sheets comprised of composites of poly (glycolic acid and collagen. Homogenous nanoparticles of cationic biodegradable natural polymer were formed by simple mixing of an aqueous solution of plasmid DNA encoded bone morphogenic protein-2 with the same volume of cationic polysaccharide, dextran-spermine. Rat bone marrow MSC were cultured on electrospun nanofiber sheets comprised of composites of poly (glycolic acid and collagen prior to the incorporation of the nanoparticles into the nanofiber sheets. Bone morphogenic protein-2 was significantly detected in MSC cultured on nanofiber sheets incorporated with nanoparticles after 2 days compared with MSC cultured on nanofiber sheets incorporated with naked plasmid DNA. We conclude that the incorporation of nanoparticles into nanofiber sheets is a very promising strategy to genetically engineer MSC and can be used for further applications in regenerative medicine therapy.Keywords: 3D culture, nanoparticles, nanofibers, polycations, tissue engineering

  11. Imperative role of dental pulp stem cells in regenerative therapies: A systematic review

    Directory of Open Access Journals (Sweden)

    Ramchandra Kabir

    2014-01-01

    Full Text Available Stem cells are primitive cells that can differentiate and regenerate organs in different parts of the body such as heart, bones, muscles and nervous system. This has been a field of great clinical interest with immense possibilities of using the stem cells in regeneration of human organ those are damaged due to disease, developmental defects and accident. The knowledge of stem cell technology is increasing quickly in all medical specialties and in dental field too. Stem cells of dental origin appears to hold the key to various cell-based therapies in regenerative medicine, but most avenues are in experimental stages and many procedures are undergoing standardization and validation. Long-term preservation of SHED cells or DPSC is becoming a popular consideration, similar to the banking of umbilical cord blood. Dental pulp stem cells (DPSCs are the adult multipotent cells that reside in the cell rich zone of the dental pulp. The multipotent nature of these DPSCs may be utilized in both dental and medical applications. A systematic review of the literature was performed using various internet based search engines (PubMed, Medline Plus, Cochrane, Medknow, Ebsco, Science Direct, Hinari, WebMD, IndMed, Embase using keywords like "dental pulp stem cells", "regeneration", "medical applications", "tissue engineering". DPSCs appears to be a promising innovation for the re-growth of tissues however, long term clinical studies need to be carried out that could establish some authentic guidelines in this perspective.

  12. The performance of dental pulp stem cells on nanofibrous PCL/gelatin/nHA scaffolds.

    NARCIS (Netherlands)

    Yang, X.; Yang, F.; Walboomers, X.F.; Bian, Z.; Fan, M.; Jansen, J.A.

    2010-01-01

    The aim of current study is to investigate the in vitro and in vivo behavior of dental pulp stem cells (DPSCs) seeded on electrospun poly(epsilon-caprolactone) (PCL)/gelatin scaffolds with or without the addition of nano-hydroxyapatite (nHA). For the in vitro evaluation, DNA content, alkaline phosph

  13. Retracted: Effects of pro-inflammatory cytokines on mineralization potential of rat dental pulp stem cells

    NARCIS (Netherlands)

    Yang, X.; Walboomers, X.F.; Bian, Z.; Jansen, J.A.; Fan, M.

    2011-01-01

    The following article from the Journal of Tissue Engineering and Regenerative Medicine, 'Effects of Pro-inflammatory Cytokines on Mineralization Potential of Rat Dental Pulp Stem Cells' by Yang X, Walboomers XF, Bian Z, Jansen JA, Fan M, published online on 11 July 2011 in Wiley Online Library (onli

  14. The effect of porosity on cell ingrowth into accurately defined, laser-made, polylactide-based 3D scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Danilevicius, Paulius; Georgiadi, Leoni [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece); Pateman, Christopher J.; Claeyssens, Frederik [Kroto Research Institute, Department of Materials Science and Engineering, University of Sheffield, Broad Lane, Sheffield S3 7HQ (United Kingdom); Chatzinikolaidou, Maria, E-mail: mchatzin@materials.uoc.gr [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece); Department of Materials Science and Technology, University of Crete, PO Box 2208, 71303 Heraklion (Greece); Farsari, Maria, E-mail: mfarsari@iesl.forth.gr [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece)

    2015-05-01

    Highlights: • We studied the porosity of laser-made 3D scaffolds on MC3T3-E1 pre-osteoblastic cells. • We made polylactide 3D scaffolds with pores 25–110 μm. - Abstract: The aim of this study is to demonstrate the accuracy required for the investigation of the role of solid scaffolds’ porosity in cell proliferation. We therefore present a qualitative investigation into the effect of porosity on MC3T3-E1 pre-osteoblastic cell ingrowth of three-dimensional (3D) scaffolds fabricated by direct femtosecond laser writing. The material we used is a purpose made photosensitive pre-polymer based on polylactide. We designed and fabricated complex, geometry-controlled 3D scaffolds with pore sizes ranging from 25 to 110 μm, representing porosities 70%, 82%, 86%, and 90%. The 70% porosity scaffolds did not support cell growth initially and in the long term. For the other porosities, we found a strong adhesion of the pre-osteoblastic cells from the first hours after seeding and a remarkable proliferation increase after 3 weeks and up to 8 weeks. The 86% porosity scaffolds exhibited a higher efficiency compared to 82% and 90%. In addition, bulk material degradation studies showed that the employed, highly-acrylated polylactide is degradable. These findings support the potential use of the proposed material and the scaffold fabrication technique in bone tissue engineering.

  15. Sustained PDGF-BB release from PHBHHx loaded nanoparticles in 3D hydrogel/stem cell model.

    Science.gov (United States)

    Dong, Cui-Ling; Webb, William R; Peng, Qiang; Tang, James Z; Forsyth, Nicholas R; Chen, Guo-Qiang; El Haj, Alicia J

    2015-01-01

    This study aimed to design a growth factor loaded copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) nanoparticles containing 3D collagen matrix to achieve growth factor sustained release for long-term stimulation of human mesenchymal stem cells (hMSCs) proliferation/differentiation for tissue engineer application. Platelet-derived growth factor-BB (PDGF-BB), which is known to enhance hMSCs proliferation in human serum, was selected as a model growth factor, and biodegradable copolyester of PHBHHx was chosen to be the sustained release vehicle. PDGF-BB phospholipid complex encapsulated PHBHHx nanoparticles were fabricated, and their effect on hMSCs proliferation was investigated via assays of CCK-8 and live-dead staining to cells inoculated in 2D tissue culture plates and 3D collagen gel scaffolds, respectively. The resulting spherical PHBHHx nanoparticles were stable in terms of their mean particle size, polydispersity index and zeta potential before and after lyophilization. In vitro study revealed a sustained release of PDGF-BB with a low burst release. Furthermore, sustained released PDGF-BB was revealed to significantly promote hMSCs proliferation in both cell monolayer and cell seeded 3D collagen scaffolds inoculated in serum-free media. Therefore, the 3D collagen matrices with locally sustained release growth factor nanoparticles hold promise to be used for stem cell tissue engineering.

  16. 3D Ultrastructural Organization of Whole Chlamydomonas reinhardtii Cells Studied by Nanoscale Soft X-Ray Tomography

    OpenAIRE

    Hummel, Eric; Guttmann, Peter; Werner, Stephan; Tarek, Basel; SCHNEIDER, Gerd; Kunz, Michael; Frangakis, Achilleas S.; Westermann, Benedikt

    2012-01-01

    The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial re...

  17. 3D ultrastructural organization of whole Chlamydomonas reinhardtii cells studied by nanoscale soft x-ray tomography

    OpenAIRE

    Hummel, Eric; Guttmann, Peter; Werner, Stephan; Tarek, Basel; SCHNEIDER, Gerd; Kunz, Michael; Frangakis, Achilleas S.; Westermann, Benedikt

    2012-01-01

    The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial re...

  18. Infrared imaging of MDA-MB-231 breast cancer cell line phenotypes in 2D and 3D cultures.

    Science.gov (United States)

    Smolina, Margarita; Goormaghtigh, Erik

    2015-04-01

    One current challenge in the field of breast cancer infrared imaging is the identification of carcinoma cell subtypes in the tissue. Neither sequencing nor immunochemistry is currently able to provide a cell by cell thorough classification. The latter is needed to build accurate statistical models capable of recognizing the diversity of breast cancer cell lines that may be present in a tissue section. One possible approach for overcoming this problem is to obtain the IR spectral signature of well-characterized tumor cell lines in culture. Cultures in three-dimensional matrices appear to generate an environment that mimics better the in vivo environment. There are, at present, series of breast cancer cell lines that have been thoroughly characterized in two- and three-dimensional (2D and 3D) cultures by full transcriptomics analyses. In this work, we describe the methods used to grow, to process, and to characterize a triple-negative breast cancer cell line, MDA-MB-231, in 3D laminin-rich extracellular matrix (lrECM) culture and compare it with traditional monolayer cultures and tissue sections. While unsupervised analyses did not completely separate spectra of cells grown in 2D from 3D lrECM cultures, a supervised statistical analysis resulted in an almost perfect separation. When IR spectral responses of epithelial tumor cells from clinical triple-negative breast carcinoma samples were added to these data, a principal component analysis indicated that they cluster closer to the spectra of 3D culture cells than to the spectra of cells grown on a flat plastic substrata. This result is encouraging because of correlating well-characterized cell line features with clinical biopsies. PMID:25568895

  19. Dental pulp stem cells and their potential roles in central nervous system regeneration and repair.

    Science.gov (United States)

    Young, Fraser; Sloan, Alastair; Song, Bing

    2013-11-01

    Functional recovery from injuries to the brain or spinal cord represents a major clinical challenge. The transplantation of stem cells, traditionally isolated from embryonic tissue, may help to reduce damage following such events and promote regeneration and repair through both direct cell replacement and neurotrophic mechanisms. However, the therapeutic potential of using embryonic stem/progenitor cells is significantly restricted by the availability of embryonic tissues and associated ethical issues. Populations of stem cells reside within the dental pulp, representing an alternative source of cells that can be isolated with minimal invasiveness, and thus should illicit fewer moral objections, as a replacement for embryonic/fetal-derived stem cells. Here we discuss the similarities between dental pulp stem cells (DPSCs) and the endogenous stem cells of the central nervous system (CNS) and their ability to differentiate into neuronal cell types. We also consider in vitro and in vivo studies demonstrating the ability of DPSCs to help protect against and repair neuronal damage, suggesting that dental pulp may provide a viable alternative source of stem cells for replacement therapy following CNS damage.

  20. Imperative Role of Dental Pulp Stem Cells in Regenerative Therapies: A Systematic Review

    Science.gov (United States)

    Kabir, Ramchandra; Gupta, Manish; Aggarwal, Avanti; Sharma, Deepak; Sarin, Anurag; Kola, Mohammed Zaheer

    2014-01-01

    Stem cells are primitive cells that can differentiate and regenerate organs in different parts of the body such as heart, bones, muscles and nervous system. This has been a field of great clinical interest with immense possibilities of using the stem cells in regeneration of human organ those are damaged due to disease, developmental defects and accident. The knowledge of stem cell technology is increasing quickly in all medical specialties and in dental field too. Stem cells of dental origin appears to hold the key to various cell-based therapies in regenerative medicine, but most avenues are in experimental stages and many procedures are undergoing standardization and validation. Long-term preservation of SHED cells or DPSC is becoming a popular consideration, similar to the banking of umbilical cord blood. Dental pulp stem cells (DPSCs) are the adult multipotent cells that reside in the cell rich zone of the dental pulp. The multipotent nature of these DPSCs may be utilized in both dental and medical applications. A systematic review of the literature was performed using various internet based search engines (PubMed, Medline Plus, Cochrane, Medknow, Ebsco, Science Direct, Hinari, WebMD, IndMed, Embase) using keywords like “dental pulp stem cells”, “regeneration”, “medical applications”, “tissue engineering”. DPSCs appears to be a promising innovation for the re-growth of tissues however, long term clinical studies need to be carried out that could establish some authentic guidelines in this perspective. PMID:24665194

  1. Effect of biomimetic 3D environment of an injectable polymeric scaffold on MG-63 osteoblastic-cell response

    International Nuclear Information System (INIS)

    Solid PLGA microspheres were fabricated and characterized in terms of their in vitro degradation behaviour. Microsphere scaffolds were then modified covalently by P-15 (GTPGPQGIAGQRGVV) to obtain a 3D bioactive collagen surrogate matrix for bone filling applications. These scaffolds were characterized for surface topography, hydrophilicity and evaluated for their effect on osteoblastic activity of MG-63 cell line vis-a-vis 2D monolayer culture. AFM and contact angle experiments indicated enhanced nano-level roughness and hydrophilicity on P-15 modification. Modified scaffolds showed enhanced cell attachment, proliferation, extracellular matrix formation, mineralization and collagen type-I expression when compared to unmodified microspheres, prerequisite for bone filling applications. On long term in vitro cell culture, however, decreased cell viability was observed which may be attributed to the acidic microenvironment generated due to polymer degradation and reduction in nutrient diffusion through the copious ECM formed in 3D scaffolds. Though a higher cell count could be obtained in 2D monolayer cell culture, it was overshadowed by weak cell attachment, poor phenotypic characteristics, decreased cell viability and low mineralization levels, over 28 day cell culture studies. Results indicate that P-15 modified microsphere scaffolds may provide a natural, biomimetic 3D environment and may be successfully exploited for non-invasive bone filling applications.

  2. Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp

    DEFF Research Database (Denmark)

    Pierdomenico, Laura; Bonsi, Laura; Calvitti, Mario;

    2005-01-01

    BACKGROUND: Bone marrow mesenchymal stem cells (MSCs) are currently being investigated in preclinical and clinical settings because of their multipotent differentiative capacity or, alternatively, their immunosuppressive function. The aim of this study was to evaluate dental pulp (DP) as a potent...... characteristics of DP-MSCs may prompt future studies aimed at using these cells in the treatment or prevention of T-cell alloreactivity in hematopoietic or solid organ allogeneic transplantation....

  3. Designing a binding interface for control of cancer cell adhesion via 3D topography and metabolic oligosaccharide engineering.

    Science.gov (United States)

    Du, Jian; Che, Pao-Lin; Wang, Zhi-Yun; Aich, Udayanath; Yarema, Kevin J

    2011-08-01

    This study combines metabolic oligosaccharide engineering (MOE), a technology where the glycocalyx of living cells is endowed with chemical features not normally found in sugars, with custom-designed three-dimensional biomaterial substrates to enhance the adhesion of cancer cells and control their morphology and gene expression. Specifically, Ac(5)ManNTGc, a thiol-bearing analog of N-acetyl-d-mannosamine (ManNAc) was used to introduce thiolated sialic acids into the glycocalyx of human Jurkat T-lymphoma derived cells. In parallel 2D films and 3D electrospun nanofibrous scaffolds were prepared from polyethersulfone (PES) and (as controls) left unmodified or aminated. Alternately, the materials were malemided or gold-coated to provide bio-orthogonal binding partners for the thiol groups newly expressed on the cell surface. Cell attachment was modulated by both the topography of the substrate surface and by the chemical compatibility of the binding interface between the cell and the substrate; a substantial increase in binding for normally non-adhesive Jurkat line for 3D scaffold compared to 2D surfaces with an added degree of adhesion resulting from chemoselective binding to malemidede-derivatived or gold-coated surfaces. In addition, the morphology of the cells attached to the 3D scaffolds via MOE-mediated adhesion was dramatically altered and the expression of genes involved in cell adhesion changed in a time-dependent manner. This study showed that cell adhesion could be enhanced, gene expression modulated, and cell fate controlled by introducing the 3D topograhical cues into the growth substrate and by creating a glycoengineered binding interface where the chemistry of both the cell surface and biomaterials scaffold was controlled to facilitate a new mode of carbohydrate-mediated adhesion. PMID:21549424

  4. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

    Energy Technology Data Exchange (ETDEWEB)

    Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany); Ansari, Nariman [Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt (Germany); Esner, Milan; Bickle, Marc [Max Planck Institute of Molecular Cell Biology and Genetics, High-Throughput Technology Development Studio (TDS), Dresden (Germany); Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H. [Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt (Germany); Parczyk, Karsten; Prechtl, Stefan [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany); Steigemann, Patrick, E-mail: Patrick.Steigemann@bayer.com [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany)

    2014-04-15

    Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high

  5. Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Anna C Luca

    Full Text Available Three-dimensional (3D tumor cell cultures grown in laminin-rich-extracellular matrix (lrECM are considered to reflect human tumors more realistic as compared to cells grown as monolayer on plastic. Here, we systematically investigated the impact of ECM on phenotype, gene expression, EGFR signaling pathway, and on EGFR inhibition in commonly used colorectal cancer (CRC cell lines. LrECM on-top (3D culture assays were performed with the CRC cell lines SW-480, HT-29, DLD-1, LOVO, CACO-2, COLO-205 and COLO-206F. Morphology of lrECM cultivated CRC cell lines was determined by phase contrast and confocal laser scanning fluorescence microscopy. Proliferation of cells was examined by MTT assay, invasive capacity of the cell lines was assayed using Matrigel-coated Boyden chambers, and migratory activity was determined employing the Fence assay. Differential gene expression was analyzed at the transcriptional level by the Agilent array platform. EGFR was inhibited by using the specific small molecule inhibitor AG1478. A specific spheroid growth pattern was observed for all investigated CRC cell lines. DLD-1, HT-29 and SW-480 and CACO-2 exhibited a clear solid tumor cell formation, while LOVO, COLO-205 and COLO-206F were characterized by forming grape-like structures. Although the occurrence of a spheroid morphology did not correlate with an altered migratory, invasive, or proliferative capacity of CRC cell lines, gene expression was clearly altered in cells grown on lrECM as compared to 2D cultures. Interestingly, in KRAS wild-type cell lines, inhibition of EGFR was less effective in lrECM (3D cultures as compared to 2D cell cultures. Thus, comparing both 2D and 3D cell culture models, our data support the influence of the ECM on cancer growth. Compared to conventional 2D cell culture, the lrECM (3D cell culture model offers the opportunity to investigate permanent CRC cell lines under more physiological conditions, i.e. in the context of molecular

  6. Microfluidics 3D gel-island chip for single cell isolation and lineage-dependent drug responses study.

    Science.gov (United States)

    Zhang, Zhixiong; Chen, Yu-Chih; Cheng, Yu-Heng; Luan, Yi; Yoon, Euisik

    2016-07-01

    3D cell culture in the extracellular matrix (ECM), which not only provides structural support to cellular constituents, but also initiates regulatory biochemical cues for a variety of important cell functions in tissue, has become more and more important in understanding cancer pathology and drug testing. Although the ECM-gel has been used in cell culture both in bulk and on-chip, previous studies focused on collective cell behavior rather than single-cell heterogeneity. To track the behavior of each individual cell, we have developed a gel-island chip, which can form thousands of islands containing single cells encapsulated by the desired ECM. Optimized by Poisson's distribution, the device can attain 34% single cell capture efficiency of the exact number of single cells per island. A good culture media exchange rate and high cell viability can be achieved in the gel-islands. The cells in the islands can be automatically counted for high-throughput analysis. As a proof of concept, we monitored the proliferation and differentiation of single Notch+ (stem-like) T47D breast cancer cells. The 3D collagen gel environment was found to be favorable for the stem-like phenotype through better self-renewal and de-differentiation (Notch- to Notch+ transition). More interestingly, we found that the Notch- de-differentiated cells were more resistant to doxorubicin and cisplatin than the Notch+ cells. Combining the 3D ECM culture and single cell resolution, the presented platform can automatically analyze the individual cell behaviors of hundreds of cells using a small amount of drug and reagents. PMID:27270563

  7. Therapeutic potential of dental pulp stem cells in regenerative medicine: An overview

    Directory of Open Access Journals (Sweden)

    Kavita Verma

    2014-01-01

    Full Text Available The purpose of this review is to gain an overview of the applications of the dental pulp stem cells (DPSCs in the treatment of various medical diseases. Stem cells have the capacity to differentiate and regenerate into various tissues. DPSCs are the adult stem cells that reside in the cell rich zone of the dental pulp. These are the multipotent cells that can be explained by their embryonic origin from the neural crest. Owing to this multipotency, these DPSCs can be used in both dental and medical applications. A review of literature has been performed using electronic and hand-searching methods for the medical applications of DPSCs. On the basis of the available information, DPSCs appear to be a promising alternative for the regeneration of tissues and treatment of various diseases, although, long-term clinical trials and studies are needed to confirm their efficacy.

  8. DNA degradation within mouse brain and dental pulp cells 72 hours postmortem

    Institute of Scientific and Technical Information of China (English)

    Jilong Zheng; Xiaona Li; Di Shan; Han Zhang; Dawei Guan

    2012-01-01

    In this study, we sought to elucidate the process of DNA degradation in brain and dental pulp cells of mice, within postmortem 0-72 hours, by using the single cell gel electrophoresis assay and professional comet image analysis and processing techniques. The frequency of comet-like cells, the percentage of tail DNA, tail length, tail moment, Olive moment and tail area increased in tandem with increasing postmortem interval. In contrast, the head radius, the percentage of head DNA and head area showed a decreasing trend. Linear regression analysis revealed a high correlation between these parameters and the postmortem interval. The findings suggest that the single cell gel electrophoresis assay is a quick and sensitive method to detect DNA degradation in brain and dental pulp cells, providing an objective and accurate new way to estimate postmortem interval.

  9. Therapeutic potential of dental pulp stem cells in regenerative medicine: An overview

    Science.gov (United States)

    Verma, Kavita; Bains, Rhythm; Bains, Vivek Kumar; Rawtiya, Manjusha; Loomba, Kapil; Srivastava, Shrish Charan

    2014-01-01

    The purpose of this review is to gain an overview of the applications of the dental pulp stem cells (DPSCs) in the treatment of various medical diseases. Stem cells have the capacity to differentiate and regenerate into various tissues. DPSCs are the adult stem cells that reside in the cell rich zone of the dental pulp. These are the multipotent cells that can be explained by their embryonic origin from the neural crest. Owing to this multipotency, these DPSCs can be used in both dental and medical applications. A review of literature has been performed using electronic and hand-searching methods for the medical applications of DPSCs. On the basis of the available information, DPSCs appear to be a promising alternative for the regeneration of tissues and treatment of various diseases, although, long-term clinical trials and studies are needed to confirm their efficacy. PMID:25097638

  10. Dental pulp stem cells (DPSCs) differentiation study by confocal Raman microscopy

    Science.gov (United States)

    Salehi, H.; Collart-Dutilleul, P.-Y.; Gergely, C.; Cuisinier, F. J. G.

    2014-03-01

    Regenerative medicine brings a huge application for Mesenchymal stem cells such as Dental Pulp Stem Cells (DPSCs). Confocal Raman microscopy, a non-invasive, label free , real time and high spatial resolution imaging technique is used to study osteogenic differentiation of DPSCs. Integrated Raman intensities in the 2800-3000 cm-1 region (C-H stretching) and 960 cm-1 peak (phosphate PO4 3-) were collected. In Dental Pulp Stem Cells 21st day differentiated in buffer solution, phosphate peaks ν1 PO4 3- (first vibrational mode) at 960cm-1 and ν2 PO4 3- at 430cm-1 and ν4 PO4 3- at 585cm-1 are obviously present. Confocal Raman microscopy enables the detection of cell differentiation and it can be used to investigate clinical stem cell research.

  11. 3D Ultrastructural organization of whole Chlamydomonas reinhardtii cells studied by nanoscale soft x-ray tomography.

    Directory of Open Access Journals (Sweden)

    Eric Hummel

    Full Text Available The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology.

  12. AlgiMatrix™ Based 3D Cell Culture System as an In-Vitro Tumor Model for Anticancer Studies

    OpenAIRE

    Godugu, Chandraiah; Patel, Apurva R.; Desai, Utkarsh; Andey, Terrick; Sams, Alexandria; Singh, Mandip

    2013-01-01

    Background Three-dimensional (3D) in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening. Methods Cancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100–300 µm. Spheroids were grown in two weeks in cultures without co...

  13. Human dental pulp stem cell is a promising autologous seed cell for bone tissue engineering

    Institute of Scientific and Technical Information of China (English)

    LI Jing-hui; LIU Da-yong; ZHANG Fang-ming; WANG Fan; ZHANG Wen-kui; ZHANG Zhen-ting

    2011-01-01

    Background The seed cell is a core problem in bone tissue engineering research.Recent research indicates that human dental pulp stem cells (hDPSCs) can differentiate into osteoblasts in vitro,which suggests that they may become a new kind of seed cells for bone tissue engineering.The aim of this study was to evaluate the osteogenic differentiation of hDPSCs in vitro and bone-like tissue formation when transplanted with three-dimensional gelatin scaffolds in vivo,and hDPSCs may become appropriate seed cells for bone tissue engineering.Methods We have utilized enzymatic digestion to obtain hDPSCs from dental pulp tissue extracted during orthodontic treatment.After culturing and expansion to three passages,the cells were seeded in 6-well plates or on three-dimensional gelatin scaffolds and cultured in osteogenic medium.After 14 days in culture,the three-dimensional gelatin scaffolds were implanted subcutaneously in nude mice for 4 weeks.In 6-well plate culture,osteogenesis was assessed by alkaline phosphatase staining,Von Kossa staining,and reverse transcription-polymerase chain reaction (RT-PCR) analysis of the osteogenesis-specific genes type I collagen (COL l),bone sialoprotein (BSP),osteocalcin (OCN),RUNX2,and osterix (OSX).In three-dimensional gelatin scaffold culture,X-rays,hematoxylin/eosin staining,and immunohistochemical staining were used to examine bone formation.Results In vitro studies revealed that hDPSCs do possess osteogenic differentiation potential.In vivo studies revealed that hDPSCs seeded on gelatin scaffolds can form bone structures in heterotopic sites of nude mice.Conclusions These findings suggested that hDPSCs may be valuable as seed cells for bone tissue engineering.As a special stem cell source,hDPSCs may blaze a new path for bone tissue engineering.

  14. Dose distribution of IMRT and 3D-CRT on treating central non-small-cell lung cancer

    International Nuclear Information System (INIS)

    3D-CRT and IMRT were used in the radiation therapy of Central Non-small-cell lung cancer (NSCLC), and the dose difference of the methods was estimated. Thirty-two patients suffering with II class NSCLC were selected. Based on CT images, each patient was given 1 3D-CRT (3 dimensional conformal radiotherapy) and 2 IMRT(intensity modulated radiation therapy) treatment plans (5 fields and 7 fields), respectively, and the dose distribution was evaluated too. The results showed that PTVDmean and the PTVmax, PTVDmax (%) and CI of IMRT were both higher than those of 3D-CRT, but the uniformity was not as good as 3D-CRT. All indexes of lung and spinal cord treated with IMRT were lower than that treated with 3D-CRT. Moreover, there was no significance of the difference between 5 fields and 7 fields. In a conclusion, IMRT could not only decrease the target dose of NSCLC, but it can protect normal tissue from radiation damage effectively. And when IMRT was used, 5 fields might be enough. (authors)

  15. A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology.

    Directory of Open Access Journals (Sweden)

    Jonathan J Campbell

    Full Text Available Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM in three dimensional (3D space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.

  16. Structural design, layout analysis and routing strategy for constructing IC standard cells using emerging 3D vertical MOSFETs

    Science.gov (United States)

    Liu, Hongyi; Hong, Chuyang; Han, Ting; Zhou, Jun; Chen, Yijian

    2016-03-01

    As optical lithography and conventional transistor structure are approaching their physical limits, 3D vertical gate-all-around (GAA) nanowire MOSFETs and double-surrounding-gate (DSG) MOSFETs are two promising device candidates for post-FinFET logic scaling owing to their superior gate control and scaling potential. However, source, drain and gate of a vertical nanowire MOSFET and DSG MOSFETs are located in different physical layers. Consequently, structural design of IC devices/circuits, layout arrangement for high-density vertical nanowires/interconnects, and routing strategy are non-trivial challenges. In this paper, we shall discuss these critical issues for constructing standard cells using 3D vertical GAA nanowire MOSFETs and DSG MOSFETs. We redesigned the standard cells in Nangate Open Cell Library for 5nm node using vertical GAA nanowire MOSFETs and DSG MOSFETs. Experimental results verify the functionality of the proposed standard cell layout design approach.

  17. Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused 3D Porous Polymer Scaffold for Liver Tissue Engineering

    DEFF Research Database (Denmark)

    Hemmingsen, Mette; Muhammad, Haseena Bashir; Mohanty, Soumyaranjan;

    differentiation of hIPS-derived definitive endoderm (DE) cells in a 3D porous polymer scaffold built-in a perfusable bioreactor. The use of a microfluidic bioreactor array enables the culture of 16 independent tissues in one experimental run and thereby an optimization study to be performed....... to limitations of primary hepatocytes regarding availability and maintenance of functionality, stem cells and especially human induced pluripotent stem cells (hIPS cells) are an attractive cell source for liver tissue engineering. The aim of this part of NanoBio4Trans is to optimize culture and hepatic...

  18. Heterogeneity of stromal cells in the human splenic white pulp. Fibroblastic reticulum cells, follicular dendritic cells and a third superficial stromal cell type

    Science.gov (United States)

    Steiniger, Birte S; Wilhelmi, Verena; Seiler, Anja; Lampp, Katrin; Stachniss, Vitus

    2014-01-01

    At least three phenotypically and morphologically distinguishable types of branched stromal cells are revealed in the human splenic white pulp by subtractive immunohistological double-staining. CD271 is expressed in fibroblastic reticulum cells of T-cell zones and in follicular dendritic cells of follicles. In addition, there is a third CD271− and CD271+/− stromal cell population surrounding T-cell zones and follicles. At the surface of follicles the third population consists of individually variable partially overlapping shells of stromal cells exhibiting CD90 (Thy-1), MAdCAM-1, CD105 (endoglin), CD141 (thrombomodulin) and smooth muscle α-actin (SMA) with expression of CD90 characterizing the broadest shell and SMA the smallest. In addition, CXCL12, CXCL13 and CCL21 are also present in third-population stromal cells and/or along fibres. Not only CD27+ and switched B lymphocytes, but also scattered IgD++ B lymphocytes and variable numbers of CD4+ T lymphocytes often occur close to the third stromal cell population or one of its subpopulations at the surface of the follicles. In contrast to human lymph nodes, neither podoplanin nor RANKL (CD254) were detected in adult human splenic white pulp stromal cells. The superficial stromal cells of the human splenic white pulp belong to a widespread cell type, which is also found at the surface of red pulp arterioles surrounded by a mixed T-cell/B-cell population. Superficial white pulp stromal cells differ from fibroblastic reticulum cells and follicular dendritic cells not only in humans, but apparently also in mice and perhaps in rats. However, the phenotype of white pulp stromal cells is species-specific and more heterogeneous than described so far. PMID:24890772

  19. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions.

    Science.gov (United States)

    Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian; Ansari, Nariman; Esner, Milan; Bickle, Marc; Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H; Parczyk, Karsten; Prechtl, Stefan; Steigemann, Patrick

    2014-04-15

    Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.

  20. Splenic red pulp macrophages are intrinsically superparamagnetic and contaminate magnetic cell isolates.

    Science.gov (United States)

    Franken, Lars; Klein, Marika; Spasova, Marina; Elsukova, Anna; Wiedwald, Ulf; Welz, Meike; Knolle, Percy; Farle, Michael; Limmer, Andreas; Kurts, Christian

    2015-01-01

    A main function of splenic red pulp macrophages is the degradation of damaged or aged erythrocytes. Here we show that these macrophages accumulate ferrimagnetic iron oxides that render them intrinsically superparamagnetic. Consequently, these cells routinely contaminate splenic cell isolates obtained with the use of MCS, a technique that has been widely used in immunological research for decades. These contaminations can profoundly alter experimental results. In mice deficient for the transcription factor SpiC, which lack red pulp macrophages, liver Kupffer cells take over the task of erythrocyte degradation and become superparamagnetic. We describe a simple additional magnetic separation step that avoids this problem and substantially improves purity of magnetic cell isolates from the spleen. PMID:26260698

  1. Gene expression changes in bioceramic paste-treated human dental pulp cells.

    Science.gov (United States)

    Torun, Deniz; Torun, Zeynep Ö; Demirkaya, Kadriye; Sarper, Meral; Elçi, Mualla P; Avcu, Ferit

    2016-01-01

    We evaluated the gene expression profiles of human dental pulp cells exposed to iRoot BP using microarray after 24 and 72 h. The results were verified using quantitative reverse transcriptase PCR analysis. Of the 36,000 transcripts arrayed, 21 were up-regulated and 15 were down-regulated by more than two fold. The largest group of up-regulated genes included those involved in nucleobase-containing compound metabolic processes, cell communication, protein metabolic processes, developmental processes, and biological regulation. The largest groups of down-regulated genes were those involved in cell communication, development, and biological regulation processes. In conclusion, iRoot BP affects the expression of genes involved in different biological processes in human dental pulp cells. (J Oral Sci 58, 307-315, 2016). PMID:27665968

  2. Culture medium modulates the behaviour of human dental pulp-derived cells: Technical Note

    Directory of Open Access Journals (Sweden)

    S Lopez-Cazaux

    2006-02-01

    Full Text Available In vitro approaches have extensively been developed to study reparative dentinogenesis. While dental pulp is a source of unidentified progenitors able to differentiate into odontoblast-like cells, we investigated the effect of two media; MEM (1.8mM Ca and 1mM Pi and RPMI 1640 (0.8mM Ca and 5mM Pi on the behaviour of human dental pulp cells. Our data indicate that MEM significantly increased cell proliferation and markedly enhanced the proportion of -smooth muscle actin positive cells, which represent a putative source of progenitors able to give rise to odontoblast-like cells. In addition, MEM strongly stimulated alkaline phosphatase activity and was found to induce expression of transcripts encoding dentin sialophosphoprotein, an odontoblastic marker, without affecting that of parathyroid hormone/parathyroid hormone related protein-receptor and osteonectin. In conclusion, these observations demonstrate that not only proliferation but also differentiation into odontoblast-like cells was induced by rich calcium and poor phosphate medium (MEM as compared to RPMI 1640. This study provides important data for the determination of the optimal culture conditions allowing odontoblast-like differentiation in human pulp cell culture.

  3. Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis

    International Nuclear Information System (INIS)

    Research highlights: → We irradiated a 3-D human oral cell culture of keratinocytes and fibroblasts with 12 and 2 Gy. → 6 h after irradiation the histopathology and apoptosis of the 3-D culture were evaluated. Microarrays were used to assess the gene expression in the irradiated 3-D tissue. → 12 Gy induced significant histopathologic changes and cellular apoptosis. → 12 Gy significantly affected genes of the NF-kB pathway, inflammatory cytokines and DAMPs. -- Abstract: Mucositis is a debilitating adverse effect of chemotherapy and radiation treatment. It is important to develop a simple and reliable in vitro model, which can routinely be used to screen new drugs for prevention and treatment of mucositis. Furthermore, identifying cell and molecular stresses especially in the initiation phase of mucositis in this model will help towards this end. We evaluated a three-dimensional (3-D) human oral cell culture that consisted of oral keratinocytes and fibroblasts as a model of oral mucositis. The 3-D cell culture model was irradiated with 12 or 2 Gy. Six hours after the irradiation we evaluated microscopic sections of the cell culture for evidence of morphologic changes including apoptosis. We used microarrays to compare the expression of several genes from the irradiated tissue with identical genes from tissue that was not irradiated. We found that irradiation with 12 Gy induced significant histopathologic effects including cellular apoptosis. Irradiation significantly affected the expression of several genes of the NF-kB pathway and several inflammatory cytokines, such as IL-1B, 1L-8, NF-kB1, and FOS compared to tissue that was not irradiated. We identified significant upregulation of several genes that belong to damage-associated molecular patterns (DAMPs) such as HMB1, S100A13, SA10014, and SA10016 in the 3-D tissues that received 12 Gy but not in tissues that received 2 Gy. In conclusion, this model quantifies radiation damage and this is an important first

  4. Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis

    Energy Technology Data Exchange (ETDEWEB)

    Lambros, Maria Polikandritou, E-mail: mlambros@westernu.edu [Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Parsa, Cyrus [Department of Clinical Sciences, College of Osteopathic Medicine, Western University of Health Sciences, Pomona, CA 91766 (United States); Mulamalla, HariChandana [Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Orlando, Robert [Department of Clinical Sciences, College of Osteopathic Medicine, Western University of Health Sciences, Pomona, CA 91766 (United States); Lau, Bernard [Center for Advancement of Drug Research and Evaluation (CADRE), Western University of Health Sciences, Pomona, CA 91766 (United States); Huang, Ying [Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Center for Advancement of Drug Research and Evaluation (CADRE), Western University of Health Sciences, Pomona, CA 91766 (United States); Pon, Doreen [Department of Pharmacy Practice and Administration, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Chow, Moses [Department of Pharmacy Practice and Administration, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Center for Advancement of Drug Research and Evaluation (CADRE), Western University of Health Sciences, Pomona, CA 91766 (United States)

    2011-02-04

    Research highlights: {yields} We irradiated a 3-D human oral cell culture of keratinocytes and fibroblasts with 12 and 2 Gy. {yields} 6 h after irradiation the histopathology and apoptosis of the 3-D culture were evaluated. Microarrays were used to assess the gene expression in the irradiated 3-D tissue. {yields} 12 Gy induced significant histopathologic changes and cellular apoptosis. {yields} 12 Gy significantly affected genes of the NF-kB pathway, inflammatory cytokines and DAMPs. -- Abstract: Mucositis is a debilitating adverse effect of chemotherapy and radiation treatment. It is important to develop a simple and reliable in vitro model, which can routinely be used to screen new drugs for prevention and treatment of mucositis. Furthermore, identifying cell and molecular stresses especially in the initiation phase of mucositis in this model will help towards this end. We evaluated a three-dimensional (3-D) human oral cell culture that consisted of oral keratinocytes and fibroblasts as a model of oral mucositis. The 3-D cell culture model was irradiated with 12 or 2 Gy. Six hours after the irradiation we evaluated microscopic sections of the cell culture for evidence of morphologic changes including apoptosis. We used microarrays to compare the expression of several genes from the irradiated tissue with identical genes from tissue that was not irradiated. We found that irradiation with 12 Gy induced significant histopathologic effects including cellular apoptosis. Irradiation significantly affected the expression of several genes of the NF-kB pathway and several inflammatory cytokines, such as IL-1B, 1L-8, NF-kB1, and FOS compared to tissue that was not irradiated. We identified significant upregulation of several genes that belong to damage-associated molecular patterns (DAMPs) such as HMB1, S100A13, SA10014, and SA10016 in the 3-D tissues that received 12 Gy but not in tissues that received 2 Gy. In conclusion, this model quantifies radiation damage and this

  5. Micro 3D cell culture systems for cellular behavior studies: Culture matrices, devices, substrates, and in-situ sensing methods.

    Science.gov (United States)

    Choi, Jonghoon; Lee, Eun Kyu; Choo, Jaebum; Yuh, Junhan; Hong, Jong Wook

    2015-09-01

    Microfabricated systems equipped with 3D cell culture devices and in-situ cellular biosensing tools can be a powerful bionanotechnology platform to investigate a variety of biomedical applications. Various construction substrates such as plastics, glass, and paper are used for microstructures. When selecting a construction substrate, a key consideration is a porous microenvironment that allows for spheroid growth and mimics the extracellular matrix (ECM) of cell aggregates. Various bio-functionalized hydrogels are ideal candidates that mimic the natural ECM for 3D cell culture. When selecting an optimal and appropriate microfabrication method, both the intended use of the system and the characteristics and restrictions of the target cells should be carefully considered. For highly sensitive and near-cell surface detection of excreted cellular compounds, SERS-based microsystems capable of dual modal imaging have the potential to be powerful tools; however, the development of optical reporters and nanoprobes remains a key challenge. We expect that the microsystems capable of both 3D cell culture and cellular response monitoring would serve as excellent tools to provide fundamental cellular behavior information for various biomedical applications such as metastasis, wound healing, high throughput screening, tissue engineering, regenerative medicine, and drug discovery and development. PMID:26358782

  6. 3D Plant Cell Architecture of Arabidopsis thaliana (Brassicaceae Using Focused Ion Beam–Scanning Electron Microscopy

    Directory of Open Access Journals (Sweden)

    Bhawana

    2014-06-01

    Full Text Available Premise of the study: Focused ion beam–scanning electron microscopy (FIB-SEM combines the ability to sequentially mill the sample surface and obtain SEM images that can be used to create 3D renderings with micron-level resolution. We have applied FIB-SEM to study Arabidopsis cell architecture. The goal was to determine the efficacy of this technique in plant tissue and cellular studies and to demonstrate its usefulness in studying cell and organelle architecture and distribution. Methods: Seed aleurone, leaf mesophyll, stem cortex, root cortex, and petal lamina from Arabidopsis were fixed and embedded for electron microscopy using protocols developed for animal tissues and modified for use with plant cells. Each sample was sectioned using the FIB and imaged with SEM. These serial images were assembled to produce 3D renderings of each cell type. Results: Organelles such as nuclei and chloroplasts were easily identifiable, and other structures such as endoplasmic reticula, lipid bodies, and starch grains were distinguishable in each tissue. Discussion: The application of FIB-SEM produced 3D renderings of five plant cell types and offered unique views of their shapes and internal content. These results demonstrate the usefulness of FIB-SEM for organelle distribution and cell architecture studies.

  7. Distribution of class ii major histocompatibility complex antigenexpressing cells in human dental pulp with carious lesions

    OpenAIRE

    Tetiana Haniastuti

    2012-01-01

    Background: Dental caries is a bacterial infection which causes destruction of the hard tissues of the tooth. Exposure of the dentin to the oral environment as a result of caries inevitably results in a cellular response in the pulp. The major histocompatibility complex (MHC) is a group of genes that code for cell-surface histocompatibility antigens. Cells expressing class II MHC molecules participate in the initial recognition and the processing of antigenic substances to serve as antigen-pr...

  8. Splenic red pulp macrophages are intrinsically superparamagnetic and contaminate magnetic cell isolates

    OpenAIRE

    Lars Franken; Marika Klein; Marina Spasova; Anna Elsukova; Ulf Wiedwald; Meike Welz; Percy Knolle; Michael Farle; Andreas Limmer; Christian Kurts

    2015-01-01

    A main function of splenic red pulp macrophages is the degradation of damaged or aged erythrocytes. Here we show that these macrophages accumulate ferrimagnetic iron oxides that render them intrinsically superparamagnetic. Consequently, these cells routinely contaminate splenic cell isolates obtained with the use of MCS, a technique that has been widely used in immunological research for decades. These contaminations can profoundly alter experimental results. In mice deficient for the transcr...

  9. Effects of GPNMB on proliferation and odontoblastic differentiation of human dental pulp cells

    OpenAIRE

    Wang, Yu-Liang; Hu, Ye-Jia; Zhang, Feng-He

    2015-01-01

    Glycoprotein (transmembrane) nonmetastatic melanoma protein b (GPNMB) plays crucial roles in odontogenesis. However, the role of GPNMB in human dental pulp cells (hDPCs) is still unclear. Therefore, in this study, we investigated the expression and function of the GPNMB in odontoblastic differentiation of hDPCs. Cells were cultured in odontoblast differentiation-inducing medium; the expression of the GPNMB was assessed by reverse transcriptase polymerase chain reaction and Western blot analys...

  10. Laser 3D printing with sub-microscale resolution of porous elastomeric scaffolds for supporting human bone stem cells.

    Science.gov (United States)

    Petrochenko, Peter E; Torgersen, Jan; Gruber, Peter; Hicks, Lucas A; Zheng, Jiwen; Kumar, Girish; Narayan, Roger J; Goering, Peter L; Liska, Robert; Stampfl, Jürgen; Ovsianikov, Aleksandr

    2015-04-01

    A reproducible method is needed to fabricate 3D scaffold constructs that results in periodic and uniform structures with precise control at sub-micrometer and micrometer length scales. In this study, fabrication of scaffolds by two-photon polymerization (2PP) of a biodegradable urethane and acrylate-based photoelastomer is demonstrated. This material supports 2PP processing with sub-micrometer spatial resolution. The high photoreactivity of the biophotoelastomer permits 2PP processing at a scanning speed of 1000 mm s(-1), facilitating rapid fabrication of relatively large structures (>5 mm(3)). These structures are custom printed for in vitro assay screening in 96-well plates and are sufficiently flexible to enable facile handling and transplantation. These results indicate that stable scaffolds with porosities of greater than 60% can be produced using 2PP. Human bone marrow stromal cells grown on 3D scaffolds exhibit increased growth and proliferation compared to smooth 2D scaffold controls. 3D scaffolds adsorb larger amounts of protein than smooth 2D scaffolds due to their larger surface area; the scaffolds also allow cells to attach in multiple planes and to completely infiltrate the porous scaffolds. The flexible photoelastomer material is biocompatible in vitro and is associated with facile handling, making it a viable candidate for further study of complex 3D-printed scaffolds.

  11. Characterizing microscale aluminum composite layer properties on silicon solar cells with hybrid 3D scanning force measurements

    Science.gov (United States)

    Bae, Sung-Kuk; Choi, Beomjoon; Chung, Haseung; Shin, Seungwon; Song, Hee-Eun; Seo, Jung Hwan

    2016-03-01

    This article presents a novel technique to estimate the mechanical properties of the aluminum composite layer on silicon solar cells by using a hybrid 3-dimensional laser scanning force measurement (3-D LSFM) system. The 3-D LSFM system measures the material properties of sub-layers constituting a solar cell. This measurement is critical for realizing high-efficient ultra-thin solar cells. The screen-printed aluminum layer, which significantly affects the bowing phenomenon, is separated from the complete solar cell by removing the silicon (Si) layer with deep reactive ion etching. An elastic modulus of ~15.1 GPa and a yield strength of ~35.0 MPa for the aluminum (Al) composite layer were obtained by the 3-D LSFM system. In experiments performed for 6-inch Si solar cells, the bowing distances decreased from 12.02 to 1.18 mm while the Si layer thicknesses increased from 90 to 190 μm. These results are in excellent agreement with the theoretical predictions for ultra-thin Si thickness (90 μm) based on the obtained Al composite layer properties.

  12. Lamellipodin promotes invasive 3D cancer cell migration via regulated interactions with Ena/VASP and SCAR/WAVE.

    Science.gov (United States)

    Carmona, G; Perera, U; Gillett, C; Naba, A; Law, A-L; Sharma, V P; Wang, J; Wyckoff, J; Balsamo, M; Mosis, F; De Piano, M; Monypenny, J; Woodman, N; McConnell, R E; Mouneimne, G; Van Hemelrijck, M; Cao, Y; Condeelis, J; Hynes, R O; Gertler, F B; Krause, M

    2016-09-29

    Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlate with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement, we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation and matrix degradation was impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not with Ena/VASP is required for random 2D cell migration. We identified a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, whereas Src-dependent phosphorylation enhances binding to Scar/WAVE but not to Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of epidermal growth factor (EGF) gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis.

  13. A photopolymerizable hydrogel for 3-D culture of human embryonic stem cell-derived cardiomyocytes and rat neonatal cardiac cells.

    Science.gov (United States)

    Shapira-Schweitzer, Keren; Habib, Manhal; Gepstein, Lior; Seliktar, Dror

    2009-02-01

    The purpose of this study was to assess the in vitro ability of two types of cardiomyocytes (cardiomyocytes derived from human embryonic stem cells (hESC-CM) and rat neonatal cardiomyocytes (rN-CM)) to survive and generate a functional cardiac syncytium in a three-dimensional in situ polymerizable hydrogel environment. Each cell type was cultured in a PEGylated fibrinogen (PF) hydrogel for up to two weeks while maturation and cardiac function were documented in terms of spontaneous contractile behavior and biomolecular organization. Quantitative contractile parameters including contraction amplitude and synchronization were measured by non-invasive image analysis. The rN-CM demonstrated the fastest maturation and the most significant spontaneous contraction. The hESC-CM maturation occurred between 10-14 days in culture, and exhibited less contraction amplitude and synchronization in comparison to the rN-CMs. The maturation of both cell types within the hydrogels was confirmed by cardiac-specific biomolecular markers, including alpha-sarcomeric actin, actinin, and connexin-43. Cellular responsiveness to isoproterenol, carbamylcholine and heptanol provided further evidence of the cardiac maturation in the 3-D PF hydrogel as well as identified a potential to use this system for in vitro drug screening. These findings indicate that the PF hydrogel biomaterial can be used as an in situ polymerizable biomaterial for stem cells and their cardiomyocyte derivatives. PMID:19027751

  14. Inhibition of matrix metalloproteinases expression in human dental pulp cells by all-trans retinoic acid

    Institute of Scientific and Technical Information of China (English)

    Jin Man Kim; Sang Wook Kang; Su-Mi Shin; Duck Su Kim; Kyong-Kyu Choi; Eun-Cheol Kim; Sun-Young Kim

    2014-01-01

    All-trans retinoic acid (ATRA) inhibits matrix metalloproteinase (MMP)-2 and MMP-9 in synovial fibroblasts, skin fibroblasts, bronchoalveolar lavage cells and cancer cells, but activates MMP-9 in neuroblast and leukemia cells. Very little is known regarding whether ATRA can activate or inhibit MMPs in human dental pulp cells (HDPCs). The purpose of this study was to determine the effects of ATRA on the production and secretion of MMP-2 and-9 in HDPCs. The productions and messenger RNA (mRNA) expressions of MMP-2 and-9 were accessed by gelatin zymography and real-time polymerase chain reaction (PCR), respectively. ATRA was found to decrease MMP-2 level in a dose-dependent manner. Significant reduction in MMP-2 mRNA expression was also observed in HDPCs treated with 25 mmol?L21 ATRA. However, HDPCs treated with ATRA had no effect on the pattern of MMP-9 produced or secreted in either cell extracts or conditioned medium fractions. Taken together, ATRA had an inhibitory effect on MMP-2 expression in HDPCs, which suggests that ATRA could be a candidate as a medicament which could control the inflammation of pulp tissue in vital pulp therapy and regenerative endodontics.

  15. CONSTRUCTION OF RECOMBINANT PLASMID pcDNA3.1/BMP-2 AND ITS INVOLVEMENT IN DIFFERENTIATION OF HUMAN DENTAL PULP-DERIVED CELLS INTO AN ODONTOBLASTIC LINEAGE

    Directory of Open Access Journals (Sweden)

    Boy M. Bachtiar

    2009-06-01

    Full Text Available Experimental studies have shown that dental pulp tissue has potential to regenerate dentine in response to adverse stimuli, such as caries and associated operative procedures. However, the potential of dental pulp regeneration seems to be limited by regenerative capacity of the cell involved. In this study, we report the effect of transfection of a recombinant plasmid containing human BMP-2 gene in proliferation and differentiation of dental pulp tissue in vitro. The regenerative capacity was analyzed by ALP production and calcium content. Results showed that the transfected dental pulp cell was able to differentiate into the odontoblast phenotype, indicating the presence of odontoblast progentitor cells in dental pulp tissue.

  16. Bladder Smooth Muscle Cells Differentiation from Dental Pulp Stem Cells: Future Potential for Bladder Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Bing Song

    2016-01-01

    Full Text Available Dental pulp stem cells (DPSCs are multipotent cells capable of differentiating into multiple cell lines, thus providing an alternative source of cell for tissue engineering. Smooth muscle cell (SMC regeneration is a crucial step in tissue engineering of the urinary bladder. It is known that DPSCs have the potential to differentiate into a smooth muscle phenotype in vitro with differentiation agents. However, most of these studies are focused on the vascular SMCs. The optimal approaches to induce human DPSCs to differentiate into bladder SMCs are still under investigation. We demonstrate in this study the ability of human DPSCs to differentiate into bladder SMCs in a growth environment containing bladder SMCs-conditioned medium with the addition of the transforming growth factor beta 1 (TGF-β1. After 14 days of exposure to this medium, the gene and protein expression of SMC-specific marker (α-SMA, desmin, and calponin increased over time. In particular, myosin was present in differentiated cells after 11 days of induction, which indicated that the cells differentiated into the mature SMCs. These data suggested that human DPSCs could be used as an alternative and less invasive source of stem cells for smooth muscle regeneration, a technology that has applications for bladder tissue engineering.

  17. Application of Induced Pluripotent Stem Cells Reprogrammed from Dental Pulp Cells: a Novel Approach for Tooth Regeneration

    Directory of Open Access Journals (Sweden)

    Xiaoyan Zhou

    2011-03-01

    Full Text Available Introduction: Candidate human dental stem/progenitor cells have been isolated and charac-terized from dental tissues and shown to hold the capability to differentiate into tooth-generating cells. However, ad-vances in engineering a whole tooth by these stem cells are hindered by various factors, such as the poor availability of human primitive tooth bud stem cells, difficulties in isolating and purifying dental mesenchymal stem cells and ethical controversies when using embryonic oral epithelium. As a result it is meaningful to find other autologous dental cells for the purpose of reconstructing a tooth.The hypothesis: Previous studies demonstrated that somatic cells can be reprogrammed into induced pluripotent stem cells by ex-ogenous expression Oct-4 and Sox-2. On the basis of these findings we can reasonably hypothesize that when transfected with specific transcription factors Oct-4 and Sox-2, dental pulp cells, the main cell in pulp, could also be reprogrammed into induced pluripotent stem cells, which are considered to be of best potential to regenerate a whole tooth. Evaluation of the hypothesis: After transfection with Oct-4 and Sox-2 into human dental pulp cells, the positive colonies are isolated and then identified according to the characteristics of iPS cells. These cells are further investigated the capability in differentiating into ameloblasts and odontoblasts and finally seeded onto the sur-face of a tooth-shaped biodegradable polymer scaffold to detect the ability of constructing a bioengineered tooth.

  18. Synthesis and Characterization of a Photoelectrode with a Novel 3D Structure for Dye-Sensitized Solar Cells

    Directory of Open Access Journals (Sweden)

    Kun-Ching Cho

    2014-01-01

    Full Text Available This study designs a novel dye-sensitized solar cell (DSSC in which the photoanode is derived from its three-dimensional (3D structure. The inside of the cell has a positive illumination structure, with the purposes of increasing the area of photoelectrode thin film and of increasing the illuminated area within a fixed area in order to achieve the objective of enhancing the photoelectric conversion efficiency of cell. For the cell structure experiment, the study uses graphite paper, carbon and platinum as counter electrode materials, and then conducts measurement with cell heights of 3 mm, 5 mm, and 7 mm. The electrolyte used is a gel polymer electrolyte. The assembly of the cell is divided into vertical assembly, inclined assembly, and tandem assembly. In the 3D tandem cell experiment, the counter electrode material is platinum. Experimental results show that when cell height is 7 mm and illuminated area is 0.28 cm2, open-loop voltage is 0.662 V, short-circuit current density is 18.42 mA/cm2, fill factor is 0.31, and the photoelectric conversion efficiency is 3.85%, which is 1.65 times that under vertical assembly (2.34% and 2.15 times that of the flat cell (1.79%.

  19. 3D exploration of light scattering from live cells in the presence of gold nanomarkers using holographic microscopy

    CERN Document Server

    Joud, Fadwa; Bun, P; Verpillat, Frédéric; Suck, Sarah Y; Tessier, G; Atlan, Michael; Desbiolles, Pierre; Coppey-Moisan, Maite; Abboud, Marie; Gross, Michel

    2011-01-01

    In this paper, we explore the 3D structure of light scattering from dark-field illuminated live 3T3 cells in the presence of 40 nm gold nanomarkers. For this purpose, we use a high resolution holographic microscope combining the off-axis heterodyne geometry and the phase-shifting acquisition of the digital holograms. A comparative study of the 3D reconstructions of the scattered fields allows us to locate the gold markers which yield, contrarily to the cell structures, well defined bright scattering patterns that are not angularly titled and clearly located along the optical axis (z). This characterization is an unambiguous signature of the presence of gold biological nanomarkers, and validates the capability of digital holographic microscopy to discriminate them from background signals in live ce

  20. Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells.

    Science.gov (United States)

    Romero-Brey, Inés; Bartenschlager, Ralf

    2015-12-01

    As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications.

  1. Epstein-Barr virus (EBV) infection of murine L cells expressing recombinant human EBV/C3d receptor.

    OpenAIRE

    Ahearn, J M; Hayward, S D; Hickey, J C; Fearon, D T

    1988-01-01

    The normal host range of Epstein-Barr virus (EBV) is limited to primate B lymphocytes and certain epithelial cells that express the C3d/EBV receptor [complement receptor 2 (CR2, CD21)]. In the present study, expansion of the tissue tropism of EBV has been accomplished by stably transfecting the murine fibroblast L cell line with pMT.CR2. neo.1, a eukaryotic expression vector promoting the transcription of a complementary DNA insert encoding human CR2. High CR2-expressing transfected L cells w...

  2. Scanning transmission and computer-aided volumic electron microscopy: 3-D modeling of entire cells by electronic imaging

    Science.gov (United States)

    Bron, Christophe; Gremillet, Philip; Launay, D.; Jourlin, Michel; Gautschi, H. P.; Baechi, Thomas; Schuepbach, Joerg

    1990-05-01

    The digital processing of electron microscopic images from serial sections containing laser-induced topographical references allows a 3-D reconstruction at a depth resolution of 30 to 40 nm of entire cells by the use of image analysis methods, as already demonstrated for Transmission Electron Microscopy (TEM) coupled with a video camera. We decided to use a Scanning Transmission Electron Microscope (STEM) to get higher contrast and better resolution at medium magnification. The scanning of our specimens at video frequencies is an attractive and easy way to link a STEM with an image processing system but the hysteresis of the electronic spools responsible for the magnetic deviation of the scanning electron beam induces deformations of images which have to be modelized and corrected before registration. Computer algorithms developed for image analysis and treatment correct the artifacts caused by the use of STEM and by serial sectioning to automatically reconstruct the third dimension of the cells. They permit the normalization of the images through logarithmic processing of the original grey level infonnation. The automatic extraction of cell limits allows to link the image analysis and treatments with image synthesis methods by minimal human intervention. The surface representation and the registered images provide an ultrastructural data base from which quantitative 3-D morphological parameters, as well as otherwise impossible visualizations, can be computed. This 3-D image processing named C.A.V.U.M. for Computer Aided Volumic Ultra-Microscopy offers a new tool for the documentation and analysis of cell ultrastructure and for 3-D morphometric studies at EM magnifications. Further, a virtual observer can be computed in such a way as to simulate a visit of the reconstructed object.

  3. Exploring natural silk protein sericin for regenerative medicine: an injectable, photoluminescent, cell-adhesive 3D hydrogel

    OpenAIRE

    Wang, Zheng; Zhang, Yeshun; Zhang, Jinxiang; Huang, Lei; Liu, Jia; Li, Yongkui; Zhang, Guozheng; Kundu, Subhas C.; Wang, Lin

    2014-01-01

    Sericin, a major component of silk, has a long history of being discarded as a waste during silk processing. The value of sericin for tissue engineering is underestimated and its potential application in regenerative medicine has just begun to be explored. Here we report the successful fabrication and characterization of a covalently-crosslinked 3D pure sericin hydrogel for delivery of cells and drugs. This hydrogel is injectable, permitting its implantation through minimally invasive approac...

  4. Cellular differentiation in 3D-bioprinted mesenchymal stem cell-loaded hydrogels with varying structural and mechanical properties

    OpenAIRE

    Duarte Campos, Daniela Filipa

    2016-01-01

    Hydrogels are a promising alternative to rigid biomaterials typically used in the field of bone tissue engineering for the treatment of musculoskeletal disorders. By hydrogel-based 3D-bioprinting, the native ornamentation of cells and matrix from bone tissue could be resembled. Herein, it was hypothesized the combination of polysaccharides (agarose, alginate) with biological components (collagen, fibrinogen) would increase mechanical stiffness of printed constructs as well as support the prin...

  5. Tuning the Mechanical Properties of Poly(Ethylene Glycol) Microgel-Based Scaffolds to Increase 3D Schwann Cell Proliferation.

    Science.gov (United States)

    Zhou, Wenda; Stukel, Jessica M; Cebull, Hannah L; Willits, Rebecca Kuntz

    2016-04-01

    2D in vitro studies have demonstrated that Schwann cells prefer scaffolds with mechanical modulus approximately 10× higher than the modulus preferred by nerves, limiting the ability of many scaffolds to promote both neuron extension and Schwann cell proliferation. Therefore, the goals of this work are to develop and characterize microgel-based scaffolds that are tuned over the stiffness range relevant to neural tissue engineering and investigate Schwann cell morphology, viability, and proliferation within 3D scaffolds. Using thiol-ene reaction, microgels with surface thiols are produced and crosslinked into hydrogels using a multiarm vinylsulfone (VS). By varying the concentration of VS, scaffold stiffness ranges from 0.13 to 0.76 kPa. Cell morphology in all groups demonstrates that cells are able to spread and interact with the scaffold through day 5. Although the viability in all groups is high, proliferation of Schwann cells within the scaffold of G* = 0.53 kPa is significantly higher than other groups. This result is ≈ 5× lower than previously reported optimal stiffnesses on 2D surfaces, demonstrating the need for correlation of 3D cell response to mechanical modulus. As proliferation is the first step in Schwann cell integration into peripheral nerve conduits, these scaffolds demonstrate that the stiffness is a critical parameter to optimizing the regenerative process.

  6. Collagen esterification enhances the function and survival of pancreatic β cells in 2D and 3D culture systems

    International Nuclear Information System (INIS)

    Collagen, one of the most important components of the extracellular matrix (ECM), may play a role in the survival of pancreatic islet cells. In addition, chemical modifications that change the collagen charge profile to a net positive charge by esterification have been shown to increase the adhesion and proliferation of various cell types. The purpose of this study was to characterize and compare the effects of native collagen (NC) and esterified collagen (EC) on β cell function and survival. After isolation by the collagenase digestion technique, rat islets were cultured with NC and EC in 2 dimensional (2D) and 3 dimensional (3D) environments for a long-term duration in vitro. The cells were assessed for islet adhesion, morphology, viability, glucose-induced insulin secretion, and mRNA expression of glucose metabolism-related genes, and visualized by scanning electron microscopy (SEM). Islet cells attached tightly in the NC group, but islet cell viability was similar in both the NC and EC groups. Glucose-stimulated insulin secretion was higher in the EC group than in the NC group in both 2D and 3D culture. Furthermore, the mRNA expression levels of glucokinase in the EC group were higher than those in the NC group and were associated with glucose metabolism and insulin secretion. Finally, SEM observation confirmed that islets had more intact component cells on EC sponges than on NC sponges. These results indicate that modification of collagen may offer opportunities to improve function and viability of islet cells. - Highlights: • We changed the collagen charge profile to a net positive charge by esterification. • Islets cultured on esterified collagen improved survival in both 2D and 3D culture. • Islets cultured on esterified collagen enhanced glucose-stimulated insulin release. • High levels of glucokinase mRNA may be associated with increased insulin release

  7. Collagen esterification enhances the function and survival of pancreatic β cells in 2D and 3D culture systems

    Energy Technology Data Exchange (ETDEWEB)

    Ko, Jae Hyung [Regenerative Medicine Research Center, Dalim Tissen Co., LTD., 383-93, Yonnam-Dong, Mapo-gu, Seoul (Korea, Republic of); Kim, Yang Hee [Regenerative Medicine Research Center, Dalim Tissen Co., LTD., 383-93, Yonnam-Dong, Mapo-gu, Seoul (Korea, Republic of); Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Jeong, Seong Hee; Lee, Song [Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Park, Si-Nae [Regenerative Medicine Research Center, Dalim Tissen Co., LTD., 383-93, Yonnam-Dong, Mapo-gu, Seoul (Korea, Republic of); Shim, In Kyong, E-mail: shimiink@gmail.com [Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Kim, Song Cheol, E-mail: drksc@amc.seoul.kr [Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Department of Surgery, University of Ulsan College of Medicine & Asan Medical Center, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of)

    2015-08-07

    Collagen, one of the most important components of the extracellular matrix (ECM), may play a role in the survival of pancreatic islet cells. In addition, chemical modifications that change the collagen charge profile to a net positive charge by esterification have been shown to increase the adhesion and proliferation of various cell types. The purpose of this study was to characterize and compare the effects of native collagen (NC) and esterified collagen (EC) on β cell function and survival. After isolation by the collagenase digestion technique, rat islets were cultured with NC and EC in 2 dimensional (2D) and 3 dimensional (3D) environments for a long-term duration in vitro. The cells were assessed for islet adhesion, morphology, viability, glucose-induced insulin secretion, and mRNA expression of glucose metabolism-related genes, and visualized by scanning electron microscopy (SEM). Islet cells attached tightly in the NC group, but islet cell viability was similar in both the NC and EC groups. Glucose-stimulated insulin secretion was higher in the EC group than in the NC group in both 2D and 3D culture. Furthermore, the mRNA expression levels of glucokinase in the EC group were higher than those in the NC group and were associated with glucose metabolism and insulin secretion. Finally, SEM observation confirmed that islets had more intact component cells on EC sponges than on NC sponges. These results indicate that modification of collagen may offer opportunities to improve function and viability of islet cells. - Highlights: • We changed the collagen charge profile to a net positive charge by esterification. • Islets cultured on esterified collagen improved survival in both 2D and 3D culture. • Islets cultured on esterified collagen enhanced glucose-stimulated insulin release. • High levels of glucokinase mRNA may be associated with increased insulin release.

  8. Trophic Effects and Regenerative Potential of Mobilized Mesenchymal Stem Cells From Bone Marrow and Adipose Tissue as Alternative Cell Sources for Pulp/Dentin Regeneration.

    Science.gov (United States)

    Murakami, Masashi; Hayashi, Yuki; Iohara, Koichiro; Osako, Yohei; Hirose, Yujiro; Nakashima, Misako

    2015-01-01

    Dental pulp stem cell (DPSC) subsets mobilized by granulocyte-colony-stimulating factor (G-CSF) are safe and efficacious for complete pulp regeneration. The supply of autologous pulp tissue, however, is very limited in the aged. Therefore, alternative sources of mesenchymal stem/progenitor cells (MSCs) are needed for the cell therapy. In this study, DPSCs, bone marrow (BM), and adipose tissue (AD)-derived stem cells of the same individual dog were isolated using G-CSF-induced mobilization (MDPSCs, MBMSCs, and MADSCs). The positive rates of CXCR4 and G-CSFR in MDPSCs were similar to MADSCs and were significantly higher than those in MBMSCs. Trophic effects of MDPSCs on angiogenesis, neurite extension, migration, and antiapoptosis were higher than those of MBMSCs and MADSCs. Pulp-like loose connective tissues were regenerated in all three MSC transplantations. Significantly higher volume of regenerated pulp and higher density of vascularization and innervation were observed in response to MDPSCs compared to MBMSC and MADSC transplantation. Collagenous matrix containing dentin sialophosphoprotein (DSPP)-positive odontoblast-like cells was the highest in MBMSCs and significantly higher in MADSCs compared to MDPSCs. MBMSCs and MADSCs, therefore, have potential for pulp regeneration, although the volume of regenerated pulp tissue, angiogenesis, and reinnervation, were less. Thus, in conclusion, an alternative cell source for dental pulp/dentin regeneration are stem cells from BM and AD tissue.

  9. Ammonium accumulation and cell death in a rat 3D brain cell model of glutaric aciduria type I.

    Directory of Open Access Journals (Sweden)

    Paris Jafari

    Full Text Available Glutaric aciduria type I (glutaryl-CoA dehydrogenase deficiency is an inborn error of metabolism that usually manifests in infancy by an acute encephalopathic crisis and often results in permanent motor handicap. Biochemical hallmarks of this disease are elevated levels of glutarate and 3-hydroxyglutarate in blood and urine. The neuropathology of this disease is still poorly understood, as low lysine diet and carnitine supplementation do not always prevent brain damage, even in early-treated patients. We used a 3D in vitro model of rat organotypic brain cell cultures in aggregates to mimic glutaric aciduria type I by repeated administration of 1 mM glutarate or 3-hydroxyglutarate at two time points representing different developmental stages. Both metabolites were deleterious for the developing brain cells, with 3-hydroxyglutarate being the most toxic metabolite in our model. Astrocytes were the cells most strongly affected by metabolite exposure. In culture medium, we observed an up to 11-fold increase of ammonium in the culture medium with a concomitant decrease of glutamine. We further observed an increase in lactate and a concomitant decrease in glucose. Exposure to 3-hydroxyglutarate led to a significantly increased cell death rate. Thus, we propose a three step model for brain damage in glutaric aciduria type I: (i 3-OHGA causes the death of astrocytes, (ii deficiency of the astrocytic enzyme glutamine synthetase leads to intracerebral ammonium accumulation, and (iii high ammonium triggers secondary death of other brain cells. These unexpected findings need to be further investigated and verified in vivo. They suggest that intracerebral ammonium accumulation might be an important target for the development of more effective treatment strategies to prevent brain damage in patients with glutaric aciduria type I.

  10. Mystery Inside the Tooth: The Dental Pulp Stem Cells

    OpenAIRE

    Masthan, K.M.K.; Sankari, S. Leena; Babu, N. Aravindha; Gopalakrishnan, T.

    2013-01-01

    Stem cells are distinguished by their ability to differentiate into different types of cells in the body and to self-replicate. During the recent years, stem cells have been used extensively in the field of medicine for the repair and regeneration of defective tissues and organs. However, the knowledge on the stem cell technology is increasing quickly in all medical disciplines and it dictates the need for new protective approaches in all fields, which include reparative dentistry. Stem cell ...

  11. Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro.

    Science.gov (United States)

    Chun, So Young; Soker, Shay; Jang, Yu-Jin; Kwon, Tae Gyun; Yoo, Eun Sang

    2016-02-01

    We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

  12. A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

    Science.gov (United States)

    Lei, Yuguo; Schaffer, David V.

    2013-12-01

    Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human- or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage, for a 1072-fold expansion over 280 d), yield (∼2.0 × 107 cells per mL of hydrogel), and purity (∼95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ∼8 × 107 dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development.

  13. AlgiMatrix™ based 3D cell culture system as an in-vitro tumor model for anticancer studies.

    Directory of Open Access Journals (Sweden)

    Chandraiah Godugu

    Full Text Available BACKGROUND: Three-dimensional (3D in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening. METHODS: Cancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100-300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin and nanoparticle (NLC were done using spheroids. RESULTS: IC(50 values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro. CONCLUSION: The results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations.

  14. Modulation of Dental Pulp Stem Cell Odontogenesis in a Tunable PEG-Fibrinogen Hydrogel System.

    Science.gov (United States)

    Lu, Qiqi; Pandya, Mirali; Rufaihah, Abdul Jalil; Rosa, Vinicius; Tong, Huei Jinn; Seliktar, Dror; Toh, Wei Seong

    2015-01-01

    Injectable hydrogels have the great potential for clinical translation of dental pulp regeneration. A recently developed PEG-fibrinogen (PF) hydrogel, which comprises a bioactive fibrinogen backbone conjugated to polyethylene glycol (PEG) side chains, can be cross-linked after injection by photopolymerization. The objective of this study was to investigate the use of this hydrogel, which allows tuning of its mechanical properties, as a scaffold for dental pulp tissue engineering. The cross-linking degree of PF hydrogels could be controlled by varying the amounts of PEG-diacrylate (PEG-DA) cross-linker. PF hydrogels are generally cytocompatible with the encapsulated dental pulp stem cells (DPSCs), yielding >85% cell viability in all hydrogels. It was found that the cell morphology of encapsulated DPSCs, odontogenic gene expression, and mineralization were strongly modulated by the hydrogel cross-linking degree and matrix stiffness. Notably, DPSCs cultured within the highest cross-linked hydrogel remained mostly rounded in aggregates and demonstrated the greatest enhancement in odontogenic gene expression. Consistently, the highest degree of mineralization was observed in the highest cross-linked hydrogel. Collectively, our results indicate that PF hydrogels can be used as a scaffold for DPSCs and offers the possibility of influencing DPSCs in ways that may be beneficial for applications in regenerative endodontics. PMID:26124841

  15. Modulation of Dental Pulp Stem Cell Odontogenesis in a Tunable PEG-Fibrinogen Hydrogel System

    Directory of Open Access Journals (Sweden)

    Qiqi Lu

    2015-01-01

    Full Text Available Injectable hydrogels have the great potential for clinical translation of dental pulp regeneration. A recently developed PEG-fibrinogen (PF hydrogel, which comprises a bioactive fibrinogen backbone conjugated to polyethylene glycol (PEG side chains, can be cross-linked after injection by photopolymerization. The objective of this study was to investigate the use of this hydrogel, which allows tuning of its mechanical properties, as a scaffold for dental pulp tissue engineering. The cross-linking degree of PF hydrogels could be controlled by varying the amounts of PEG-diacrylate (PEG-DA cross-linker. PF hydrogels are generally cytocompatible with the encapsulated dental pulp stem cells (DPSCs, yielding >85% cell viability in all hydrogels. It was found that the cell morphology of encapsulated DPSCs, odontogenic gene expression, and mineralization were strongly modulated by the hydrogel cross-linking degree and matrix stiffness. Notably, DPSCs cultured within the highest cross-linked hydrogel remained mostly rounded in aggregates and demonstrated the greatest enhancement in odontogenic gene expression. Consistently, the highest degree of mineralization was observed in the highest cross-linked hydrogel. Collectively, our results indicate that PF hydrogels can be used as a scaffold for DPSCs and offers the possibility of influencing DPSCs in ways that may be beneficial for applications in regenerative endodontics.

  16. Histone deacetylase inhibitors epigenetically promote reparative events in primary dental pulp cells

    Energy Technology Data Exchange (ETDEWEB)

    Duncan, Henry F., E-mail: Hal.Duncan@dental.tcd.ie [Division of Restorative Dentistry and Periodontology, Dublin Dental University Hospital, Trinity College Dublin, Lincoln Place, Dublin 2 (Ireland); Smith, Anthony J. [Oral Biology, School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, Birmingham (United Kingdom); Fleming, Garry J.P. [Material Science Unit, Division of Oral Biosciences, Dublin Dental University Hospital, Trinity College Dublin, Dublin (Ireland); Cooper, Paul R. [Oral Biology, School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, Birmingham (United Kingdom)

    2013-06-10

    Application of histone deacetylase inhibitors (HDACi) to cells epigenetically alters their chromatin structure and induces transcriptional and cellular reparative events. This study investigated the application of two HDACi, valproic acid (VPA) and trichostatin A (TSA) on the induction of repair-associated responses in primary dental pulp cell (DPC) cultures. Flow cytometry demonstrated that TSA (100 nM, 400 nM) significantly increased cell viability. Neither HDACi was cytotoxic, although cell growth analysis revealed significant anti-proliferative effects at higher concentrations for VPA (>0.5 mM) and TSA (>50 nM). While high-content-analysis demonstrated that HDACi did not significantly induce caspase-3 or p21 activity, p53-expression was increased by VPA (3 mM, 5 mM) at 48 h. HDACi-exposure induced mineralization per cell dose-dependently to a plateau level (VPA-0.125 mM and TSA-25 nM) with accompanying increases in mineralization/dentinogenic-associated gene expression at 5 days (DMP-1, BMP-2/-4, Nestin) and 10 days (DSPP, BMP-2/-4). Both HDACis, at a range of concentrations, significantly stimulated osteopontin and BMP-2 protein expression at 10 and 14 days further supporting the ability of HDACi to promote differentiation. HDACi exert different effects on primary compared with transformed DPCs and promote mineralization and differentiation events without cytotoxic effects. These novel data now highlight the potential in restorative dentistry for applying low concentrations of HDACi in vital pulp treatment. -- Highlights: • Valproic acid and trichostatin A promoted mineralization in primary pulp cells. • Cell viability, apoptosis, caspase-3, p21 unaltered; p53 increased by valproic acid. • Trichostatin A increased cell viability at 24 h at selected concentrations. • Altered cell toxicity and differentiation between primary and transformed cells. • HDACi-induced the differentiation marker proteins osteopontin and BMP-2.

  17. Controlled positioning of cells in biomaterials - approaches towards 3D tissue printing

    OpenAIRE

    Sandra Hofmann; Ralph Müller; Silke Wüst

    2011-01-01

    Current tissue engineering techniques have various drawbacks: they often incorporate uncontrolled and imprecise scaffold geometries, whereas the current conventional cell seeding techniques result mostly in random cell placement rather than uniform cell distribution. For the successful reconstruction of deficient tissue, new material engineering approaches have to be considered to overcome current limitations. An emerging method to produce complex biological products including cells or extrac...

  18. Multiscale modeling of mechanosensing channels on vesicles and cell membranes in 3D constricted flows and shear flows

    Science.gov (United States)

    Peng, Zhangli; Pak, On Shun; Young, Yuan-Nan; Liu, Allen; Stone, Howard

    2015-11-01

    We investigate the gating of mechanosensing channels (Mscls) on vesicles and cell membranes under different flow conditions using a multiscale approach. At the cell level (microns), the membrane tension is calculated using a 3D two-component whole-cell membrane model based on dissipative particle dynamics (DPD), including the cortex cytoskeleton and its interactions with the lipid bilayer. At the Mscl level (nanometers), we predict the relation between channel gating and the membrane tension obtained from a cell-level model using a semi-analytical model based on the bilayer hydrophobic mismatch energy. We systematically study the gating of Mscls of vesicles and cell membranes in constricted channel flows and shear flows, and explore the dependence of the gating on flow rate, cell shape and size. The results provide guidance for future experiments in inducing Mscl opening for various purposes such as drug delivery.

  19. Apoptosis and survivability of human dental pulp cells under exposure to Bis-GMA

    Directory of Open Access Journals (Sweden)

    Junya Yano

    2011-06-01

    Full Text Available OBJECTIVE: In the present study, we examined whether 2, 2-bis [4-(2-hydroxy-3-methacryloxypropoxy phenyl] propane (Bis-GMA has effects on LSC2 cells, human dental pulp cell line. MATERIAL AND METHODS: The viability, cell cycle, and morphology of LSC2 cells were analyzed after exposure to several different concentrations of Bis-GMA. The recovery of viability of Bis-GMA exposed cells was also analyzed in the condition without Bis-GMA. Further, penetration of Bis-GMA to dentin disc was examined using isocratic high-performance liquid chromatography. RESULTS: There was a concentration-dependent decrease in cell proliferation and an increase in cell number in the sub-G1 population after exposure to Bis-GMA. Furthermore, the cells showed typical characteristics of apoptotic cells after the exposure to high concentration of Bis-GMA. In contrast, cells exposed to lower concentrations of Bis-GMA recovered their viability after being cultured without Bis-GMA. We also found that Bis-GMA is capable of penetrating 1-mm-thick dentin discs, though the penetrated concentration was lower than that showing cytotoxicity. CONCLUSION: These results suggest that Bis-GMA has cytotoxic effects, though dental pulp exposed to lower concentrations is able to recover their viability when Bis-GMA is removed.

  20. Super-resolved 3-D imaging of live cells organelles from bright-field photon transmission micrographs

    CERN Document Server

    Rychtarikova, Renata; Shi, Kevin; Malakhova, Daria; Machacek, Petr; Smaha, Rebecca; Urban, Jan; Stys, Dalibor

    2016-01-01

    Current biological and medical research is aimed at obtaining a detailed spatiotemporal map of a live cell's interior to describe and predict cell's physiological state. We present here an algorithm for complete 3-D modelling of cellular structures from a z-stack of images obtained using label-free wide-field bright-field light-transmitted microscopy. The method visualizes 3-D objects with a volume equivalent to the area of a camera pixel multiplied by the z-height. The computation is based on finding pixels of unchanged intensities between two consecutive images of an object spread function. These pixels represent strongly light-diffracting, light-absorbing, or light-emitting objects. To accomplish this, variables derived from R\\'{e}nyi entropy are used to suppress camera noise. Using this algorithm, the detection limit of objects is only limited by the technical specifications of the microscope setup--we achieve the detection of objects of the size of one camera pixel. This method allows us to obtain 3-D re...

  1. Electricity Generation with the Novel 3D Electrode from Swim Wastewater in a Dual-chamber Microbial Fuel Cell

    Directory of Open Access Journals (Sweden)

    Lai Mei-Feng

    2016-01-01

    Full Text Available The swine wastewater has the characteristics of high concentration of organic matter, suspended solids and more high ammonia nitrogen, odor, complex pollution ingredient and large emissions. Microbial fuel cells (MFC is an electrochemical and biological systems related to chemical energy into electrical energy. A two-chambered cubic microbial fuel cell was used to evaluate the effect of a novel 3D electrode which made of iron and copper on the electricity generation. The swine wastewater containing total chemical oxygen demand (TCOD 3300±300 mg/L was used as the feedstock in anode chamber, and the potassium ferricyanide was used as electron acceptor in cathode chamber. The MFC reactor was incubated with the initial pH 7.0 in a air-shaker with a temperature (ca. 35°C and 100 rpm in fed-batch mode. A fixed external resistance (R of 100 Ω was connected between the electrodes and the closed circuit potentials of the MFCs were recorded every 2 min. The results show that using iron 3D electrode has the peak electricity generation of 176 mV at the first two day and maintained the stable electricity voltage of 110 mV during the 5th to 15th days. The COD removal efficiency could reach 80%. Using copper 3D electrode only can generate the peak electricity of 33.1 mV and stable electricity of 27 mV with the COD removal efficiency of 70%.

  2. 3D printed sample holder for in-operando EPR spectroscopy on high temperature polymer electrolyte fuel cells.

    Science.gov (United States)

    Niemöller, Arvid; Jakes, Peter; Kayser, Steffen; Lin, Yu; Lehnert, Werner; Granwehr, Josef

    2016-08-01

    Electrochemical cells contain electrically conductive components, which causes various problems if such a cell is analyzed during operation in an EPR resonator. The optimum cell design strongly depends on the application and it is necessary to make certain compromises that need to be individually arranged. Rapid prototyping presents a straightforward option to implement a variable cell design that can be easily adapted to changing requirements. In this communication, it is demonstrated that sample containers produced by 3D printing are suitable for EPR applications, with a particular emphasis on electrochemical applications. The housing of a high temperature polymer electrolyte fuel cell (HT-PEFC) with a phosphoric acid doped polybenzimidazole membrane was prepared from polycarbonate by 3D printing. Using a custom glass Dewar, this fuel cell could be operated at temperatures up to 140°C in a standard EPR cavity. The carbon-based gas diffusion layer showed an EPR signal with a characteristic Dysonian line shape, whose evolution could be monitored in-operando in a non-invasive manner.

  3. 3D printed sample holder for in-operando EPR spectroscopy on high temperature polymer electrolyte fuel cells

    Science.gov (United States)

    Niemöller, Arvid; Jakes, Peter; Kayser, Steffen; Lin, Yu; Lehnert, Werner; Granwehr, Josef

    2016-08-01

    Electrochemical cells contain electrically conductive components, which causes various problems if such a cell is analyzed during operation in an EPR resonator. The optimum cell design strongly depends on the application and it is necessary to make certain compromises that need to be individually arranged. Rapid prototyping presents a straightforward option to implement a variable cell design that can be easily adapted to changing requirements. In this communication, it is demonstrated that sample containers produced by 3D printing are suitable for EPR applications, with a particular emphasis on electrochemical applications. The housing of a high temperature polymer electrolyte fuel cell (HT-PEFC) with a phosphoric acid doped polybenzimidazole membrane was prepared from polycarbonate by 3D printing. Using a custom glass Dewar, this fuel cell could be operated at temperatures up to 140 °C in a standard EPR cavity. The carbon-based gas diffusion layer showed an EPR signal with a characteristic Dysonian line shape, whose evolution could be monitored in-operando in a non-invasive manner.

  4. 3D printed sample holder for in-operando EPR spectroscopy on high temperature polymer electrolyte fuel cells.

    Science.gov (United States)

    Niemöller, Arvid; Jakes, Peter; Kayser, Steffen; Lin, Yu; Lehnert, Werner; Granwehr, Josef

    2016-08-01

    Electrochemical cells contain electrically conductive components, which causes various problems if such a cell is analyzed during operation in an EPR resonator. The optimum cell design strongly depends on the application and it is necessary to make certain compromises that need to be individually arranged. Rapid prototyping presents a straightforward option to implement a variable cell design that can be easily adapted to changing requirements. In this communication, it is demonstrated that sample containers produced by 3D printing are suitable for EPR applications, with a particular emphasis on electrochemical applications. The housing of a high temperature polymer electrolyte fuel cell (HT-PEFC) with a phosphoric acid doped polybenzimidazole membrane was prepared from polycarbonate by 3D printing. Using a custom glass Dewar, this fuel cell could be operated at temperatures up to 140°C in a standard EPR cavity. The carbon-based gas diffusion layer showed an EPR signal with a characteristic Dysonian line shape, whose evolution could be monitored in-operando in a non-invasive manner. PMID:27323280

  5. Optimum dose of 2-hydroxyethyl methacrylate based bonding material on pulp cells toxicity

    Directory of Open Access Journals (Sweden)

    Widya Saraswati

    2010-06-01

    Full Text Available Background: 2-hydroxyethyl methacrylate (HEMA, one type of resins commonly used as bonding base material, is commonly used due to its advantageous chemical characteristics. Several preliminary studies indicated that resin is a material capable to induce damage in dentin-pulp complex. It is necessary to perform further investigation related with its biological safety for hard and soft tissues in oral cavity. Purpose: The author performed an in vitro test to find optimum dose of HEMA resin monomer that may induce toxicity in pulp fibroblast cells. Method: The method of this study was experimental laboratory with post test control group design. Primary cell culture was made from dental pulp fibroblast cells, and was given with HEMA resin bonding material in various concentrations (5 µg/ml–2560 µg/ml, and then subjected to toxicity test (MTT assay. Result: HEMA optimum concentration was 320 µg/ml to induce cytotoxicity in pulp fibroblast cells. Conclusion: The used of HEMA - base bonding material with the concentration of 200 µg/ml may induced pulp fibroblas cell toxicity.Latar belakang: Keberhasilan suatu bahan bonding secara klinis tergantung pada kandungan fisik, kimia dan keamanan secara biologis. HEMA (2-hydroxyethyl methacrylate adalah bahan resin yang paling banyak digunakan karena memiliki sifat fisik-kimia yang baik. Beberapa penelitian pendahuluan menyebutkan bahwa resin merupakan bahan yang mampu menyebabkan gangguan pada kompleks dentin pulpa sehingga perlu dilakukan penelitian lebih lanjut menyangkut segi keamanan secara biologis bagi jaringan keras dan jaringan lunak di rongga mulut. Tujuan: Penelitian ini akan menguji secara in vitro (pada kultur sel fibroblas pulpa gigi untuk mengetahui dosis optimal monomer resin HEMA yang dapat menyebabkan toksisitas pada sel fibroblas pulpa. Metode: Metode penelitian ini adalah eksperimental laboratoris dengan rancangan penelitian post test control group design. Kultur sel primer dibuat dari

  6. A new method to address unmet needs for extracting individual cell migration features from a large number of cells embedded in 3D volumes.

    Directory of Open Access Journals (Sweden)

    Ivan Adanja

    Full Text Available BACKGROUND: In vitro cell observation has been widely used by biologists and pharmacologists for screening molecule-induced effects on cancer cells. Computer-assisted time-lapse microscopy enables automated live cell imaging in vitro, enabling cell behavior characterization through image analysis, in particular regarding cell migration. In this context, 3D cell assays in transparent matrix gels have been developed to provide more realistic in vitro 3D environments for monitoring cell migration (fundamentally different from cell motility behavior observed in 2D, which is related to the spread of cancer and metastases. METHODOLOGY/PRINCIPAL FINDINGS: In this paper we propose an improved automated tracking method that is designed to robustly and individually follow a large number of unlabeled cells observed under phase-contrast microscopy in 3D gels. The method automatically detects and tracks individual cells across a sequence of acquired volumes, using a template matching filtering method that in turn allows for robust detection and mean-shift tracking. The robustness of the method results from detecting and managing the cases where two cell (mean-shift trackers converge to the same point. The resulting trajectories quantify cell migration through statistical analysis of 3D trajectory descriptors. We manually validated the method and observed efficient cell detection and a low tracking error rate (6%. We also applied the method in a real biological experiment where the pro-migratory effects of hyaluronic acid (HA were analyzed on brain cancer cells. Using collagen gels with increased HA proportions, we were able to evidence a dose-response effect on cell migration abilities. CONCLUSIONS/SIGNIFICANCE: The developed method enables biomedical researchers to automatically and robustly quantify the pro- or anti-migratory effects of different experimental conditions on unlabeled cell cultures in a 3D environment.

  7. Quantitative 3-D imaging of eukaryotic cells using soft x-ray tomography

    OpenAIRE

    Parkinson, Dilworth Y.; McDermott, Gerry; Etkin, Laurence D.; Le Gros, Mark A.; Larabell, Carolyn A.

    2008-01-01

    Imaging has long been one of the principal techniques used in biological and biomedical research. Indeed, the field of cell biology grew out of the first electron microscopy images of organelles in a cell. Since this landmark event, much work has been carried out to image and classify the organelles in eukaryotic cells using electron microscopy. Fluorescently labeled organelles can now be tracked in live cells, and recently, powerful light microscope techniques have pushed the limit of optica...

  8. 3D Imaging of mammalian cells with ion-abrasion scanning electron microscopy

    OpenAIRE

    Heymann, Jurgen A. W.; Shi, Dan; Kim, Sang; Bliss, Donald; Milne, Jacqueline L. S.; Subramaniam, Sriram

    2008-01-01

    Understanding the hierarchical organization of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. We are using ion-abrasion scanning electron microscopy (IA-SEM) to visualize this hierarchical organization in an approach that combines focused ion-beam milling with scanning electron microscopy. Here, we extend our previous studies on imaging yeast cells to image subcellular architecture in human melanoma cells and mela...

  9. The cell composition of the red pulp of mice. Pt. 1

    International Nuclear Information System (INIS)

    A cytokinetic and morphological cell analysis was performed, with autoradiograms of thin sections in red pulp of untreated mice. The following cells could be differentiated in the thin preparations: 1) basophilic blasts (large and small), 2) erythroblasts (basophilic and poly- and orthochromatic), 3) lymphocytes, 4) plasma cells, 5) reticulum cells (without and with phagocytosis), 6) endothelial cells, 7) myelopoietic cells, and 8) megakaryocytes. Erythropoiesis essentially takes place in the interfollicular zones whilst myelopoiesis predominates in the subcapsular and peritrabecular zones. The autoradiographic study has suggested that in the interfollicular zone, basophilic blasts and erythroblasts can be classified as locally arising cells, whilst small lymphocytes, reticulum cells without and with phagocytosis (macrophages) can be regarded as migrating cells. (orig.)

  10. A new 3D tracking method for cell mechanics investigation exploiting the capabilities of digital holography in microscopy

    Science.gov (United States)

    Miccio, L.; Memmolo, P.; Merola, F.; Fusco, S.; Netti, P. A.; Ferraro, P.

    2014-03-01

    A method for 3D tracking has been developed exploiting Digital Holography features in Microscopy (DHM). In the framework of self-consistent platform for manipulation and measurement of biological specimen we use DHM for quantitative and completely label free analysis of samples with low amplitude contrast. Tracking capability extend the potentiality of DHM allowing to monitor the motion of appropriate probes and correlate it with sample properties. Complete 3D tracking has been obtained for the probes avoiding the amplitude refocusing in traditional tracking processes. Moreover, in biology and biomedical research fields one of the main topic is the understanding of morphology and mechanics of cells and microorganisms. Biological samples present low amplitude contrast that limits the information that can be retrieved through optical bright-field microscope measurements. The main effect on light propagating in such objects is in phase. This is known as phase-retardation or phase-shift. DHM is an innovative and alternative approach in microscopy, it's a good candidate for no-invasive and complete specimen analysis because its main characteristic is the possibility to discern between intensity and phase information performing quantitative mapping of the Optical Path Length. In this paper, the flexibility of DH is employed to analyze cell mechanics of unstained cells subjected to appropriate stimuli. DHM is used to measure all the parameters useful to understand the deformations induced by external and controlled stresses on in-vitro cells. Our configuration allows 3D tracking of micro-particles and, simultaneously, furnish quantitative phase-contrast maps. Experimental results are presented and discussed for in vitro cells.

  11. Role of Mechanical Cues in Cell Differentiation and Proliferation: A 3D Numerical Model.

    Directory of Open Access Journals (Sweden)

    Seyed Jamaleddin Mousavi

    Full Text Available Cell differentiation, proliferation and migration are essential processes in tissue regeneration. Experimental evidence confirms that cell differentiation or proliferation can be regulated according to the extracellular matrix stiffness. For instance, mesenchymal stem cells (MSCs can differentiate to neuroblast, chondrocyte or osteoblast within matrices mimicking the stiffness of their native substrate. However, the precise mechanisms by which the substrate stiffness governs cell differentiation or proliferation are not well known. Therefore, a mechano-sensing computational model is here developed to elucidate how substrate stiffness regulates cell differentiation and/or proliferation during cell migration. In agreement with experimental observations, it is assumed that internal deformation of the cell (a mechanical signal together with the cell maturation state directly coordinates cell differentiation and/or proliferation. Our findings indicate that MSC differentiation to neurogenic, chondrogenic or osteogenic lineage specifications occurs within soft (0.1-1 kPa, intermediate (20-25 kPa or hard (30-45 kPa substrates, respectively. These results are consistent with well-known experimental observations. Remarkably, when a MSC differentiate to a compatible phenotype, the average net traction force depends on the substrate stiffness in such a way that it might increase in intermediate and hard substrates but it would reduce in a soft matrix. However, in all cases the average net traction force considerably increases at the instant of cell proliferation because of cell-cell interaction. Moreover cell differentiation and proliferation accelerate with increasing substrate stiffness due to the decrease in the cell maturation time. Thus, the model provides insights to explain the hypothesis that substrate stiffness plays a key role in regulating cell fate during mechanotaxis.

  12. Role of Mechanical Cues in Cell Differentiation and Proliferation: A 3D Numerical Model

    Science.gov (United States)

    Mousavi, Seyed Jamaleddin; Hamdy Doweidar, Mohamed

    2015-01-01

    Cell differentiation, proliferation and migration are essential processes in tissue regeneration. Experimental evidence confirms that cell differentiation or proliferation can be regulated according to the extracellular matrix stiffness. For instance, mesenchymal stem cells (MSCs) can differentiate to neuroblast, chondrocyte or osteoblast within matrices mimicking the stiffness of their native substrate. However, the precise mechanisms by which the substrate stiffness governs cell differentiation or proliferation are not well known. Therefore, a mechano-sensing computational model is here developed to elucidate how substrate stiffness regulates cell differentiation and/or proliferation during cell migration. In agreement with experimental observations, it is assumed that internal deformation of the cell (a mechanical signal) together with the cell maturation state directly coordinates cell differentiation and/or proliferation. Our findings indicate that MSC differentiation to neurogenic, chondrogenic or osteogenic lineage specifications occurs within soft (0.1-1 kPa), intermediate (20-25 kPa) or hard (30-45 kPa) substrates, respectively. These results are consistent with well-known experimental observations. Remarkably, when a MSC differentiate to a compatible phenotype, the average net traction force depends on the substrate stiffness in such a way that it might increase in intermediate and hard substrates but it would reduce in a soft matrix. However, in all cases the average net traction force considerably increases at the instant of cell proliferation because of cell-cell interaction. Moreover cell differentiation and proliferation accelerate with increasing substrate stiffness due to the decrease in the cell maturation time. Thus, the model provides insights to explain the hypothesis that substrate stiffness plays a key role in regulating cell fate during mechanotaxis. PMID:25933372

  13. An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.

    Science.gov (United States)

    Bohórquez, Diego V; Samsa, Leigh A; Roholt, Andrew; Medicetty, Satish; Chandra, Rashmi; Liddle, Rodger A

    2014-01-01

    The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells. PMID:24587096

  14. An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.

    Directory of Open Access Journals (Sweden)

    Diego V Bohórquez

    Full Text Available The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM. Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.

  15. Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

    Science.gov (United States)

    Itano, Michelle S; Bleck, Marina; Johnson, Daniel S; Simon, Sanford M

    2016-02-01

    Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

  16. Odontoblastic inductive potential of epithelial cells derived from human deciduous dental pulp.

    Science.gov (United States)

    Lee, Hye-Kyung; Park, Ji-Won; Seo, You-Mi; Kim, Ha Hoon; Lee, Gene; Bae, Hyun-Sook; Park, Joo-Cheol

    2016-06-01

    For the dentin regeneration, dental epithelial cells are indispensible and must possess odontoblastic induction capability. Epithelial cell-like stem cells were recently identified in human deciduous dental pulp (DPESCs). However, their cellular characteristics remain poorly defined. The purpose of this study was to characterize DPESCs compared to HAT-7 ameloblastic cells. Expression levels of ameloblast-specific markers [odontogenic ameloblast-associated protein (Odam), matrix metalloproteinase (Mmp)-20, amelogenin, and ameloblastin] were detected in DPESCs. Co-culturing odontoblastic MDPC-23 cells with DPESCs increased expression of odontoblast differentiation markers (Dmp1 and Dspp) from days 4 to 10, while the expression of bone sialoprotein rapidly decreased. MDPC-23 cells cultured in DPESC-conditioned medium (CM) showed increased Dspp promoter activity compared with control MDPC-23 cultures. Mineralization was first observed in the CM groups from day 4 and proceeded rapidly until day 14, whereas mineralized nodules were found from day 7 in control media-cultured cells. In conclusion, DPESCs in human deciduous pulp possess ameloblast-like characteristics and differentiation properties, and substances derived from DPESCs promote odontoblastic differentiation. Thus, our results indicate that DPESCs can be a realistic epithelial source for use in odontoblastic induction and dentin formation of dental mesenchymal cells. PMID:27098651

  17. New process for fuel cell fabrication. 3D screen printing of metal bipolar plates; Neues Verfahren zur Brennstoffzellenfertigung. 3D-Siebdruck von metallischen Bipolarplatten

    Energy Technology Data Exchange (ETDEWEB)

    Studnitzky, Thomas [Fraunhofer-Institut fuer Fertigungstechnik und Angewandte Materialforschung (IFAM), Dresden (Germany); Helm, Peter; Heinzel, Angelika [Zentrum fuer BrennstoffzellenTechnik GmbH (ZBT), Duisburg (Germany)

    2011-01-15

    Minimization of space requirements, weight, and production cost is one of the key preconditions for successful launching of the polymer electrolyte membrane fuel cell (PEM). In the stacks constructed from single PEM cells, the bipolar plate is a central component. It determines the weight and volume of the stack and accounts for more than 30 percent of the overall cost, depending on the fabrication process. It is therefore important for producers of fuel cells to develop a process that combines free design, high functionality and low cost in serial production.

  18. Development of a randomized 3D cell model for Monte Carlo microdosimetry simulations

    Energy Technology Data Exchange (ETDEWEB)

    Douglass, Michael; Bezak, Eva; Penfold, Scott [School of Chemistry and Physics, University of Adelaide, North Terrace, Adelaide 5005, South Australia (Australia) and Department of Medical Physics, Royal Adelaide Hospital, North Terrace, Adelaide 5000, South Australia (Australia)

    2012-06-15

    Purpose: The objective of the current work was to develop an algorithm for growing a macroscopic tumor volume from individual randomized quasi-realistic cells. The major physical and chemical components of the cell need to be modeled. It is intended to import the tumor volume into GEANT4 (and potentially other Monte Carlo packages) to simulate ionization events within the cell regions. Methods: A MATLAB Copyright-Sign code was developed to produce a tumor coordinate system consisting of individual ellipsoidal cells randomized in their spatial coordinates, sizes, and rotations. An eigenvalue method using a mathematical equation to represent individual cells was used to detect overlapping cells. GEANT4 code was then developed to import the coordinate system into GEANT4 and populate it with individual cells of varying sizes and composed of the membrane, cytoplasm, reticulum, nucleus, and nucleolus. Each region is composed of chemically realistic materials. Results: The in-house developed MATLAB Copyright-Sign code was able to grow semi-realistic cell distributions ({approx}2 Multiplication-Sign 10{sup 8} cells in 1 cm{sup 3}) in under 36 h. The cell distribution can be used in any number of Monte Carlo particle tracking toolkits including GEANT4, which has been demonstrated in this work. Conclusions: Using the cell distribution and GEANT4, the authors were able to simulate ionization events in the individual cell components resulting from 80 keV gamma radiation (the code is applicable to other particles and a wide range of energies). This virtual microdosimetry tool will allow for a more complete picture of cell damage to be developed.

  19. Characterization of deciduous teeth stem cells isolated from crown dental pulp

    Directory of Open Access Journals (Sweden)

    Debeljak-Martačić Jasmina

    2014-01-01

    Full Text Available Background/Aim. The last decade has been profoundly marked by persistent attempts to use ex vivo expanded and manipulated mesenchymal stem cells (MSCs, as a tool in different types of regenerative therapy. In the present study we described immunophenotype and the proliferative and differentiation potential of cells isolated from pulp remnants of exfoliated deciduous teeth in the final phase of root resorption. Methods. The initial adherent cell population from five donors was obtained by the outgrowth method. Colony forming unit-fibroblast (CFU-F assay was performed in passage one. Cell expansion was performed until passage three and all tests were done until passage eight. Cells were labeled for early mesenchymal stem cells markers and analysis have been done using flow cytometry. The proliferative potential was assessed by cell counting in defined time points and population doubling time was calculated. Commercial media were used to induce osteoblastic, chondrogenic and adipogenic differentiation. Cytology and histology methods were used for analysis of differentiated cell morphology and extracellular matrix characteristics. Results. According to immunophenotype analyses all undifferentiated cells were positive for the mesenchymal stem cell markers: CD29 and CD73. Some cells expressed CD146 and CD106. The hematopoietic cell marker, CD34, was not detected. In passage one, incidence of CFU-F was 4.7 ± 0.5/100. Population doubling time did not change significantly during cell subcultivation and was in average 25 h. After induction of differentiation, the multicolony derived cell population had a tri-lineage differentiation potential, since mineralized matrix, cartilage-like tissue and adipocytes were successfully formed after three weeks of incubation. Conclusion. Altogether, these data suggest that remnants of deciduous teeth dental pulp contained cell populations with mesenchymal stem cell-like features, with a high proliferation and

  20. Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: an improved approach in cartilage tissue engineering.

    Science.gov (United States)

    Moroz, Andrei; Bittencourt, Renata Aparecida Camargo; Almeida, Renan Padron; Felisbino, Sérgio Luis; Deffune, Elenice

    2013-01-01

    Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n = 5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1 × 10(5)) were than encapsulated inside 60 µl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI.

  1. Cytotoxicity Testing of Temporary Luting Cements with Two- and Three-Dimensional Cultures of Bovine Dental Pulp-Derived Cells

    Directory of Open Access Journals (Sweden)

    Hayriye Esra Ülker

    2013-01-01

    Full Text Available This study evaluated the cytotoxicity of eugenol-containing and eugenol-free temporary luting cements. For cytotoxicity testing, bovine pulp-derived cells transfected with Simian virus 40 Large T antigen were exposed to extracts of eugenol-containing (Rely X Temp E and eugenol-free (Provicol, PreVISION CEM, and Rely X Temp NE temporary luting cements for 24 h. The cytotoxicity of the same materials was also evaluated in a dentin barrier test device using three-dimensional cell cultures of bovine pulp-derived cells. The results of the cytotoxicity studies with two-dimensional cultures of bovine dental pulp-derived cells revealed that cell survival with the extracts of Rely X Temp E, Provicol, PreVISION CEM, and Rely X Temp NE was 89.1%, 84.9%, 92.3%, and 66.8%, respectively. Rely X Temp NE and Provicol showed cytotoxic effects on bovine dental pulp-derived cells (. The results of the dentin barrier test revealed that cell survival with the above-mentioned temporary cement was 101.5%, 91.9%, 93.5%, and 90.6%, respectively. None of the temporary luting cements significantly reduced cell survival compared with the negative control in the dentin barrier test (. Biologically active materials released from temporary luting cements may not influence the dentine-pulp complex if the residual dentine layer is at least 0.5 mm thick.

  2. Enabling Lorentz boosted frame particle-in-cell simulations of laser wakefield acceleration in quasi-3D geometry

    Science.gov (United States)

    Yu, Peicheng; Xu, Xinlu; Davidson, Asher; Tableman, Adam; Dalichaouch, Thamine; Li, Fei; Meyers, Michael D.; An, Weiming; Tsung, Frank S.; Decyk, Viktor K.; Fiuza, Frederico; Vieira, Jorge; Fonseca, Ricardo A.; Lu, Wei; Silva, Luis O.; Mori, Warren B.

    2016-07-01

    When modeling laser wakefield acceleration (LWFA) using the particle-in-cell (PIC) algorithm in a Lorentz boosted frame, the plasma is drifting relativistically at βb c towards the laser, which can lead to a computational speedup of ∼ γSUB>/bSUB>2 = (1 -space-time distribution of the LWFA data in the lab and boosted frame, we propose to use a moving window to follow the drifting plasma, instead of following the laser driver as is done in the LWFA lab frame simulations, in order to further reduce the computational loads. We describe the details of how the NCI is mitigated for the quasi-3D geometry, the setups for simulations which combine the Lorentz boosted frame, quasi-3D geometry, and the use of a moving window, and compare the results from these simulations against their corresponding lab frame cases. Good agreement is obtained among these sample simulations, particularly when there is no self-trapping, which demonstrates it is possible to combine the Lorentz boosted frame and the quasi-3D algorithms when modeling LWFA. We also discuss the preliminary speedups achieved in these sample simulations.

  3. A general approach for time-supersampling of 3D-PIV data by the vortex-in-cell method

    Science.gov (United States)

    Scarano, Fulvio; Schneiders, Jan; Dwight, Richard; Aerospace Engineering/Aerodynamics Team

    2013-11-01

    Advancements of tomographic PIV [1] have led into 3D time-resolved experiments to study the dynamical evolution of 3D turbulent flows [2]. The known bottleneck of Tomo-PIV is the high laser power required to illuminate large volumes in airflows, which becomes critical beyond 10 kHz. Time-super-sampling is an approach to reduce the sampling rate, proven for frozen turbulence where the advection model yields a significant increase of temporal resolution [3]. Instead, in separated flows, the advection principle yields unacceptable distortions. The use of Navier-Stokes numerical calculations with the vortex-in-cell (VIC) method is proposed herein. The assumption is made of inviscid incompressible flow [4]. The spatial-resolution of the data is exploited to increase the temporal resolution. The dynamical evolution of the vorticity and velocity field between subsequent snapshots in the 3D domain is numerically evaluated. The verification with fully time resolved data of a circular jet indicates a substantial increase of temporal resolution. Interestingly, data sampled below the Nyquist limit could be reconstructed faithfully, indicating the potential of VIC in alleviating requirements on PIV measurement rate. Work supported by the European Research Council grant 202887.

  4. Longitudinal, label-free, quantitative tracking of cell death and viability in a 3D tumor model with OCT

    Science.gov (United States)

    Jung, Yookyung; Klein, Oliver J.; Wang, Hequn; Evans, Conor L.

    2016-06-01

    Three-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer. Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems. Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth. Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response. We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer. OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards. Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed. OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time. We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation.

  5. An Innovative Hybrid 3D Analytic-Numerical Approach for System Level Modelling of PEM Fuel Cells

    Directory of Open Access Journals (Sweden)

    Gregor Tavčar

    2013-10-01

    Full Text Available The PEM fuel cell model presented in this paper is based on modelling species transport and coupling electrochemical reactions to species transport in an innovative way. Species transport is modelled by obtaining a 2D analytic solution for species concentration distribution in the plane perpendicular to the gas-flow and coupling consecutive 2D solutions by means of a 1D numerical gas-flow model. The 2D solution is devised on a jigsaw puzzle of multiple coupled domains which enables the modelling of parallel straight channel fuel cells with realistic geometries. Electrochemical and other nonlinear phenomena are coupled to the species transport by a routine that uses derivative approximation with prediction-iteration. A hybrid 3D analytic-numerical fuel cell model of a laboratory test fuel cell is presented and evaluated against a professional 3D computational fluid dynamic (CFD simulation tool. This comparative evaluation shows very good agreement between results of the presented model and those of the CFD simulation. Furthermore, high accuracy results are achieved at computational times short enough to be suitable for system level simulations. This computational efficiency is owed to the semi-analytic nature of its species transport modelling and to the efficient computational coupling of electrochemical kinetics and species transport.

  6. Enamel Matrix Derivative Promote Primary Human Pulp Cell Differentiation and Mineralization

    Directory of Open Access Journals (Sweden)

    Elisabeth Aurstad Riksen

    2014-05-01

    Full Text Available Enamel matrix derivative (EMD has been found to induce reactive dentin formation; however the molecular mechanisms involved are unclear. The effect of EMD (5–50 μg/mL on primary human pulp cells were compared to untreated cells and cells incubated with 10−8 M dexamethasone (DEX for 1, 2, 3, 7, and 14 days in culture. Expression analysis using Affymetrix microchips demonstrated that 10 μg/mL EMD regulated several hundred genes and stimulated the gene expression of proteins involved in mesenchymal proliferation and differentiation. Both EMD and DEX enhanced the expression of amelogenin (amel, and the dentinogenic markers dentin sialophosphoprotein (DSSP and dentin matrix acidic phosphoprotein 1 (DMP1, as well as the osteogenic markers osteocalcin (OC, BGLAP and collagen type 1 (COL1A1. Whereas, only EMD had effect on alkaline phosphatase (ALP mRNA expression, the stimulatory effect were verified by enhanced secretion of OC and COL1A from EMD treated cells, and increased ALP activity in cell culture medium after EMD treatment. Increased levels of interleukin-6 (IL-6, interleukin-8 (IL-8, and monocyte chemoattractant proteins (MCP-1 in the cell culture medium were also found. Consequently, the suggested effect of EMD is to promote differentiation of pulp cells and increases the potential for pulpal mineralization to favor reactive dentine formation.

  7. Odontoblasts: Specialized hard-tissue-forming cells in the dentin-pulp complex.

    Science.gov (United States)

    Kawashima, Nobuyuki; Okiji, Takashi

    2016-07-01

    Odontoblasts are specialized cells that produce dentin and exhibit unique morphological characteristics; i.e., they extend cytoplasmic processes into dentinal tubules. While osteoblasts, which are typical hard-tissue-forming cells, are generated from mesenchymal stem cells during normal and pathological bone metabolism, the induction of odontoblasts only occurs once during tooth development, and odontoblasts survive throughout the lives of healthy teeth. During the differentiation of odontoblasts, signaling molecules from the inner enamel epithelium are considered necessary for the differentiation of odontoblast precursors, i.e., peripheral dental papilla cells. If odontoblasts are destroyed by severe external stimuli, such as deep caries, the differentiation of dental pulp stem cells into odontoblast-like cells is induced. Various bioactive molecules, such as non-collagenous proteins, might be involved in this process, although the precise mechanisms responsible for odontoblast differentiation have not been fully elucidated. Recently, our knowledge about the other functional activities of odontoblasts (apart from dentin formation) has increased. For example, it has been suggested that odontoblasts might act as nociceptive receptors, and surveillance cells that detect the invasion of exogenous pathogens. The regeneration of the dentin-pulp complex has recently gained much attention as a promising future treatment modality that could increase the longevity of pulpless teeth. Finally, congenital dentin anomalies, which are concerned with the disturbance of odontoblast functions, are summarized. PMID:27131345

  8. Survival of Dental Pulp Stem Cells: The effect of Soymilk and Milk

    Directory of Open Access Journals (Sweden)

    Fatemeh Sholehvar

    2015-11-01

    Full Text Available Background & Objectives: Dental pulp stem cells (DPSCs are alternate source of mesenchymal stem cells. Subsequent to tooth avulsion and fracture, DPSCs can play a prominent role in tissue regeneration. This study was conducted to evaluate the effect of soymilk and milk on survival of dental pulp stem cells. Materials & Methods: DPSCs were isolated from 16 freshly extracted incisors of 5 rabbits. The 3rd passage was seeded in 24 well plates, and after 3 days, soymilk, cow milk, HBSS, and distilled water were replaced with culture media. After 45 minutes, 1.5, 3, and 6 hours, the viability of DPSCs were investigated. Mesenchymal nature of stem cells was investigated by RT-PCR. The cell viability was determined by Trypan blue exclusion. Karyotyping was done to evaluate the cytogenetic stability of cells.  Results: The viability of DPSCs in all media were significantly more than distilled water at all intervals. After 6 h, the viability of DPSCs in soymilk, cow milk, and HBSS were 100,000±0.00, 100,000±0.00, and 74.74±5.70, respectively. After 6 h, both soymilk and cow milk maintained cells significantly better than HBSS. Conclusion: Like cow milk, soymilk is a suitable alternative transfer media for avulsed and broken teeth that can increase the survival of DPSCs.   

  9. 3D structure determination of a protein in living cells using paramagnetic NMR spectroscopy.

    Science.gov (United States)

    Pan, Bin-Bin; Yang, Feng; Ye, Yansheng; Wu, Qiong; Li, Conggang; Huber, Thomas; Su, Xun-Cheng

    2016-08-11

    Determining the three-dimensional structure of a protein in living cells remains particularly challenging. We demonstrated that the integration of site-specific tagging proteins and GPS-Rosetta calculations provides a fast and effective way of determining the structures of proteins in living cells, and in principle the interactions and dynamics of protein-ligand complexes. PMID:27470136

  10. Biofabrication of tissue constructs by 3D bioprinting of cell-laden microcarriers

    International Nuclear Information System (INIS)

    Bioprinting allows the fabrication of living constructs with custom-made architectures by spatially controlled deposition of multiple bioinks. This is important for the generation of tissue, such as osteochondral tissue, which displays a zonal composition in the cartilage domain supported by the underlying subchondral bone. Challenges in fabricating functional grafts of clinically relevant size include the incorporation of cues to guide specific cell differentiation and the generation of sufficient cells, which is hard to obtain with conventional cell culture techniques. A novel strategy to address these demands is to combine bioprinting with microcarrier technology. This technology allows for the extensive expansion of cells, while they form multi-cellular aggregates, and their phenotype can be controlled. In this work, living constructs were fabricated via bioprinting of cell-laden microcarriers. Mesenchymal stromal cell (MSC)-laden polylactic acid microcarriers, obtained via static culture or spinner flask expansion, were encapsulated in gelatin methacrylamide-gellan gum bioinks, and the printability of the composite material was studied. This bioprinting approach allowed for the fabrication of constructs with high cell concentration and viability. Microcarrier encapsulation improved the compressive modulus of the hydrogel constructs, facilitated cell adhesion, and supported osteogenic differentiation and bone matrix deposition by MSCs. Bilayered osteochondral models were fabricated using microcarrier-laden bioink for the bone compartment. These findings underscore the potential of this new microcarrier-based biofabrication approach for bone and osteochondral constructs. (paper)

  11. In-chip fabrication of free-form 3D constructs for directed cell migration analysis

    DEFF Research Database (Denmark)

    Olsen, Mark Holm; Hjortø, Gertrud Malene; Hansen, Morten;

    2013-01-01

    with a range of pore sizes from 5 × 5 μm to 15 × 15 μm and prefilled with fibrillar collagen. Dendritic cells seeded into the polymer chip in a concentration gradient of the chemoattractant CCL21 efficiently negotiated the microporous maze structure for pore sizes of 8 × 8 μm or larger. The cells migrating...

  12. A genetically modified protein-based hydrogel for 3D culture of AD293 cells.

    Science.gov (United States)

    Du, Xiao; Wang, Jingyu; Diao, Wentao; Wang, Ling; Long, Jiafu; Zhou, Hao

    2014-01-01

    Hydrogels have strong application prospects for drug delivery, tissue engineering and cell therapy because of their excellent biocompatibility and abundant availability as scaffolds for drugs and cells. In this study, we created hybrid hydrogels based on a genetically modified tax interactive protein-1 (TIP1) by introducing two or four cysteine residues in the primary structure of TIP1. The introduced cysteine residues were crosslinked with a four-armed poly (ethylene glycol) having their arm ends capped with maleimide residues (4-armed-PEG-Mal) to form hydrogels. In one form of the genetically modification, we incorporated a peptide sequence 'GRGDSP' to introduce bioactivity to the protein, and the resultant hydrogel could provide an excellent environment for a three dimensional cell culture of AD293 cells. The AD293 cells continued to divide and displayed a polyhedron or spindle-shape during the 3-day culture period. Besides, AD293 cells could be easily separated from the cell-gel constructs for future large-scale culture after being cultured for 3 days and treating hydrogel with trypsinase. This work significantly expands the toolbox of recombinant proteins for hydrogel formation, and we believe that our hydrogel will be of considerable interest to those working in cell therapy and controlled drug delivery. PMID:25233088

  13. A genetically modified protein-based hydrogel for 3D culture of AD293 cells.

    Directory of Open Access Journals (Sweden)

    Xiao Du

    Full Text Available Hydrogels have strong application prospects for drug delivery, tissue engineering and cell therapy because of their excellent biocompatibility and abundant availability as scaffolds for drugs and cells. In this study, we created hybrid hydrogels based on a genetically modified tax interactive protein-1 (TIP1 by introducing two or four cysteine residues in the primary structure of TIP1. The introduced cysteine residues were crosslinked with a four-armed poly (ethylene glycol having their arm ends capped with maleimide residues (4-armed-PEG-Mal to form hydrogels. In one form of the genetically modification, we incorporated a peptide sequence 'GRGDSP' to introduce bioactivity to the protein, and the resultant hydrogel could provide an excellent environment for a three dimensional cell culture of AD293 cells. The AD293 cells continued to divide and displayed a polyhedron or spindle-shape during the 3-day culture period. Besides, AD293 cells could be easily separated from the cell-gel constructs for future large-scale culture after being cultured for 3 days and treating hydrogel with trypsinase. This work significantly expands the toolbox of recombinant proteins for hydrogel formation, and we believe that our hydrogel will be of considerable interest to those working in cell therapy and controlled drug delivery.

  14. Electric transport in 3D photonic crystal intermediate reflectors for micromorph thin-film tandem solar cells

    Science.gov (United States)

    Üpping, J.; Bielawny, A.; Lee, S.; Knez, M.; Carius, R.; Wehrspohn, R. B.

    2009-08-01

    The progress of 3D photonic intermediate reflectors for micromorph silicon tandem cells towards a first prototype cell is presented. Intermediate reflectors enhance the absorption of spectrally-selected light in the top cell and decrease the current mismatch between both junctions. A numerical method to predict filter properties for optimal current matching is presented. Our device is an inverted opal structure made of ZnO and fabricated using self-organized nanoparticles and atomic layer deposition for conformal coating. In particular, the influence of ZnO-doping and replicated cracks during drying of the opal is discussed with respect to conductivity and optical properties. A first prototype is compared to a state-of-the-art reference cell.

  15. Enrichment of diluted cell populations from large sample volumes using 3D carbon-electrode dielectrophoresis.

    Science.gov (United States)

    Islam, Monsur; Natu, Rucha; Larraga-Martinez, Maria Fernanda; Martinez-Duarte, Rodrigo

    2016-05-01

    Here, we report on an enrichment protocol using carbon electrode dielectrophoresis to isolate and purify a targeted cell population from sample volumes up to 4 ml. We aim at trapping, washing, and recovering an enriched cell fraction that will facilitate downstream analysis. We used an increasingly diluted sample of yeast, 10(6)-10(2) cells/ml, to demonstrate the isolation and enrichment of few cells at increasing flow rates. A maximum average enrichment of 154.2 ± 23.7 times was achieved when the sample flow rate was 10 μl/min and yeast cells were suspended in low electrically conductive media that maximizes dielectrophoresis trapping. A COMSOL Multiphysics model allowed for the comparison between experimental and simulation results. Discussion is conducted on the discrepancies between such results and how the model can be further improved. PMID:27375816

  16. Enrichment of diluted cell populations from large sample volumes using 3D carbon-electrode dielectrophoresis.

    Science.gov (United States)

    Islam, Monsur; Natu, Rucha; Larraga-Martinez, Maria Fernanda; Martinez-Duarte, Rodrigo

    2016-05-01

    Here, we report on an enrichment protocol using carbon electrode dielectrophoresis to isolate and purify a targeted cell population from sample volumes up to 4 ml. We aim at trapping, washing, and recovering an enriched cell fraction that will facilitate downstream analysis. We used an increasingly diluted sample of yeast, 10(6)-10(2) cells/ml, to demonstrate the isolation and enrichment of few cells at increasing flow rates. A maximum average enrichment of 154.2 ± 23.7 times was achieved when the sample flow rate was 10 μl/min and yeast cells were suspended in low electrically conductive media that maximizes dielectrophoresis trapping. A COMSOL Multiphysics model allowed for the comparison between experimental and simulation results. Discussion is conducted on the discrepancies between such results and how the model can be further improved.

  17. Differentiation of Dental Pulp Stem Cells on Gutta-Percha Scaffolds

    Directory of Open Access Journals (Sweden)

    Liudi Zhang

    2016-05-01

    Full Text Available Advances in treatment of tooth injury have shown that tooth regeneration from the pulp was a viable alternative of root canal therapy. In this study, we demonstrated that Gutta-percha, nanocomposites primarily used for obturation of the canal, are not cytotoxic and can induce differentiation of dental pulp stem cells (DPSC in the absence of soluble mediators. Flat scaffolds were obtained by spin coating Si wafers with three Gutta-percha compounds: GuttaCore™, ProTaper™, and Lexicon™. The images of annealed surfaces showed that the nanoparticles were encapsulated, forming surfaces with root mean square (RMS roughness of 136–211 nm. Then, by culturing DPSC on these substrates we found that after some initial difficulty in adhesion, confluent tissues were formed after 21 days. Imaging of the polyisoprene (PI surfaces showed that biomineral deposition only occurred when dexamethasone was present in the media. Spectra obtained from the minerals was consistent with that of hydroxyapatite (HA. In contrast, HA deposition was observed on all Gutta-percha scaffolds regardless of the presence or absence of dexamethasone, implying that surface roughness may be an enabling factor in the differentiation process. These results indicate that Gutta-percha nanocomposites may be good candidates for pulp regeneration therapy.

  18. Topographical guidance of 3D tumor cell migration at an interface of collagen densities

    International Nuclear Information System (INIS)

    During cancer progression, metastatic cells leave the primary tumor and invade into the fibrous extracellular matrix (ECM) within the surrounding stroma. This ECM network is highly heterogeneous, and interest in understanding how this network can affect cell behavior has increased in the past several decades. However, replicating this heterogeneity has proven challenging. Here, we designed and utilized a method to create a well-defined interface between two distinct regions of high- and low-density collagen gels to mimic the heterogeneities in density found in the tumor stroma. We show that cells will invade preferentially from the high-density side into the low-density side. We also demonstrate that the net cell migration is a function of the density of the collagen in which the cells are embedded, and the difference in density between the two regions has minimal effect on cell net displacement and distance travelled. Our data further indicate that a low-to-high density interface promotes directional migration and induces formation of focal adhesion on the interface surface. Together, the current results demonstrate how ECM heterogeneities, in the form of interfacial boundaries, can affect cell migration. (paper)

  19. Development of nanocellulose scaffolds with tunable structures to support 3D cell culture.

    Science.gov (United States)

    Liu, Jun; Cheng, Fang; Grénman, Henrik; Spoljaric, Steven; Seppälä, Jukka; E Eriksson, John; Willför, Stefan; Xu, Chunlin

    2016-09-01

    Swollen three-dimensional nanocellulose films and their resultant aerogels were prepared as scaffolds towards tissue engineering application. The nanocellulose hydrogels with various swelling degree (up to 500 times) and the resultant aerogels with desired porosity (porosity up to 99.7% and specific surface area up to 308m(2)/g) were prepared by tuning the nanocellulose charge density, the swelling media conditions, and the material processing approach. Representative cell-based assays were applied to assess the material biocompatibility and efficacy of the human extracellular matrix (ECM)-mimicking nanocellulose scaffolds. The effects of charge density and porosity of the scaffolds on the biological tests were investigated for the first time. The results reveal that the nanocellulose scaffolds could promote the survival and proliferation of tumor cells, and enhance the transfection of exogenous DNA into the cells. These results suggest the usefulness of the nanocellulose-based matrices in supporting crucial cellular processes during cell growth and proliferation. PMID:27185139

  20. BMP7 transfection induces in-vitro osteogenic differentiation of dental pulp mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Ka Po John Yau

    2013-01-01

    Full Text Available Objective: To assess whether in-vitro osteogenic differentiation of human dental pulp mesenchymal stem cells can be induced by transient transfection with the gene encoding human bone morphogenic protein 7 (BMP7. Materials and Methods: A mesenchymal stem cell population was isolated from the dental pulp of two extracted permanent premolars, expanded and characterized. The human BMP7 gene, as a recombinant pcDNA3.1/V5-His-TOPO-BMP7 plasmid, was transfected into the cells. Three negative controls were used: No plasmid, empty vector, and an unrelated vector encoding green fluorescent protein. After the interval of 24 and 48 h, mRNA levels of alkaline phosphatase and osteocalcin as markers of in-vitro osteogenic differentiation were measured by real-time polymerase chain reaction and standardized against β-actin mRNA levels. Results: The level of alkaline phosphatase mRNA was significantly higher for the BMP7 group than for all three negative controls 48 h after transfection (706.9 vs. 11.24 for untransfected cells, 78.05 for empty vector, and 73.10 for green fluorescent protein vector. The level of osteocalcin mRNA was significantly higher for the BMP7 group than for all three negative controls 24 h after transfection (1.0, however, decreased after another 24 h. Conclusions: In-vitro osteoblastic differentiation of human dental pulp mesenchymal stem cells, as indicated by expression of alkaline phosphatase and osteocalcin, can be induced by transient transfection with the BMP7 gene.

  1. A 3-D Model of Signaling and Transport Pathways in Epithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Quong, A A; Westbrook, C K

    2005-04-01

    A 3-dimensional computer model was developed to simulate the spatial and chemical evolution of calcium ions inside an array of human epithelial kidney cells. This is a prototype model, intended to develop a methodology to incorporate much more complex interactions of metabolic and other processes within many types of cells and lead to increased ability to predict cellular responses to disease as well as to chemical and biological warfare situations. Preliminary tests of the model are described.

  2. Impact of adjustable cryogel properties on the performance of prostate cancer cells in 3D

    OpenAIRE

    Bäcker, A.; Göppert, B.; Sturm, S.; Abaffy, P.; Sollich, T.; Gruhl, F. J.

    2016-01-01

    Background Biochemical and physical characteristics of extracellular environment play a key role in assisting cell behavior over different molecular pathways. In this study, we investigated how the presence of chemical binding sites, the pore network and the stiffness of designed scaffolds affected prostate cancer cells. Methods A blend of poly hydroxyethyl methacrylate–alginate–gelatin scaffold was synthesized by cryogelation process using polyethyleneglycol diacrylate (PEGda) and...

  3. The influence of plasma technology coupled to chemical grafting on the cell growth compliance of 3D hydroxyapatite scaffolds.

    Science.gov (United States)

    Russo, Laura; Zanini, Stefano; Giannoni, Paolo; Landi, Elena; Villa, Anna; Sandri, Monica; Riccardi, Claudia; Quarto, Rodolfo; Doglia, Silvia M; Nicotra, Francesco; Cipolla, Laura

    2012-11-01

    The development of advanced materials with biomimetic features in order to elicit desired biological responses and to guarantee tissue biocompatibility is recently gaining attention for tissue engineering applications. Bioceramics, such as hydroxyapatite-based biomaterials are now used in a number of different applications throughout the body, covering all areas of the skeleton, due to their biological and chemical similarity to the inorganic phases of bones. When bioactive sintered hydroxyapatite (HA) is desired, biomolecular modification of these materials is needed. In the present work, we investigated the influence of plasma surface modification coupled to chemical grafting on the cell growth compliance of HA 3D scaffolds.

  4. Passivation of ZnO Nanowire Guests and 3D Inverse Opal Host Photoanodes for Dye-Sensitized Solar Cells

    KAUST Repository

    Labouchere, Philippe

    2014-04-23

    A hierarchical host-guest nanostructured photoanode is reported for dye-sensitized solar cells. It is composed of ZnO nanowires grown in situ into the macropores of a 3D ZnO inverse opal structure, which acts both as a seed layer and as a conductive backbone host. Using a combination of self-assembly, hydrothermal or electrodeposition of single crystalline ZnO nanowires and TiO2 passivation, a novel photoanode with scattering capability for optimal light harvesting is fabricated. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Efficient animal-serum free 3D cultivation method for adult human neural crest-derived stem cell therapeutics

    Directory of Open Access Journals (Sweden)

    JFW Greiner

    2011-12-01

    Full Text Available Due to their broad differentiation potential and their persistence into adulthood, human neural crest-derived stem cells (NCSCs harbour great potential for autologous cellular therapies, which include the treatment of neurodegenerative diseases and replacement of complex tissues containing various cell types, as in the case of musculoskeletal injuries. The use of serum-free approaches often results in insufficient proliferation of stem cells and foetal calf serum implicates the use of xenogenic medium components. Thus, there is much need for alternative cultivation strategies. In this study we describe for the first time a novel, human blood plasma based semi-solid medium for cultivation of human NCSCs. We cultivated human neural crest-derived inferior turbinate stem cells (ITSCs within a blood plasma matrix, where they revealed higher proliferation rates compared to a standard serum-free approach. Three-dimensionality of the matrix was investigated using helium ion microscopy. ITSCs grew within the matrix as revealed by laser scanning microscopy. Genetic stability and maintenance of stemness characteristics were assured in 3D cultivated ITSCs, as demonstrated by unchanged expression profile and the capability for self-renewal. ITSCs pre-cultivated in the 3D matrix differentiated efficiently into ectodermal and mesodermal cell types, particularly including osteogenic cell types. Furthermore, ITSCs cultivated as described here could be easily infected with lentiviruses directly in substrate for potential tracing or gene therapeutic approaches. Taken together, the use of human blood plasma as an additive for a completely defined medium points towards a personalisable and autologous cultivation of human neural crest-derived stem cells under clinical grade conditions.

  6. Sub-100 nm biodegradable nanoparticles: in vitro release features and toxicity testing in 2D and 3D cell cultures

    International Nuclear Information System (INIS)

    A big challenge in tumor targeting by nanoparticles (NPs), taking advantage of the enhanced permeability and retention effect, is the fabrication of small size devices for enhanced tumor penetration, which is considered fundamental to improve chemotherapy efficacy. The purposes of this study are (i) to engineer the formulation of doxorubicin-loaded poly(d,l-lactic-co-glycolic acid) (PLGA)–block–poly(ethylene glycol) (PEG) NPs to obtain <100 nm devices and (ii) to translate standard 2D cytotoxicity studies to 3D collagen systems in which an initial step gradient of the NPs is present. Doxorubicin release can be prolonged for days to weeks depending on the NP formulation and the pH of the release medium. Sub-100 nm NPs are effectively internalized by HeLa cells in 2D and are less cytotoxic than free doxorubicin. In 3D, <100 nm NPs are significantly more toxic than larger ones towards HeLa cells, and the cell death rate is affected by the contributions of drug release and device transport through collagen. Thus, the reduction of NP size is a fundamental feature from both a technological and a biological point of view and must be properly engineered to optimize the tumor response to the NPs. (paper)

  7. Multiplex profiling of cellular invasion in 3D cell culture models.

    Directory of Open Access Journals (Sweden)

    Gerald Burgstaller

    Full Text Available To-date, most invasion or migration assays use a modified Boyden chamber-like design to assess migration as single-cell or scratch assays on coated or uncoated planar plastic surfaces. Here, we describe a 96-well microplate-based, high-content, three-dimensional cell culture assay capable of assessing invasion dynamics and molecular signatures thereof. On applying our invasion assay, we were able to demonstrate significant effects on the invasion capacity of fibroblast cell lines, as well as primary lung fibroblasts. Administration of epidermal growth factor resulted in a substantial increase of cellular invasion, thus making this technique suitable for high-throughput pharmacological screening of novel compounds regulating invasive and migratory pathways of primary cells. Our assay also correlates cellular invasiveness to molecular events. Thus, we argue of having developed a powerful and versatile toolbox for an extensive profiling of invasive cells in a 96-well format. This will have a major impact on research in disease areas like fibrosis, metastatic cancers, or chronic inflammatory states.

  8. Response of microscale cell/matrix constructs to successive force application in a 3D environment

    Science.gov (United States)

    Liu, Alan; Chen, Christopher; Reich, Daniel

    2014-03-01

    Mechanical dilation of arteries by pulsatile blood flow is directly opposed by coordinated contraction of a band of smooth muscle tissue that envelops the vessels. This mechanical adaptation of smooth muscle cells to external loading is a critical feature of normal blood vessel function. While most previous studies on biomechanical systems have focused on single cells or large excised tissue, we utilize a device to apply forces to engineered smooth muscle microtissues. This device consists of arrayed pairs of elastomeric micro-cantilevers capable of magnetic actuation. Tissues are formed through self-assembly following the introduction of cell-infused collagen gel to the array. With this system, we are able to dynamically stretch and relax these sub-millimeter sized tissues. The timing and magnitude of the force application can be precisely controlled and thus can be used to mimic a wide range of physiological behavior. In particular, we will discuss results that show that the interval between successive force applications mediates the both the subsequent mechanical and active dynamics of the cell/matrix composite system. Understanding this process will lead to better understanding of the interplay between cell and extracellular matrix responses to mechanical stimulus at a novel length scale.

  9. Role of MSX1 in Osteogenic Differentiation of Human Dental Pulp Stem Cells

    Directory of Open Access Journals (Sweden)

    Noriko Goto

    2016-01-01

    Full Text Available Msh homeobox 1 (MSX1 encodes a transcription factor implicated in embryonic development of limbs and craniofacial tissues including bone and teeth. Although MSX1 regulates osteoblast differentiation in the cranial bone of young animal, little is known about the contribution of MSX1 to the osteogenic potential of human cells. In the present study, we investigate the role of MSX1 in osteogenic differentiation of human dental pulp stem cells isolated from deciduous teeth. When these cells were exposed to osteogenesis-induction medium, runt-related transcription factor-2 (RUNX2, bone morphogenetic protein-2 (BMP2, alkaline phosphatase (ALPL, and osteocalcin (OCN mRNA levels, as well as alkaline phosphatase activity, increased on days 4–12, and thereafter the matrix was calcified on day 14. However, knockdown of MSX1 with small interfering RNA abolished the induction of the osteoblast-related gene expression, alkaline phosphatase activity, and calcification. Interestingly, DNA microarray and PCR analyses revealed that MSX1 knockdown induced the sterol regulatory element-binding protein 2 (SREBP2 transcriptional factor and its downstream target genes in the cholesterol synthesis pathway. Inhibition of cholesterol synthesis enhances osteoblast differentiation of various mesenchymal cells. Thus, MSX1 may downregulate the cholesterol synthesis-related genes to ensure osteoblast differentiation of human dental pulp stem cells.

  10. Pore architecture and cell viability on freeze dried 3D recombinant human collagen-peptide (RHC)–chitosan scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jing; Zhou, Aimei; Deng, Aipeng [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Yang, Yang [Faculty of Engineering, University of Nottingham, Nottingham NG7 2RD (United Kingdom); Gao, Lihu; Zhong, Zhaocai [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Yang, Shulin, E-mail: yshulin@njust.edu.cn [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China)

    2015-04-01

    Pore architecture of 3D scaffolds used in tissue engineering plays a critical role in the maintenance of cell survival, proliferation and further promotion of tissue regeneration. We investigated the pore size and structure, porosity, swelling as well as cell viability of a series of recombinant human collagen-peptide–chitosan (RHCC) scaffolds fabricated by lyophilization. In this paper, freezing regime containing a final temperature of freezing (T{sub f}) and cooling rates was applied to obtain scaffolds with pore size ranging from 100 μm to 120 μm. Other protocols of RHC/chitosan suspension concentration and ratio modification were studied to produce more homogenous and appropriate structural scaffolds. The mean pore size decreased along with the decline of T{sub f} at a slow cooling rate of 0.7 °C/min; a more rapid cooling rate under 5 °C/min resulted to a smaller pore size and more homogenous microstructure. High concentration could reduce pore size and lead to thick well of scaffold, while improved the ratio of RHC, lamellar and fiber structure coexisted with cellular pores. Human umbilical vein endothelial cells (HUVECs) were seeded on these manufactured scaffolds, the cell viability represented a negative correlation to the pore size. This study provides an alternative method to fabricate 3D RHC–chitosan scaffolds with appropriate pores for potential tissue engineering. - Highlights: • Fabrication of recombinant human collagen-chitosan scaffolds by freezing drying • Influence of freeze drying protocols on lyophilized scaffolds • Pore size, microstructure, porosity, swelling and cell viability were compared. • The optimized porous scaffold is suitable for cell (HUVEC) seeding.

  11. Pore architecture and cell viability on freeze dried 3D recombinant human collagen-peptide (RHC)–chitosan scaffolds

    International Nuclear Information System (INIS)

    Pore architecture of 3D scaffolds used in tissue engineering plays a critical role in the maintenance of cell survival, proliferation and further promotion of tissue regeneration. We investigated the pore size and structure, porosity, swelling as well as cell viability of a series of recombinant human collagen-peptide–chitosan (RHCC) scaffolds fabricated by lyophilization. In this paper, freezing regime containing a final temperature of freezing (Tf) and cooling rates was applied to obtain scaffolds with pore size ranging from 100 μm to 120 μm. Other protocols of RHC/chitosan suspension concentration and ratio modification were studied to produce more homogenous and appropriate structural scaffolds. The mean pore size decreased along with the decline of Tf at a slow cooling rate of 0.7 °C/min; a more rapid cooling rate under 5 °C/min resulted to a smaller pore size and more homogenous microstructure. High concentration could reduce pore size and lead to thick well of scaffold, while improved the ratio of RHC, lamellar and fiber structure coexisted with cellular pores. Human umbilical vein endothelial cells (HUVECs) were seeded on these manufactured scaffolds, the cell viability represented a negative correlation to the pore size. This study provides an alternative method to fabricate 3D RHC–chitosan scaffolds with appropriate pores for potential tissue engineering. - Highlights: • Fabrication of recombinant human collagen-chitosan scaffolds by freezing drying • Influence of freeze drying protocols on lyophilized scaffolds • Pore size, microstructure, porosity, swelling and cell viability were compared. • The optimized porous scaffold is suitable for cell (HUVEC) seeding

  12. 3D Cell Culture in a Self-Assembled Nanofiber Environment.

    Science.gov (United States)

    Chai, Yi Wen; Lee, Eu Han; Gubbe, John D; Brekke, John H

    2016-01-01

    The development and utilization of three-dimensional cell culture platforms has been gaining more traction. Three-dimensional culture platforms are capable of mimicking in vivo microenvironments, which provide greater physiological relevance in comparison to conventional two-dimensional cultures. The majority of three-dimensional culture platforms are challenged by the lack of cell attachment, long polymerization times, and inclusion of undefined xenobiotics, and cytotoxic cross-linkers. In this study, we review the use of a highly defined material composed of naturally occurring compounds, hyaluronic acid and chitosan, known as Cell-Mate3DTM. Moreover, we provide an original measurement of Young's modulus using a uniaxial unconfined compression method to elucidate the difference in microenvironment rigidity for acellular and cellular conditions. When hydrated into a tissue-like hybrid hydrocolloid/hydrogel, Cell-Mate3DTM is a highly versatile three-dimensional culture platform that enables downstream applications such as flow cytometry, immunostaining, histological staining, and functional studies to be applied with relative ease. PMID:27632425

  13. 3D Cell Culture in a Self-Assembled Nanofiber Environment

    Science.gov (United States)

    Gubbe, John D.; Brekke, John H.

    2016-01-01

    The development and utilization of three-dimensional cell culture platforms has been gaining more traction. Three-dimensional culture platforms are capable of mimicking in vivo microenvironments, which provide greater physiological relevance in comparison to conventional two-dimensional cultures. The majority of three-dimensional culture platforms are challenged by the lack of cell attachment, long polymerization times, and inclusion of undefined xenobiotics, and cytotoxic cross-linkers. In this study, we review the use of a highly defined material composed of naturally occurring compounds, hyaluronic acid and chitosan, known as Cell-Mate3DTM. Moreover, we provide an original measurement of Young’s modulus using a uniaxial unconfined compression method to elucidate the difference in microenvironment rigidity for acellular and cellular conditions. When hydrated into a tissue-like hybrid hydrocolloid/hydrogel, Cell-Mate3DTM is a highly versatile three-dimensional culture platform that enables downstream applications such as flow cytometry, immunostaining, histological staining, and functional studies to be applied with relative ease. PMID:27632425

  14. Decrease of reactive oxygen species-related biomarkers in the tissue-mimic 3D spheroid culture of human lung cells exposed to zinc oxide nanoparticles.

    Science.gov (United States)

    Kim, Eunjoo; Jeon, Won Bae; Kim, Soonhyun; Lee, Soo-Keun

    2014-05-01

    Common 2-dimensional (2D) cell cultures do not adequately represent cell-cell and cell-matrix signaling and substantially different diffusion/transport pathways. To obtain tissue-mimic information on nanoparticle toxicity from in vitro cell tests, we used a 3-dimensional (3D) culture of human lung cells (A549) prepared with elastin-like peptides modified with an arginine-glycine-aspartate motif. The 3D cells showed different cellular phenotypes, gene expression profiles, and functionalities compared to the 2D cultured cells. In gene array analysis, 3D cells displayed the induced extracellular matrix (ECM)-related biological functions such as cell-to-cell signaling and interaction, cellular function and maintenance, connective tissue development and function, molecular transport, and tissue morphology. Additionally, the expression of ECM-related molecules, such as laminin, fibronectin, and insulin-like growth factor binding protein 3 (IGFBP3), was simultaneously induced at both mRNA and protein levels. When 0.08-50 microg/ml zinc oxide nanoparticles (ZnO-NPs) were administered to 2D and 3D cells, the cell proliferation was not significantly changed. The level of molecular markers for oxidative stress, such as superoxide dismutase (SOD), Bcl-2, ATP synthase, and Complex IV (cytochrome C oxidase), was significantly reduced in 2D culture when exposed to 10 microg/ml ZnO-NPs, but no significant decrease was detected in 3D culture when exposed to the same concentration of ZnO-NPs. In conclusion, the tissue-mimic phenotype and functionality of 3D cells could be achieved through the elevated expression of ECM components. The 3D cells were expected to help to better predict the nanotoxicity of ZnO-NPs at tissue-level by increased cell-cell and cell-ECM adhesion and signaling. The tissue-mimic morphology would also be useful to simulate the diffusion/transport of the nanoparticles in vitro.

  15. Improving organic tandem solar cells based on water-processed nanoparticles by quantitative 3D nanoimaging

    DEFF Research Database (Denmark)

    Pedersen, Emil Bøje Lind; Angmo, Dechan; Dam, Henrik Friis;

    2015-01-01

    Organic solar cells have great potential for upscaling due to roll-to-roll processing and a low energy payback time, making them an attractive sustainable energy source for the future. Active layers coated with water-dispersible Landfester particles enable greater control of the layer formation...... and easier access to the printing industry, which has reduced the use of organic solvents since the 1980s. Through ptychographic X-ray computed tomography (PXCT), we image quantitatively a roll-to-roll coated photovoltaic tandem stack consisting of one bulk heterojunction active layer and one Landfester...... the increased performance larger than 1 V expected for a tandem cell. This study highlights that quantitative imaging of weakly scattering stacked layers of organic materials has become feasible by PXCT, and that this information cannot be obtained by other methods. In the present study, this technique...

  16. Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

    OpenAIRE

    Chuang, Han-Ning; Lohaus, Raphaela; Hanisch, Uwe-Karsten; Binder, Claudia; Dehghani, Faramarz; Pukrop, Tobias

    2013-01-01

    Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macro...

  17. Visible light cured thiol-vinyl hydrogels with tunable degradation for 3D cell culture

    OpenAIRE

    Hao, Yiting; Shih, Han; Muňoz, Zachary; Kemp, Arika; Lin, Chien-Chi

    2013-01-01

    We report here a synthetically simple yet highly tunable and diverse visible light mediated thiol-vinyl gelation system for fabricating cell-instructive hydrogels. Gelation was achieved via a mixed-mode step-and-chain-growth photopolymerization using functionalized 4-arm poly(ethylene glycol) as backbone macromer, eosin-Y as photosensitizer, and di-thiol containing molecule as dual purpose co-initiator/cross-linker. N-vinylpyrrolidone (NVP) was used to accelerate gelation kinetics and to adju...

  18. Osteogenic Differentiation of Miniature Pig Mesenchymal Stem Cells in 2D and 3D Environment

    OpenAIRE

    Juhásová, J. (Jana); Juhás, Š. (Štefan); Klíma, J.; Strnádel, J. (Ján); Holubová, M. (Monika); Motlík, J. (Jan)

    2011-01-01

    Mesenchymal stem cells (MSCs) have been repeatedly shown to be able to repair bone defects. The aim of this study was to characterize the osteogenic differentiation of miniature pig MSCs and markers of this differentiation in vitro. Flow-cytometrically characterized MSCs were seeded on cultivation plastic (collagen I and vitronectin coated/uncoated) or plasma clot (PC)/plasma-alginate clot (PAC) scaffolds and differentiated in osteogenic medium. During three weeks of differentiation, the form...

  19. Effect of 3D stall-cells on the pressure distribution of a laminar NACA64-418 wing

    Science.gov (United States)

    Ragni, Daniele; Ferreira, Carlos

    2016-08-01

    A 3D stall-cell flow-field has been studied in a 4.8 aspect-ratio wing obtained by linear extrusion of a laminar NACA64-418 airfoil profile. The span-wise change in the velocity and pressure distribution along the wing has been quantified with respect to the development of cellular structures from 8° to 20° angle of attack. Oil-flow visualizations help localizing the regular cellular pattern in function of the angle of attack. Multi-plane stereoscopic PIV measurements obtained by traversing the entire setup along the wing span show that the flow separation is not span-wise uniform. The combination of different stereoscopic fields into a 3D volume of velocity data allows studying the global effect of the stall-cell pattern on the wing flow. Integration of the experimentally computed pressure gradient from the Navier-Stokes equation is employed to compute the span-wise distribution of the mean surface pressure. Comparison of the results with the ones obtained from pressure taps installed in the wing evidences a span-wise periodic loading on the wing. The periodic loading has maxima confined in the stream-wise direction between the location of the highest airfoil curvature and the one of the airfoil flow separation. Estimation of the periodic loading is found within 2-6 % of the sectional wing lift.

  20. Automated detection of retinal cell nuclei in 3D micro-CT images of zebrafish using support vector machine classification

    Science.gov (United States)

    Ding, Yifu; Tavolara, Thomas; Cheng, Keith

    2016-03-01

    Our group is developing a method to examine biological specimens in cellular detail using synchrotron microCT. The method can acquire 3D images of tissue at micrometer-scale resolutions, allowing for individual cell types to be visualized in the context of the entire specimen. For model organism research, this tool will enable the rapid characterization of tissue architecture and cellular morphology from every organ system. This characterization is critical for proposed and ongoing "phenome" projects that aim to phenotype whole-organism mutants and diseased tissues from different organisms including humans. With the envisioned collection of hundreds to thousands of images for a phenome project, it is important to develop quantitative image analysis tools for the automated scoring of organism phenotypes across organ systems. Here we present a first step towards that goal, demonstrating the use of support vector machines (SVM) in detecting retinal cell nuclei in 3D images of wild-type zebrafish. In addition, we apply the SVM classifier on a mutant zebrafish to examine whether SVMs can be used to capture phenotypic differences in these images. The longterm goal of this work is to allow cellular and tissue morphology to be characterized quantitatively for many organ systems, at the level of the whole-organism.

  1. An innovative hybrid 3D analytic-numerical model for air breathing parallel channel counter-flow PEM fuel cells.

    Science.gov (United States)

    Tavčar, Gregor; Katrašnik, Tomaž

    2014-01-01

    The parallel straight channel PEM fuel cell model presented in this paper extends the innovative hybrid 3D analytic-numerical (HAN) approach previously published by the authors with capabilities to address ternary diffusion systems and counter-flow configurations. The model's core principle is modelling species transport by obtaining a 2D analytic solution for species concentration distribution in the plane perpendicular to the cannel gas-flow and coupling consecutive 2D solutions by means of a 1D numerical pipe-flow model. Electrochemical and other nonlinear phenomena are coupled to the species transport by a routine that uses derivative approximation with prediction-iteration. The latter is also the core of the counter-flow computation algorithm. A HAN model of a laboratory test fuel cell is presented and evaluated against a professional 3D CFD simulation tool showing very good agreement between results of the presented model and those of the CFD simulation. Furthermore, high accuracy results are achieved at moderate computational times, which is owed to the semi-analytic nature and to the efficient computational coupling of electrochemical kinetics and species transport. PMID:25125112

  2. Inhibition of B cell growth factor (BCGF) by monoclonal antibodies directed against the C3d receptor (CR2).

    Science.gov (United States)

    Perri, R T; Wilson, B S; Kay, N E

    1986-04-01

    Normal human B cell proliferation is controlled by various immunoregulatory signals including the T cell-derived lymphokine B cell growth factor (BCGF). Human BCGF provides the final proliferative signal to normal, activated B cells. We herein show that anti-CR2 monoclonal antibodies inhibit human B cell responsiveness to purified BCGF. Addition of anti-CR2 antibody, AB5, was capable of completely inhibiting BCGF-mediated enhancement of either anti-mu or staphylococcal protein A-activated human B cells (191 +/- 21 cpm vs. 3942 +/- 622 cpm, mean +/- SEM). Inhibition of B cell response to BCGF by AB5 occurred in a dose-dependent manner. Monoclonal antibody anti-B2, which recognizes the same 140-kDa glycoprotein as AB5, in comparable concentrations also inhibited B cell responsiveness to BCGF. Monoclonal antibodies of the same subclass (IgG1) showed no inhibitory effect on BCGF enhancement of B cell proliferation. The F(ab')2 fragment of AB5 generated by pepsin digestion was similarly inhibitory as was the intact Ig. AB5-mediated inhibition was independent of the target B cell state of activation. Both resting and activated B cells (anti-mu or staphylococcal protein A activated) incubated with similar concentrations of AB5 were unresponsive to BCGF. The ability of anti-CR2 antibodies to block BCGF-dependent B cell proliferation suggests that occupancy of C3d membrane receptors may result in modulation of B cell proliferation in physiologic or clinical disease states. PMID:2938967

  3. Isolation of Mesenchymal Stem Cells From Dental Pulp of Exfoliated Human Deciduous Teeth

    Directory of Open Access Journals (Sweden)

    N. Nourbakhsh

    2008-01-01

    Full Text Available Objective: The exfoliated human deciduous tooth (SHED contain multipotent stemcells that identified to be a population of highly proliferative and clonogenic .Thesecells are capable of differentiating into a variety of cell types including neural cells,adipocytes, and odontoblasts.Material and Methods: Normal exfoliated human deciduous incisors collected fromsix- to nine-years-old children. The pulp was separated from the crown and digestedwith collagenase .Single cell solutions were cultivated in α-MEM supplemented withES-FCS. After two to three days, the cells reached confluency and were trypsinizedand cultured for further passages. The passage-4 cells were analyzed with CD34,CD45, CD105, CD166, CD31, CD90 and CD146 markers that indicated these cellshad a mesenchymal stem cell (MSC identity. We examined the cells for AlkalinePhosphatase activity to investigate the mesenchymal (stromal nature.Finally, thecells were differentiated into the osteoblastic and adipocytic lineages in differentsubcultures and analysed by RT-PCR and different staining protocols.Results: Viable cells growing out of the explants showed elongated shapes inclusters. These cells showed alkaline phosphatase activity. Flow cytometry resultsrevealed high expression of pluripotent stem cell markers .In some area of theosteoinductive cultures nodule-like structures were observed that showed redmineralizing area upon staining with Alizarin Red.In adipogenic cultures lipid vesiclesappeared after five weeks of induction with Oil Red.Conclusion: This study show that pulp contains cells with high plasticity andproliferation capacity and can be easily isolated without any serious intervention.

  4. Dose distribution and tumor control probability in out-of-field lymph node stations in intensity modulated radiotherapy (IMRT) vs 3D-conformal radiotherapy (3D-CRT) of non-small-cell lung cancer: an in silico analysis

    OpenAIRE

    Fleckenstein, Jochen; Eschler, Andrea; Kremp, Katharina; Kremp, Stephanie; Rübe, Christian

    2015-01-01

    Background The advent of IMRT and image-guided radiotherapy (IGRT) in combination with involved-field radiotherapy (IF-RT) in inoperable non-small-cell lung cancer results in a decreased incidental dose deposition in elective nodal stations. While incidental nodal irradiation is considered a relevant by-product of 3D-CRT to control microscopic disease this planning study analyzed the impact of IMRT on dosimetric parameters and tumor control probabilities (TCP) in elective nodal stations in di...

  5. Exosomes from Human Dental Pulp Stem Cells Suppress Carrageenan-Induced Acute Inflammation in Mice.

    Science.gov (United States)

    Pivoraitė, Ugnė; Jarmalavičiūtė, Akvilė; Tunaitis, Virginijus; Ramanauskaitė, Giedrė; Vaitkuvienė, Aida; Kašėta, Vytautas; Biziulevičienė, Genė; Venalis, Algirdas; Pivoriūnas, Augustas

    2015-10-01

    The primary goal of this study was to examine the effects of human dental pulp stem cell-derived exosomes on the carrageenan-induced acute inflammation in mice. Exosomes were purified by differential ultracentrifugation from the supernatants of stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) cultivated in serum-free medium. At 1 h post-carrageenan injection, exosomes derived from supernatants of 2 × 10(6) SHEDs were administered by intraplantar injection to BALB/c mice; 30 mg/kg of prednisolone and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. Edema was measured at 6, 24, and 48 h after carrageenan injection. For the in vivo imaging experiments, AngioSPARK750, Cat B 750 FAST, and MMPSense 750 FAST were administered into the mouse tail vein 2 h post-carrageenan injection. Fluorescence images were acquired at 6, 24, and 48 h after edema induction by IVIS Spectrum in vivo imaging system. Exosomes significantly reduced the carrageenan-induced edema at all the time points studied (by 39.5, 41.6, and 25.6% at 6, 24, and 48 h after injection, respectively), to similar levels seen with the positive control (prednisolone). In vivo imaging experiments revealed that, both exosomes and prednisolone suppress activities of cathepsin B and matrix metalloproteinases (MMPs) at the site of carrageenan-induced acute inflammation, showing more prominent effects of prednisolone at the early stages, while exosomes exerted their suppressive effects gradually and at later time points. Our study demonstrates for the first time that exosomes derived from human dental pulp stem cells suppress carrageenan-induced acute inflammation in mice.

  6. Enhanced penetration into 3D cell culture using two and three layered gold nanoparticles

    Directory of Open Access Journals (Sweden)

    Engl

    2013-10-01

    Full Text Available Christopher G England,1 Thomas Priest,2 Guandong Zhang,2 Xinghua Sun,2 Dhruvinkumar N Patel,2 Lacey R McNally,3,4 Victor van Berkel,4,5 André M Gobin,2 Hermann B Frieboes1,2,41Department of Pharmacology and Toxicology, 2Department of Bioengineering, 3Department of Medicine, 4James Graham Brown Cancer Center, 5Department of Surgery, University of Louisville, KY, USAAbstract: Nano-scale particles sized 10–400 nm administered systemically preferentially extravasate from tumor vasculature due to the enhanced permeability and retention effect. Therapeutic success remains elusive, however, because of inhomogeneous particle distribution within tumor tissue. Insufficient tumor vascularization limits particle transport and also results in avascular hypoxic regions with non-proliferating cells, which can regenerate tissue after nanoparticle-delivered cytotoxicity or thermal ablation. Nanoparticle surface modifications provide for increasing tumor targeting and uptake while decreasing immunogenicity and toxicity. Herein, we created novel two layer gold-nanoshell particles coated with alkanethiol and phosphatidylcholine, and three layer nanoshells additionally coated with high-density-lipoprotein. We hypothesize that these particles have enhanced penetration into 3-dimensional cell cultures modeling avascular tissue when compared to standard poly(ethylene glycol (PEG-coated nanoshells. Particle uptake and distribution in liver, lung, and pancreatic tumor cell cultures were evaluated using silver-enhancement staining and hyperspectral imaging with dark field microscopy. Two layer nanoshells exhibited significantly higher uptake compared to PEGylated nanoshells. This multilayer formulation may help overcome transport barriers presented by tumor vasculature, and could be further investigated in vivo as a platform for targeted cancer therapies.Keywords: cancer nanotherapy, tumor hypoxia, nanovector transport

  7. Comparative proteomic analyses demonstrate enhanced Interferon and STAT-1 activation in reovirus T3D-infected HeLa cells

    Directory of Open Access Journals (Sweden)

    Peyman eEzzati

    2015-04-01

    Full Text Available As obligate intracellular parasites, viruses are exclusively and intimately dependent upon their host cells for replication. During replication viruses induce profound changes within cells, including: induction of signaling pathways, morphological changes, and cell death. Many such cellular perturbations have been analyzed at the transcriptomic level by gene arrays and recent efforts have begun to analyze cellular proteomic responses. We recently described comparative stable isotopic (SILAC analyses of reovirus, strain type 3 Dearing (T3D-infected HeLa cells. For the present study we employed the complementary labeling strategy of iTRAQ (isobaric tags for relative and absolute quantitation to examine HeLa cell changes induced by T3D, another reovirus strain, type 1 Lang, and UV-inactivated T3D (UV-T3D. Triplicate replicates of cytosolic and nuclear fractions identified a total of 2375 proteins, of which 50, 57, and 46 were significantly up-regulated, and 37, 26 and 44 were significantly down-regulated by T1L, T3D and UV-T3D, respectively. Several pathways, most notably the Interferon signaling pathway and the EIF2 and ILK signaling pathways, were induced by virus infection. Western blots confirmed that cells were more strongly activated by live T3D as demonstrated by elevated levels of key proteins like STAT-1, ISG-15, IFIT-1, IFIT-3 and Mx1. This study expands our understanding of reovirus-induced host responses.

  8. CONTROLLING THE 3D NANOSCALE ORGANIZATION OF BULK HETEROJUNCTION POLYMER SOLAR CELLS

    Institute of Scientific and Technical Information of China (English)

    Svetlana S. Van Bavel; Erwan Sourty; Gijsbertus de With; Joachim Loos

    2009-01-01

    In this study,the three dimensional nanoscale organization in the photoactive layers of poly(3-hexylthiophene)(P3HT) and a methanofullerene derivative (PCBM) is revealed by transmission electron tomography.After annealing treatment,either at elevated temperature or during slow solvent evaporation,nanoscale interpenetrating networks are formed with high crystalline order and favorable concentration gradients of both components through the thickness of the photoactive layer.Such a tailored morphology accounts for the considerable increase of the power conversion efficiency in corresponding solar cell devices.

  9. A novel stem cell source for vasculogenesis in ischemia: subfraction of side population cells from dental pulp.

    Science.gov (United States)

    Iohara, Koichiro; Zheng, Li; Wake, Hiroaki; Ito, Masataka; Nabekura, Junichi; Wakita, Hideaki; Nakamura, Hiroshi; Into, Takeshi; Matsushita, Kenji; Nakashima, Misako

    2008-09-01

    Cell therapy with stem cells and endothelial progenitor cells (EPCs) to stimulate vasculogenesis as a potential treatment for ischemic disease is an exciting area of research in regenerative medicine. EPCs are present in bone marrow, peripheral blood, and adipose tissue. Autologous EPCs, however, are obtained by invasive biopsy, a potentially painful procedure. An alternative approach is proposed in this investigation. Permanent and deciduous pulp tissue is easily available from teeth after extraction without ethical issues and has potential for clinical use. We isolated a highly vasculogenic subfraction of side population (SP) cells based on CD31 and CD146, from dental pulp. The CD31(-);CD146(-) SP cells, demonstrating CD34+ and vascular endothelial growth factor-2 (VEGFR2)/Flk1+, were similar to EPCs. These cells were distinct from the hematopoietic lineage as CD11b, CD14, and CD45 mRNA were not expressed. They showed high proliferation and migration activities and multilineage differentiation potential including vasculogenic potential. In models of mouse hind limb ischemia, local transplantation of this subfraction of SP cells resulted in successful engraftment and an increase in the blood flow including high density of capillary formation. The transplanted cells were in proximity of the newly formed vasculature and expressed several proangiogenic factors, such as VEGF-A, G-CSF, GM-CSF, and MMP3. Conditioned medium from this subfraction showed the mitogenic and antiapoptotic activity on human umbilical vein endothelial cells. In conclusion, subfraction of SP cells from dental pulp is a new stem cell source for cell-based therapy to stimulate angiogenesis/vasculogenesis during tissue regeneration. PMID:18583536

  10. A novel stem cell source for vasculogenesis in ischemia: subfraction of side population cells from dental pulp.

    Science.gov (United States)

    Iohara, Koichiro; Zheng, Li; Wake, Hiroaki; Ito, Masataka; Nabekura, Junichi; Wakita, Hideaki; Nakamura, Hiroshi; Into, Takeshi; Matsushita, Kenji; Nakashima, Misako

    2008-09-01

    Cell therapy with stem cells and endothelial progenitor cells (EPCs) to stimulate vasculogenesis as a potential treatment for ischemic disease is an exciting area of research in regenerative medicine. EPCs are present in bone marrow, peripheral blood, and adipose tissue. Autologous EPCs, however, are obtained by invasive biopsy, a potentially painful procedure. An alternative approach is proposed in this investigation. Permanent and deciduous pulp tissue is easily available from teeth after extraction without ethical issues and has potential for clinical use. We isolated a highly vasculogenic subfraction of side population (SP) cells based on CD31 and CD146, from dental pulp. The CD31(-);CD146(-) SP cells, demonstrating CD34+ and vascular endothelial growth factor-2 (VEGFR2)/Flk1+, were similar to EPCs. These cells were distinct from the hematopoietic lineage as CD11b, CD14, and CD45 mRNA were not expressed. They showed high proliferation and migration activities and multilineage differentiation potential including vasculogenic potential. In models of mouse hind limb ischemia, local transplantation of this subfraction of SP cells resulted in successful engraftment and an increase in the blood flow including high density of capillary formation. The transplanted cells were in proximity of the newly formed vasculature and expressed several proangiogenic factors, such as VEGF-A, G-CSF, GM-CSF, and MMP3. Conditioned medium from this subfraction showed the mitogenic and antiapoptotic activity on human umbilical vein endothelial cells. In conclusion, subfraction of SP cells from dental pulp is a new stem cell source for cell-based therapy to stimulate angiogenesis/vasculogenesis during tissue regeneration.

  11. Retracted: Effects of pro-inflammatory cytokines on mineralization potential of rat dental pulp stem cells.

    Science.gov (United States)

    2011-10-01

    The following article from the Journal of Tissue Engineering and Regenerative Medicine, 'Effects of Pro-inflammatory Cytokines on Mineralization Potential of Rat Dental Pulp Stem Cells' by Yang X, Walboomers XF, Bian Z, Jansen JA, Fan M, published online on 11 July 2011 in Wiley Online Library (onlinelibrary.wiley.com), has been retracted by agreement between the authors, the journal Editor-in-Chief, and John Wiley & Sons, Ltd. The retraction has been agreed due to two authors (Walboomers XF, and Jansen JA) not having been involved in the research described, nor made aware of their names being listed on the manuscript, nor told of its submission to the journal.

  12. Cell therapy, 3D culture systems and tissue engineering for cardiac regeneration.

    Science.gov (United States)

    Emmert, Maximilian Y; Hitchcock, Robert W; Hoerstrup, Simon P

    2014-04-01

    Ischemic Heart Disease (IHD) still represents the "Number One Killer" worldwide accounting for the death of numerous patients. However the capacity for self-regeneration of the adult heart is very limited and the loss of cardiomyocytes in the infarcted heart leads to continuous adverse cardiac-remodeling which often leads to heart-failure (HF). The concept of regenerative medicine comprising cell-based therapies, bio-engineering technologies and hybrid solutions has been proposed as a promising next-generation approach to address IHD and HF. Numerous strategies are under investigation evaluating the potential of regenerative medicine on the failing myocardium including classical cell-therapy concepts, three-dimensional culture techniques and tissue-engineering approaches. While most of these regenerative strategies have shown great potential in experimental studies, the translation into a clinical setting has either been limited or too rapid leaving many key questions unanswered. This review summarizes the current state-of-the-art, important challenges and future research directions as to regenerative approaches addressing IHD and resulting HF.

  13. Biocompatibility and bioactivity of calcium silicate-based endodontic sealers in human dental pulp cells

    Directory of Open Access Journals (Sweden)

    Leticia Boldrin MESTIERI

    2015-10-01

    Full Text Available Mineral Trioxide Aggregate (MTA is a calcium silicate-based material. New sealers have been developed based on calcium silicate as MTA Fillapex and MTA Plus.Objective The aim of this study was to evaluate biocompatibility and bioactivity of these two calcium silicate-based sealers in culture of human dental pulp cells (hDPCs.Material and Methods The cells were isolated from third molars extracted from a 16-year-old patient. Pulp tissue was sectioned into fragments with approximately 1 mm3 and kept in supplemented medium to obtain hDPCs adherent cultures. Cell characterization assays were performed to prove the osteogenic potential. The evaluated materials were: MTA Plus (MTAP; MTA Fillapex (MTAF and FillCanal (FC. Biocompatibility was evaluated with MTT and Neutral Red (NR assays, after hDPCs exposure for 24 h to different dilutions of each sealer extract (1:2, 1:3 and 1:4. Unexposed cells were the positive control (CT. Bioactivity was assessed by alkaline phosphatase (ALP enzymatic assay in cells exposed for one and three days to sealer extracts (1:4 dilution. All data were analyzed by ANOVA and Tukey post-test (p≤0.05%.Results MTT and NR results showed suitable cell viability rates for MTAP at all dilutions (90-135%. Cells exposed to MTAF and FC (1:2 and 1:4 dilutions showed significant low viability rate when compared to CT in MTT. The NR results demonstrated cell viability for all materials tested. In MTAP group, the cells ALP activity was similar to CT in one and three days of exposure to the material. MTAF and FC groups demonstrated a decrease in ALP activity when compared to CT at both periods of cell exposure.Conclusions The hDPCs were suitable for the evaluation of new endodontic materialsin vitro. MTAP may be considered a promising material for endodontic treatments.

  14. Concurrent gemcitabine and 3D radiotherapy in patients with stage III unresectable non-small cell lung cancer

    International Nuclear Information System (INIS)

    Stage III unresectable non-small cell lung cancer (NSCLC) is preferably treated with concurrent schedules of chemoradiotherapy, but none is clearly superior Gemcitabine is a radiosensitizing cytotoxic drug that has been studied in phase 1 and 2 studies in this setting. The aim of this study was to describe outcome and toxicity of low-dose weekly gemcitabine combined with concurrent 3-dimensional conformal radiotherapy (3D-CRT). Treatment consisted of two cycles of a cisplatin and gemcitabine followed by weekly gemcitabine 300 mg/m2 during 5 weeks of 3D-CRT, 60 Gy in 5 weeks (hypofractionated-accelerated). Overall survival (OS), progression-free survival (PFS), and treatment related toxicity according to Common Toxicity Criteria of Adverse Events (CTCAE) version 3.0 were assessed. Between February 2002 and August 2008, 318 patients were treated. Median age was 64 years (range 36–86); 72% were male, WHO PS 0/1/2 was 44/53/3%. Median PFS was 15.5 months (95% confidence interval [CI], 12.9-18.1) and median OS was 24.6 months (95% CI., 21.0-28.1). Main toxicity (CTCAE grade ≥3) was dysphagia (12.6%), esophagitis (9.6%), followed by radiation pneumonitis (3.0%). There were five treatment related deaths (1.6%), two due to esophagitis and three due to radiation pneumonitis. Concurrent low-dose gemcitabine and 3D-CRT provides a comparable survival and toxicity profile to other available treatment schemes for unresectable stage III

  15. Inkjet printing of carbon supported platinum 3-D catalyst layers for use in fuel cells

    Science.gov (United States)

    Taylor, André D.; Kim, Edward Y.; Humes, Virgil P.; Kizuka, Jeremy; Thompson, Levi T.

    We present a method of using inkjet printing (IJP) to deposit catalyst materials onto gas diffusion layers (GDLs) that are made into membrane electrode assemblies (MEAs) for polymer electrolyte fuel cell (PEMFC). Existing ink deposition methods such as spray painting or screen printing are not well suited for ultra low (Monarch 700, Black Pearls 2000, etc.). Our ink jet printed MEAs with catalyst loadings of 0.020 mg Pt cm -2 have shown Pt utilizations in excess of 16,000 mW mg -1 Pt which is higher than our traditional screen printed MEAs (800 mW mg -1 Pt). As a further demonstration of IJP versatility, we present results of a graded distribution of Pt/C catalyst structure using standard Johnson Matthey (JM) catalyst. Compared to a continuous catalyst layer of JM Pt/C (20% Pt), the graded catalyst structure showed enhanced performance.

  16. Culture phases, cytotoxicity and protein expressions of agarose hydrogel induced Sp2/0, A549, MCF-7 cell line 3D cultures.

    Science.gov (United States)

    Ravi, Maddaly; Kaviya, S R; Paramesh, V

    2016-05-01

    Advancements in cell cultures are occurring at a rapid pace, an important direction is culturing cells in 3D conditions. We demonstrate the usefulness of agarose hydrogels in obtaining 3 dimensional aggregates of three cell lines, A549, MCF-7 and Sp2/0. The differences in culture phases, susceptibility to cisplatin-induced cytotoxicity are studied. Also, the 3D aggregates of the three cell lines were reverted into 2D cultures and the protein profile differences among the 2D, 3D and revert cultures were studied. The analysis of protein profile differences using UniProt data base further augment the usefulness of agarose hydrogels for obtaining 3D cell cultures.

  17. Distribution of class ii major histocompatibility complex antigenexpressing cells in human dental pulp with carious lesions

    Directory of Open Access Journals (Sweden)

    Tetiana Haniastuti

    2012-09-01

    Full Text Available Background: Dental caries is a bacterial infection which causes destruction of the hard tissues of the tooth. Exposure of the dentin to the oral environment as a result of caries inevitably results in a cellular response in the pulp. The major histocompatibility complex (MHC is a group of genes that code for cell-surface histocompatibility antigens. Cells expressing class II MHC molecules participate in the initial recognition and the processing of antigenic substances to serve as antigen-presenting cells. Purpose: The aim of the study was to elucidate the alteration in the distribution of class II MHC antigen-expressing cells in human dental pulp as carious lesions progressed toward the pulp. Methods: Fifteen third molars with caries at the occlusal site at various stages of decay and 5 intact third molars were extracted and used in this study. Before decalcifying with 10% EDTA solution (pH 7.4, all the samples were observed by micro-computed tomography to confirm the lesion condition three-dimensionally. The specimens were then processed for cryosection and immunohistochemistry using an anti-MHC class II monoclonal antibody. Results: Class II MHC antigen-expressing cells were found both in normal and carious specimens. In normal tooth, the class II MHC-immunopositive cells were observed mainly at the periphery of the pulp tissue. In teeth with caries, class II MHC-immunopositive cells were located predominantly subjacent to the carious lesions. As the caries progressed, the number of class II MHC antigen-expressing cells was increased. Conclusion: The depth of carious lesions affects the distribution of class II MHC antigen-expressing cells in the dental pulp.Latar belakang: Karies merupakan penyakit infeksi bakteri yang mengakibatkan destruksi jaringan keras gigi. Dentin yang terbuka akibat karies akan menginduksi respon imun seluler pada pulpa. Kompleks histokompatibilitas utama (MHC merupakan sekumpulan gen yang mengkode histokompatibilitas

  18. Inhibitors of Rho kinase (ROCK) signaling revert the malignant phenotype of breast cancer cells in 3D context.

    Science.gov (United States)

    Matsubara, Masahiro; Bissell, Mina J

    2016-05-31

    Loss of polarity and quiescence along with increased cellular invasiveness are associated with breast tumor progression. ROCK plays a central role in actin-cytoskeletal rearrangement. We used physiologically relevant 3D cultures of nonmalignant and cancer cells in gels made of laminin-rich extracellular matrix, to investigate ROCK function. Whereas expression levels of ROCK1 and ROCK2 were elevated in cancer cells compared to nonmalignant cells, this was not observed in 2D cultures. Malignant cells showed increased phosphorylation of MLC, corresponding to disorganized F-actin. Inhibition of ROCK signaling restored polarity, decreased disorganization of F-actin, and led to reduction of proliferation. Inhibition of ROCK also decreased EGFR and Integrinβ1 levels, and consequently suppressed activation of Akt, MAPK and FAK as well as GLUT3 and LDHA levels. Again, ROCK inhibition did not inhibit these molecules in 2D. A triple negative breast cancer cell line, which lacks E-cadherin, had high levels of ROCK but was less sensitive to ROCK inhibitors. Exogenous overexpression of E-cadherin, however, rendered these cells strikingly sensitive to ROCK inhibition. Our results add to the growing literature that demonstrate the importance of context and tissue architecture in determining not only regulation of normal and malignant phenotypes but also drug response.

  19. TET1 knockdown inhibits the odontogenic differentiation potential of human dental pulp cells.

    Science.gov (United States)

    Rao, Li-Jia; Yi, Bai-Cheng; Li, Qi-Meng; Xu, Qiong

    2016-01-01

    Human dental pulp cells (hDPCs) possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten-eleven translocation 1 (TET1) is a novel DNA methyldioxygenase that plays an important role in the promotion of DNA demethylation and transcriptional regulation in several cell lines. However, the role of TET1 in the biological functions of hDPCs is unknown. To investigate the effect of TET1 on the proliferation and odontogenic differentiation potential of hDPCs, a recombinant shRNA lentiviral vector was used to knock down TET1 expression in hDPCs. Following TET1 knockdown, TET1 was significantly downregulated at both the mRNA and protein levels. Proliferation of the hDPCs was suppressed in the TET1 knockdown groups. Alkaline phosphatase activity, the formation of mineralized nodules, and the expression levels of DSPP and DMP1 were all reduced in the TET1-knockdown hDPCs undergoing odontogenic differentiation. Based on these results, we concluded that TET1 knockdown can prevent the proliferation and odontogenic differentiation of hDPCs, which suggests that TET1 may play an important role in dental pulp repair and regeneration. PMID:27357322

  20. Effects of Dental Methacrylates on Oxygen Consumption and Redox Status of Human Pulp Cells

    Directory of Open Access Journals (Sweden)

    Giuseppina Nocca

    2014-01-01

    Full Text Available Several studies have already demonstrated that the incomplete polymerization of resin-based dental materials causes the release of monomers which might affect cell metabolism. The aim of this study was to investigate the effects of triethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, urethane dimethacrylate, and 2-hydroxyethyl methacrylate on (1 cellular energy metabolism, evaluating oxygen consumption rate, glucose consumption, glucose 6-phosphate dehydrogenase activity, and lactate production, and (2 cellular redox status, through the evaluation of glutathione concentration and of the activities of enzymes regulating glutathione metabolism. Methods. Human pulp cells were used and oxygen consumption was measured by means of a Clark electrode. Moreover, reactive oxygen species production was quantified. Enzymatic activity and glucose and lactate concentrations were determined through a specific kit. Results. Triethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, and 2-hydroxyethyl methacrylate induced a decrease in oxygen consumption rate, an enhancement of glucose consumption, and lactate production, whilst glucose 6-phosphate dehydrogenase and glutathione reductase activity were not significantly modified. Moreover, the monomers induced an increase of reactive oxygen species production with a consequent increase of superoxide dismutase and catalase enzymatic activities. A depletion of both reduced and total glutathione was also observed. Conclusion. The obtained results indicate that dental monomers might alter energy metabolism and glutathione redox balance in human pulp cells.

  1. Spanish patterns of care for 3D radiotherapy in non-small-cell lung cancer

    International Nuclear Information System (INIS)

    Purpose: Curative radiotherapy for non-small-cell lung cancer is a difficult challenge, despite the use of conformal radiotherapy. Optimal three-dimensional delineation of treatment volumes is essential for improvement of local control and for limiting of tissue toxicity. Material and Methods: A planning course on clinical practice of lung cancer was held in Barcelona. A questionnaire was given concerning (1) patient positioning, (2) planning-computed tomography scan, (3) accounting for tumor mobility, (4) investigative-procedure respiration-gated radiotherapy and breath-holding maneuvers, (5) generation of target volumes, (6) treatment planning, and (7) treatment delivery. This questionnaire was made to determine the Spanish application of European recommendations. Results: On the negative side, 1 hospital did not use three-dimensional tools, less than 50% used immobilization devices, and 55.6% used computed tomography slices of greater than 5 mm. On the positive side, 70.4% did not use standard margins for gross target volume derived from a computed tomography scan, 92.6% agreed with the inclusion of Naruke anatomic criteria of 1 cm or more in gross target volume planning, and 75% used V20 to estimate the risk of pneumonitis. Conclusions: This study is the first validation of European recommendations for treatment planning and execution of radiotherapy in lung cancer. The main conclusion is the need to improve the negative aspects determined

  2. Cytotoxic effects of bulk fill composite resins on human dental pulp stem cells.

    Science.gov (United States)

    Şişman, Reyhan; Aksoy, Ayça; Yalçın, Muhammet; Karaöz, Erdal

    2016-01-01

    Five bulk fill composite resins, including SDR, Tetric EvoCeram Bulk Fill (TEC), X-trafil (XTF), Sonic Fill (SF), Filtek Bulk Fill (FBF), were used in this study. Human dental pulp stem cells were cultured in 12-well culture dishes (3 × 104 cells per cm(2)) and stored in an incubator at 37°C and 5% CO2 for 1 day. On days 1, 7, 14, and 21 of co-culture, viable cells were measured using a WST-1 assay. Lower cell viability was observed with XTF and SDR bulk fill composite resins compared to the control group during the WST-1 assay. Although bulk fill composite resins provide advantages in practical applications, they are limited by their cytotoxic properties. (J Oral Sci 58, 299-305, 2016).

  3. Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells.

    Directory of Open Access Journals (Sweden)

    Elena Gaberman

    Full Text Available Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×10(6 cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat increased the survival of the irradiated mice from ∼27% to 68% (P<0.001, while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD increased the survival to ∼98% (P<0.0001. The dose modifying factor of this treatment for both 50% and 37% survival (DMF50 and DMF37 was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05. A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective "off the shelf" therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia.

  4. Polymeric vs hydroxyapatite-based scaffolds on dental pulp stem cell proliferation and differentiation

    Institute of Scientific and Technical Information of China (English)

    Arash; Khojasteh; Saeed; Reza; Motamedian; Maryam; Rezai; Rad; Mehrnoosh; Hasan; Shahriari; Nasser; Nadjmi

    2015-01-01

    AIM: To evaluate adhesion, proliferation and differentiation of human dental pulp stem cells(h DPSCs) on four commercially available scaffold biomaterials. METHODS: hD PSCs were isolated from human dental pulp tissues of extracted wisdom teeth and established in stem cell growth medium. h DPSCs at passage 3-5 were seeded on four commercially available scaffold biomaterials, SureO ss(Allograft), Cerabone(Xenograft), PLLA(Synthetic), and OSTEON Ⅱ Collagen(Composite), for 7 and 14 d in osteogenic medium. Cell adhesion and morphology to the scaffolds were evaluated by scanning electron microscopy(SEM). Cell proliferation and differentiation into osteogenic lineage were evaluated using DNA counting and alkaline phosphatase(ALP) activity assay, respectively. RESULTS: All scaffold biomaterials except Sure Oss(Allograft) supported h DPSC adhesion, proliferation and differentiation. hD PSCs seeded on PLLA(Synthetic) scaffold showed the highest cell proliferation and attachment as indicated with both SEM and DNA counting assay. Evaluating the osteogenic differentiation capability of hD PSCs on different scaffold biomaterials with ALP activity assay showed high level of ALP activity on cells cultured on PLLA(Synthetic) and OSTEON ⅡCollagen(Composite) scaffolds. SEM micrographs also showed that in the presence of Cerabone(Xenograft) and OSTEON Ⅱ Collagen(Composite) scaffolds, the h DPSCs demonstrated the fibroblastic phenotype with several cytoplasmic extension, while the cells on PLLA scaffold showed the osteoblastic-like morphology, round-like shape. CONCLUSION: PLLA scaffold supports adhesion, proliferation and osteogenic differentiation of hD PSCs. Hence, it may be useful in combination with hD PSCs for cell-based reconstructive therapy.

  5. Isolation and characterization of neural crest-derived stem cells from dental pulp of neonatal mice.

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    Kajohnkiart Janebodin

    Full Text Available Dental pulp stem cells (DPSCs are shown to reside within the tooth and play an important role in dentin regeneration. DPSCs were first isolated and characterized from human teeth and most studies have focused on using this adult stem cell for clinical applications. However, mouse DPSCs have not been well characterized and their origin(s have not yet been elucidated. Herein we examined if murine DPSCs are neural crest derived and determined their in vitro and in vivo capacity. DPSCs from neonatal murine tooth pulp expressed embryonic stem cell and neural crest related genes, but lacked expression of mesodermal genes. Cells isolated from the Wnt1-Cre/R26R-LacZ model, a reporter of neural crest-derived tissues, indicated that DPSCs were Wnt1-marked and therefore of neural crest origin. Clonal DPSCs showed multi-differentiation in neural crest lineage for odontoblasts, chondrocytes, adipocytes, neurons, and smooth muscles. Following in vivo subcutaneous transplantation with hydroxyapatite/tricalcium phosphate, based on tissue/cell morphology and specific antibody staining, the clones differentiated into odontoblast-like cells and produced dentin-like structure. Conversely, bone marrow stromal cells (BMSCs gave rise to osteoblast-like cells and generated bone-like structure. Interestingly, the capillary distribution in the DPSC transplants showed close proximity to odontoblasts whereas in the BMSC transplants bone condensations were distant to capillaries resembling dentinogenesis in the former vs. osteogenesis in the latter. Thus we demonstrate the existence of neural crest-derived DPSCs with differentiation capacity into cranial mesenchymal tissues and other neural crest-derived tissues. In turn, DPSCs hold promise as a source for regenerating cranial mesenchyme and other neural crest derived tissues.

  6. Acemannan, an extracted product from Aloe vera, stimulates dental pulp cell proliferation, differentiation, mineralization, and dentin formation.

    Science.gov (United States)

    Jittapiromsak, Nawaporn; Sahawat, Dusida; Banlunara, Wijit; Sangvanich, Polkit; Thunyakitpisal, Pasutha

    2010-06-01

    This study investigated the effect of acemannan (Aloe vera gel polysaccharide) on dentin formation. Primary human dental pulp cells were treated with acemannan. New DNA synthesis, bone morphogenetic protein-2, alkaline phosphatase activity, dentin sialoprotein expression, and mineralization were determined by [(3)H]-thymidine incorporation, enzyme-linked immunosorbent assay, biochemical assay, western blotting, and Alizarin Red staining, respectively. Then the upper first molars of 24 male Sprague Dawley rats were intentionally exposed and capped with either acemannan or calcium hydroxide. At day 28, the teeth were histopathologically examined and evaluated for the degree of inflammation, dentin bridge formation, and pulp tissue organization. The results revealed that acemannan significantly increased pulp cell proliferation, bone morphogenetic protein-2, alkaline phosphatase activity, dentin sialoprotein expression, and mineralization, compared with the untreated group. The acemannan-treated group also exhibited a complete homogeneous calcified dentin bridge and good pulp tissue organization, whereas neither was detected in the calcium hydroxide-treated and sham groups. In the acemannan-treated group, either mild or no inflammation was found, whereas the other groups had various degrees of inflammation. The data suggest that acemannan promotes dentin formation by stimulating primary human dental pulp cell proliferation, differentiation, extracellular matrix formation, and mineralization. Acemannan also has pulpal biocompatibility and promotes soft tissue organization.

  7. Genotoxicity assessment of reactive and disperse textile dyes using human dermal equivalent (3D cell culture system).

    Science.gov (United States)

    Leme, Daniela Morais; Primo, Fernando Lucas; Gobo, Graciely Gomides; da Costa, Cleber Rafael Vieira; Tedesco, Antonio Claudio; de Oliveira, Danielle Palma

    2015-01-01

    Thousands of dyes are marketed daily for different purposes, including textile dyeing. However, there are several studies reporting attributing to dyes deleterious human effects such as DNA damage. Humans may be exposed to toxic dyes through either ingestion of contaminated waters or dermal contact with colored garments. With respect to dermal exposure, human skin equivalents are promising tools to assess in vitro genotoxicity of dermally applied chemicals using a three-dimensional (3D) model to mimic tissue behavior. This study investigated the sensitivity of an in-house human dermal equivalent (DE) for detecting genotoxicity of textile dyes. Two azo (reactive green 19 [RG19] and disperse red 1[DR1]) dyes and one anthraquinone (reactive blue 2 [RB2]) dye were analyzed. RG19 was genotoxic for DE in a dose-responsive manner, whereas RB2 and DR1 were nongenotoxic under the conditions tested. These findings are not in agreement with previous genotoxicological assessment of these dyes carried out using two-dimensional (2D) cell cultures, which showed that DR1 was genotoxic in human hepatoma cells (HepG2) and RG19 was nongenotoxic for normal human dermal fibroblasts (NHDF). These discrepant results probably may be due to differences between metabolic activities of each cell type (organ-specific genotoxicity, HepG2 and fibroblasts) and the test setup systems used in each study (fibroblasts cultured at 2D and three-dimensional [3D] culture systems). Genotoxicological assessment of textile dyes in context of organ-specific genotoxicity and using in vitro models that more closely resemble in vivo tissue architecture and physiology may provide more reliable estimates of genotoxic potential of these chemicals.

  8. Drug-releasing nano-engineered titanium implants: therapeutic efficacy in 3D cell culture model, controlled release and stability.

    Science.gov (United States)

    Gulati, Karan; Kogawa, Masakazu; Prideaux, Matthew; Findlay, David M; Atkins, Gerald J; Losic, Dusan

    2016-12-01

    There is an ongoing demand for new approaches for treating localized bone pathologies. Here we propose a new strategy for treatment of such conditions, via local delivery of hormones/drugs to the trauma site using drug releasing nano-engineered implants. The proposed implants were prepared in the form of small Ti wires/needles with a nano-engineered oxide layer composed of array of titania nanotubes (TNTs). TNTs implants were inserted into a 3D collagen gel matrix containing human osteoblast-like, and the results confirmed cell migration onto the implants and their attachment and spread. To investigate therapeutic efficacy, TNTs/Ti wires loaded with parathyroid hormone (PTH), an approved anabolic therapeutic for the treatment of severe bone fractures, were inserted into 3D gels containing osteoblast-like cells. Gene expression studies revealed a suppression of SOST (sclerostin) and an increase in RANKL (receptor activator of nuclear factor kappa-B ligand) mRNA expression, confirming the release of PTH from TNTs at concentrations sufficient to alter cell function. The performance of the TNTs wire implants using an example of a drug needed at relatively higher concentrations, the anti-inflammatory drug indomethacin, is also demonstrated. Finally, the mechanical stability of the prepared implants was tested by their insertion into bovine trabecular bone cores ex vivo followed by retrieval, which confirmed the robustness of the TNT structures. This study provides proof of principle for the suitability of the TNT/Ti wire implants for localized bone therapy, which can be customized to cater for specific therapeutic requirements. PMID:27612777

  9. Epigenetic modulation of dental pulp stem cells: implications for regenerative endodontics.

    Science.gov (United States)

    Duncan, H F; Smith, A J; Fleming, G J P; Cooper, P R

    2016-05-01

    Dental pulp stem cells (DPSCs) offer significant potential for use in regenerative endodontics, and therefore, identifying cellular regulators that control stem cell fate is critical to devising novel treatment strategies. Stem cell lineage commitment and differentiation are regulated by an intricate range of host and environmental factors of which epigenetic influence is considered vital. Epigenetic modification of DNA and DNA-associated histone proteins has been demonstrated to control cell phenotype and regulate the renewal and pluripotency of stem cell populations. The activities of the nuclear enzymes, histone deacetylases, are increasingly being recognized as potential targets for pharmacologically inducing stem cell differentiation and dedifferentiation. Depending on cell maturity and niche in vitro, low concentration histone deacetylase inhibitor (HDACi) application can promote dedifferentiation of several post-natal and mouse embryonic stem cell populations and conversely increase differentiation and accelerate mineralization in DPSC populations, whilst animal studies have shown an HDACi-induced increase in stem cell marker expression during organ regeneration. Notably, both HDAC and DNA methyltransferase inhibitors have also been demonstrated to dramatically increase the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) for use in regenerative therapeutic procedures. As the regulation of cell fate will likely remain the subject of intense future research activity, this review aims to describe the current knowledge relating to stem cell epigenetic modification, focusing on the role of HDACi on alteration of DPSC phenotype, whilst presenting the potential for therapeutic application as part of regenerative endodontic regimens. PMID:26011759

  10. On the Quality of Velocity Interpolation Schemes for Marker-In-Cell Methods on 3-D Staggered Grids

    Science.gov (United States)

    Kaus, B.; Pusok, A. E.; Popov, A.

    2015-12-01

    The marker-in-cell method is generally considered to be a flexible and robust method to model advection of heterogenous non-diffusive properties (i.e. rock type or composition) in geodynamic problems or incompressible Stokes problems. In this method, Lagrangian points carrying compositional information are advected with the ambient velocity field on an immobile, Eulerian grid. However, velocity interpolation from grid points to marker locations is often performed without preserving the zero divergence of the velocity field at the interpolated locations (i.e. non-conservative). Such interpolation schemes can induce non-physical clustering of markers when strong velocity gradients are present (Jenny et al., 2001) and this may, eventually, result in empty grid cells, a serious numerical violation of the marker-in-cell method. Solutions to this problem include: using larger mesh resolutions and/or marker densities, or repeatedly controlling the marker distribution (i.e. inject/delete), but which does not have an established physical background. To remedy this at low computational costs, Jenny et al. (2001) and Meyer and Jenny (2004) proposed a simple, conservative velocity interpolation (CVI) scheme for 2-D staggered grid, while Wang et al. (2015) extended the formulation to 3-D finite element methods. Here, we follow up with these studies and report on the quality of velocity interpolation methods for 2-D and 3-D staggered grids. We adapt the formulations from both Jenny et al. (2001) and Wang et al. (2015) for use on 3-D staggered grids, where the velocity components have different node locations as compared to finite element, where they share the same node location. We test the different interpolation schemes (CVI and non-CVI) in combination with different advection schemes (Euler, RK2 and RK4) and with/out marker control on Stokes problems with strong velocity gradients, which are discretized using a finite difference method. We show that a conservative formulation

  11. The Experimental Study of the Performance of Nano-Thin Polyelectrolyte Shell for Dental Pulp Stem Cells Immobilization.

    Science.gov (United States)

    Grzeczkowicz, A; Granicka, L H; Maciejewska, I; Strawski, M; Szklarczyk, M; Borkowska, M

    2015-12-01

    Carious is the most frequent disease of mineralized dental tissues which might result in dental pulp inflammation and mortality. In such cases an endodontic treatment is the only option to prolong tooth functioning in the oral cavity; however, in the cases of severe pulpitis, especially when complicated with periodontal tissue inflammation, the endodontic treatment might not be enough to protect against tooth loss. Thus, keeping the dental pulp viable and/or possibility of the reconstruction of a viable dental pulp complex, appears to become a critical factor for carious and/or pulp inflammation treatment. The nowadays technologies, which allow handling dental pulp stem cells (DPSC), seem to bring us closer to the usage of dental stem cells for tooth tissues reconstruction. Thus, DPSC immobilized within nano-thin polymeric shells, allowing for a diffusion of produced factors and separation from bacteria, may be considered as a cover system supporting technology of dental pulp reconstruction. The DPSC were immobilized using a layer-by-layer technique within nano-thin polymeric shells constructed and modified by nanostructure involvement to ensure the layers stability and integrity as well as separation from bacterial cells. The cytotoxity of the material used for membrane production was assessed on the model of adherent cells. The performance of DPSC nano-coating was assessed in vitro. Membrane coatings showed no cytotoxicity on the immobilized cells. The presence of coating shell was confirmed with flow cytometry, atomic force microscopy and visualized with fluorescent microscopy. The transfer of immobilized DPSC within the membrane system ensuring cells integrity, viability and protection from bacteria should be considered as an alternative method for dental tissues transportation and regeneration. PMID:26682375

  12. Exploring natural silk protein sericin for regenerative medicine: an injectable, photoluminescent, cell-adhesive 3D hydrogel

    Science.gov (United States)

    Wang, Zheng; Zhang, Yeshun; Zhang, Jinxiang; Huang, Lei; Liu, Jia; Li, Yongkui; Zhang, Guozheng; Kundu, Subhas C.; Wang, Lin

    2014-11-01

    Sericin, a major component of silk, has a long history of being discarded as a waste during silk processing. The value of sericin for tissue engineering is underestimated and its potential application in regenerative medicine has just begun to be explored. Here we report the successful fabrication and characterization of a covalently-crosslinked 3D pure sericin hydrogel for delivery of cells and drugs. This hydrogel is injectable, permitting its implantation through minimally invasive approaches. Notably, this hydrogel is found to exhibit photoluminescence, enabling bioimaging and in vivo tracking. Moreover, this hydrogel system possesses excellent cell-adhesive capability, effectively promoting cell attachment, proliferation and long-term survival of various types of cells. Further, the sericin hydrogel releases bioactive reagents in a sustained manner. Additionally, this hydrogel demonstrates good elasticity, high porosity, and pH-dependent degradation dynamics, which are advantageous for this sericin hydrogel to serve as a delivery vehicle for cells and therapeutic drugs. With all these unique features, it is expected that this sericin hydrogel will have wide utility in the areas of tissue engineering and regenerative medicine.

  13. CAD/CAM-designed 3D-printed electroanalytical cell for the evaluation of nanostructured gas-diffusion electrodes

    Science.gov (United States)

    Chervin, Christopher N.; Parker, Joseph F.; Nelson, Eric S.; Rolison, Debra R.; Long, Jeffrey W.

    2016-04-01

    The ability to effectively screen and validate gas-diffusion electrodes is critical to the development of next-generation metal-air batteries and regenerative fuel cells. The limiting electrode in a classic two-terminal device such as a battery or fuel cell is difficult to discern without an internal reference electrode, but the flooded electrolyte characteristic of three-electrode electroanalytical cells negates the prime function of an air electrode—a void volume freely accessible to gases. The nanostructured catalysts that drive the energy-conversion reactions (e.g., oxygen reduction and evolution in the air electrode of metal-air batteries) are best evaluated in the electrode structure as-used in the practical device. We have designed, 3D-printed, and characterized an air-breathing, thermodynamically referenced electroanalytical cell that allows us to mimic the Janus arrangement of the gas-diffusion electrode in a metal-air cell: one face freely exposed to gases, the other wetted by electrolyte.

  14. Calcium Electroporation: Evidence for Differential Effects in Normal and Malignant Cell Lines, Evaluated in a 3D Spheroid Model.

    Directory of Open Access Journals (Sweden)

    Stine Krog Frandsen

    Full Text Available Calcium electroporation describes the use of high voltage electric pulses to introduce supraphysiological calcium concentrations into cells. This promising method is currently in clinical trial as an anti-cancer treatment. One very important issue is the relation between tumor cell kill efficacy-and normal cell sensitivity.Using a 3D spheroid cell culture model we have tested the effect of calcium electroporation and electrochemotherapy using bleomycin on three different human cancer cell lines: a colorectal adenocarcinoma (HT29, a bladder transitional cell carcinoma (SW780, and a breast adenocarcinoma (MDA-MB231, as well as on primary normal human dermal fibroblasts (HDF-n.The results showed a clear reduction in spheroid size in all three cancer cell spheroids three days after treatment with respectively calcium electroporation (p<0.0001 or electrochemotherapy using bleomycin (p<0.0001. Strikingly, the size of normal fibroblast spheroids was neither affected after calcium electroporation nor electrochemotherapy using bleomycin, indicating that calcium electroporation, like electrochemotherapy, will have limited adverse effects on the surrounding normal tissue when treating with calcium electroporation. The intracellular ATP level, which has previously been shown to be depleted after calcium electroporation, was measured in the spheroids after treatment. The results showed a dramatic decrease in the intracellular ATP level (p<0.01 in all four spheroid types-malignant as well as normal.In conclusion, calcium electroporation seems to be more effective in inducing cell death in cancer cell spheroids than in a normal fibroblast spheroid, even though intracellular ATP level is depleted in all spheroid types after treatment. These results may indicate an important therapeutic window for this therapy; although further studies are needed in vivo and in patients to investigate the effect of calcium electroporation on surrounding normal tissue when

  15. Low-intensity laser phototherapy enhances the proliferation of dental pulp stem cells under nutritional deficiency

    Directory of Open Access Journals (Sweden)

    Cacio MOURA NETTO

    2016-01-01

    Full Text Available Abstract Dental trauma in immature permanent teeth can damage pulp vascularization, which leads to necrosis and cessation of apexogenesis. Studies on tissue engineering using stem cells from human exfoliated deciduous teeth (SHEDs have yielded promising results. Laser phototherapy (LPT is able to influence the proliferation and differentiation of these cells, which could improve tissue engineering. SHEDs (eighth passage were seeded into 96-well culture plates (103 cells/well and were grown in culture medium supplemented with 15% defined fetal bovine serum (FBS for 12 h. After determining the appropriate nutrition deficiency status (5% FBS, the cells were assigned into four groups: 1 G1 – 15% FBS (positive control; 2 G2 – 5% FBS (negative control; 3 G3 – 5% FBS+LPT 3 J/cm2; and 4 G4 – 5% FBS+LPT 5 J/cm2. For the LPT groups, two laser irradiations at 6 h intervals were performed using a continuous wave InGaAlP diode laser (660 nm, with a spot size of 0.028 cm2, 10 mW in punctual and contact mode. Cell viability was assessed via an MTT reduction assay immediately after the second laser irradiation (0 h and 24, 48, and 72 h later. We found that G3 and G4 presented a significantly higher cell growth rate when compared with G2 (p 0.05. These findings indicate that LPT with 5 J/cm2 can enhance the growth of SHEDs during situations of nutritional deficiency. Therefore, LPT could be a valuable adjunct treatment in tissue engineering when using stem cells derived from the dental pulp of primary teeth.

  16. Dental pulp stem cells differentiation reveals new insights in Oct4A dynamics.

    Directory of Open Access Journals (Sweden)

    Federico Ferro

    Full Text Available Although the role played by the core transcription factor network, which includes c-Myc, Klf4, Nanog, and Oct4, in the maintenance of embryonic stem cell (ES pluripotency and in the reprogramming of adult cells is well established, its persistence and function in adult stem cells are still debated. To verify its persistence and clarify the role played by these molecules in adult stem cell function, we investigated the expression pattern of embryonic and adult stem cell markers in undifferentiated and fully differentiated dental pulp stem cells (DPSC. A particular attention was devoted to the expression pattern and intracellular localization of the stemness-associated isoform A of Oct4 (Oct4A. Our data demonstrate that: Oct4, Nanog, Klf4 and c-Myc are expressed in adult stem cells and, with the exception of c-Myc, they are significantly down-regulated following differentiation. Cell differentiation was also associated with a significant reduction in the fraction of DPSC expressing the stem cell markers CD10, CD29 and CD117. Moreover, a nuclear to cytoplasm shuttling of Oct4A was identified in differentiated cells, which was associated with Oct4A phosphorylation. The present study would highlight the importance of the post-translational modifications in DPSC stemness maintenance, by which stem cells balance self-renewal versus differentiation. Understanding and controlling these mechanisms may be of great importance for stemness maintenance and stem cells clinical use, as well as for cancer research.

  17. Hard tissue formation in a porous HA/TCP ceramic scaffold loaded with stromal cells derived from dental pulp and bone marrow.

    NARCIS (Netherlands)

    Zhang, W.; Walboomers, X.F.; Osch, G.J.V.M. van; Dolder, J. van den; Jansen, J.A.

    2008-01-01

    The aim of this study was to compare the ability of hard tissue regeneration of four types of stem cells or precursors under both in vitro and in vivo situations. Primary cultures of rat bone marrow, rat dental pulp, human bone marrow, and human dental pulp cells were seeded onto a porous ceramic sc

  18. The Effects of Matrix Stiffness and RhoA on the Phenotypic Plasticity of Smooth Muscle Cells in a 3-D Biosynthetic Hydrogel System

    OpenAIRE

    Peyton, Shelly R.; Kim, Peter D.; Ghajar, Cyrus M.; Seliktar, Dror; Putnam, Andrew J.

    2008-01-01

    Studies using 2-D cultures have shown that the mechanical properties of the extracellular matrix (ECM) influence cell migration, spreading, proliferation, and differentiation; however, cellular mechanosensing in 3-D remains under-explored. To investigate this topic, a unique biomaterial system based on poly(ethylene glycol)-conjugated fibrinogen was adapted to study phenotypic plasticity in smooth muscle cells (SMCs) as a function of ECM mechanics in 3-D. Tuning compressive modulus between 44...

  19. 3D-dynamic visualization of complex molecular cell biology processes : 1-year university students' understanding of visualizations of signal transduction

    OpenAIRE

    Jacobsson, Johan Lars Henrik

    2008-01-01

    This study deals with the use of 3D-dynamic visualizations for teaching complex molecular cell biology concepts. The focus is on signal transduction, which is a concept that constitutes an important part of biological systems. 3D-dynamic visualizations (animations) were produced and shown for a total of 24 students attending a course in molecular cell biology at Karlstad University, Sweden. Data were collected by questionnaires and interviews which were structured around the understandability...

  20. Cell wall carbohydrates from fruit pulp of Argania spinosa: structural analysis of pectin and xyloglucan polysaccharides.

    Science.gov (United States)

    Aboughe-Angone, Sophie; Nguema-Ona, Eric; Ghosh, Partha; Lerouge, Patrice; Ishii, Tadashi; Ray, Bimalendu; Driouich, Azeddine

    2008-01-14

    Isolated cell walls of Argania spinosa fruit pulp were fractionated into their polysaccharide constituents and the resulting fractions were analysed for monosaccharide composition and chemical structure. The data reveal the presence of homogalacturonan, rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) in the pectic fraction. RG-I is abundant and contains high amounts of Ara and Gal, indicative of an important branching in this polysaccharide. RG-II is less abundant than RG-I and exists as a dimer. Structural characterisation of xyloglucan using enzymatic hydrolysis, gas chromatography, MALDI-TOF-MS and methylation analysis shows that XXGG, XXXG, XXLG and XLLG are the major subunit oligosaccharides in the ratio of 0.6:1:1.2:1.6. This finding demonstrates that the major neutral hemicellulosic polysaccharide is a galacto-xyloglucan. In addition, Argania fruit xyloglucan has no XUFG, a novel xyloglucan motif recently discovered in Argania leaf cell walls. Finally, the isolation and analysis of arabinogalactan-proteins showed that Argania fruit pulp is rich in these proteoglycans. PMID:18005949

  1. Heat- and pH-induced BSA conformational changes, hydrogel formation and application as 3D cell scaffold.

    Science.gov (United States)

    Navarra, Giovanna; Peres, Chiara; Contardi, Marco; Picone, Pasquale; San Biagio, Pier Luigi; Di Carlo, Marta; Giacomazza, Daniela; Militello, Valeria

    2016-09-15

    Aggregation and gelation of globular proteins can be an advantage to generate new forms of nanoscale biomaterials based on the fibrillar architecture. Here, we report results obtained by exploiting the proteins' natural tendency to self-organize in 3D network, for the production of new material based on BSA for medical application. In particular, at five different pH values the conformational and structural changes of the BSA during all the steps of the thermal aggregation and gelation have been analyzed by FTIR spectroscopy. The macroscopic mechanical properties of these hydrogels have been obtained by rheological measurements. The microscopic structure of the gels have been studied by AFM and SEM images to have a picture of their different spatial arrangement. Finally, the use of the BSA hydrogels as scaffold has been tested in two different cell cultures.

  2. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution.

    Science.gov (United States)

    Meddens, Marjolein B M; Liu, Sheng; Finnegan, Patrick S; Edwards, Thayne L; James, Conrad D; Lidke, Keith A

    2016-06-01

    We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet. PMID:27375939

  3. Carbon nanotubes film preparation on 3D structured silicon substrates by spray coating technique for application in solar cells

    International Nuclear Information System (INIS)

    This paper firstly reports the preparation of carbon nanotubes (CNTs) film on silicon substrate of three-dimensional (3D) inverted pyramid structure (IPS) by spray coating. The effect of different substrate temperatures, spraying times and opening sizes on CNTs sidewall covering properties were investigated. The results show that the CNTs covering ratio of sidewall is much lower than that of flat surface and gradually decrease with depth. 40μm×40μm opening obtained the best sidewall covering by CNTs suspension of 40μg/ml at 120°C after 30min spraying so that the CNTs can reach the bottom of IPS and cover about 68.9% sidewall area. At last, it is demonstrated that the output power of the CNTs film-Si solar cell can be enhanced 5.7 times by this method compared to that of the plane structure

  4. Mechanosensitivity of dental pulp stem cells is related to their osteogenic maturity

    DEFF Research Database (Denmark)

    Kraft, David C E; Bindslev, Dorthe A; Melsen, Birte;

    2010-01-01

    For engineering bone tissue, mechanosensitive cells are needed for bone (re)modelling. Local bone mass and architecture are affected by mechanical loading, which provokes a cellular response via loading-induced interstitial fluid flow. We studied whether human dental pulp-derived mesenchymal stem...... cells (PDSCs) portraying mature (PDSC-mature) or immature (PDSC-immature) bone cell characteristics are responsive to pulsating fluid flow (PFF) in vitro. We also assessed bone formation by PDSCs on hydroxyapatite-tricalcium phosphate granules after subcutaneous implantation in mice. Cultured PDSC...... expression was higher than in PDSC-immature. Implantation of PDSC-mature resulted in more osteoid deposition and lamellar bone formation than PDSC-immature. We conclude that PDSCs with a mature osteogenic phenotype are more responsive to pulsating fluid shear stress than osteogenically immature PDSCs...

  5. Nanostructured p-type CZTS thin films prepared by a facile solution process for 3D p-n junction solar cells.

    Science.gov (United States)

    Park, Si-Nae; Sung, Shi-Joon; Sim, Jun-Hyoung; Yang, Kee-Jeong; Hwang, Dae-Kue; Kim, JunHo; Kim, Gee Yeong; Jo, William; Kim, Dae-Hwan; Kang, Jin-Kyu

    2015-07-01

    Nanoporous p-type semiconductor thin films prepared by a simple solution-based process with appropriate thermal treatment and three-dimensional (3D) p-n junction solar cells fabricated by depositing n-type semiconductor layers onto the nanoporous p-type thin films show considerable photovoltaic performance compared with conventional thin film p-n junction solar cells. Spin-coated p-type Cu2ZnSnS4 (CZTS) thin films prepared using metal chlorides and thiourea show unique nanoporous thin film morphology, which is composed of a cluster of CZTS nanograins of 50-500 nm, and the obvious 3D p-n junction structure is fabricated by the deposition of n-type CdS on the nanoporous CZTS thin films by chemical bath deposition. The photovoltaic properties of 3D p-n junction CZTS solar cells are predominantly affected by the scale of CZTS nanograins, which is easily controlled by the sulfurization temperature of CZTS precursor films. The scale of CZTS nanograins determines the minority carrier transportation within the 3D p-n junction between CZTS and CdS, which are closely related with the photocurrent of series resistance of 3D p-n junction solar cells. 3D p-n junction CZTS solar cells with nanograins below 100 nm show power conversion efficiency of 5.02%, which is comparable with conventional CZTS thin film solar cells. PMID:26061271

  6. Generating 3D tissue constructs with mesenchymal stem cells and a cancellous bone graft for orthopaedic applications

    Energy Technology Data Exchange (ETDEWEB)

    Arca, Turkan; Genever, Paul [Department of Biology, University of York, York, YO10 5DD (United Kingdom); Proffitt, Joanne, E-mail: paul.genever@york.ac.uk [TSL Centre of Biologics, Covidien, Allerton Bywater, Castleford, WF10 2DB (United Kingdom)

    2011-04-15

    Bone matrix (BM) is an acellular crosslinked porcine-derived cancellous bone graft, and therefore may provide advantages over other synthetic and naturally derived materials for use in orthopaedic surgery. Here, we analysed the potential of BM to support the growth and differentiation of primary human multipotent stromal cells/mesenchymal stem cells (MSCs) in order to predict in vivo bone regeneration events. Imaging with laser scanning confocal microscopy and scanning electron microscopy showed that 1 day after static seeding, a dense population of viable MSCs could be achieved on scaffolds suggesting they could be used for in vivo delivery of cells to the implant site. Long-term growth analysis by confocal imaging and histology demonstrated that BM was permissive to the growth and the 3D population of primary MSCs and an enhanced green fluorescent protein expressing osteosarcoma cell line, eGFP.MG63s, over several days in culture. Measurement of alkaline phosphatase (ALP) activities and mRNA expression levels of osteogenic markers (Runx-2, ALP, collagen type I, osteonectin, osteocalcin and osteopontin) indicated that BM supported osteogenesis of MSCs when supplemented with osteogenic stimulants. Upregulation of some of these osteogenic markers on BM, but not on tissue culture plastic, under non-osteogenic conditions suggested that BM also had osteoinductive capacities.

  7. The Synergistic Effects of Matrix Stiffness and Composition on the Response of Chondroprogenitor Cells in a 3D Precondensation Microenvironment.

    Science.gov (United States)

    Carrion, Bita; Souzanchi, Mohammad F; Wang, Victor T; Tiruchinapally, Gopinath; Shikanov, Ariella; Putnam, Andrew J; Coleman, Rhima M

    2016-05-01

    Improve functional quality of cartilage tissue engineered from stem cells requires a better understanding of the functional evolution of native cartilage tissue. Therefore, a biosynthetic hydrogel was developed containing RGD, hyaluronic acid and/or type-I collagen conjugated to poly(ethylene glycol) acrylate to recapitulate the precondensation microenvironment of the developing limb. Conjugation of any combination of the three ligands did not alter the shear moduli or diffusion properties of the PEG hydrogels; thus, the influence of ligand composition on chondrogenesis could be investigated in the context of varying matrix stiffness. Gene expression of ligand receptors (CD44 and the b1-integrin) as well as markers of condensation (cell clustering and N-cadherin gene expression) and chondrogenesis (Col2a1 gene expression and sGAG production) by chondroprogenitor cells in this system were modulated by both matrix stiffness and ligand composition, with the highest gene expression occurring in softer hydrogels containing all three ligands. Cell proliferation in these 3D matrices for 7 d prior to chondrogenic induction increased the rate of sGAG production in a stiffness-dependent manner. This biosynthetic hydrogel supports the features of early limb-bud condensation and chondrogenesis and is a novel platform in which the influence of the matrix physicochemical properties on these processes can be elucidated.

  8. Implementation and Performance Modeling of Deterministic Particle Transport (Sweep3D on the IBM Cell/B.E.

    Directory of Open Access Journals (Sweden)

    Olaf Lubeck

    2009-01-01

    Full Text Available The IBM Cell Broadband Engine (BE is a novel multi-core chip with the potential for the demanding floating point performance that is required for high-fidelity scientific simulations. However, data movement within the chip can be a major challenge to realizing the benefits of the peak floating point rates. In this paper, we present the results of implementing Sweep3D on the Cell/B.E. using an intra-chip message passing model that minimizes data movement. We compare the advantages/disadvantages of this programming model with a previous implementation using a master–worker threading strategy. We apply a previously validated micro-architecture performance model for the application executing on the Cell/B.E. (based on our previous work in Monte Carlo performance models, that predicts overall CPI (cycles per instruction, and gives a detailed breakdown of processor stalls. Finally, we use the micro-architecture model to assess the performance of future design parameters for the Cell/B.E. micro-architecture. The methodologies and results have broader implications that extend to multi-core architectures.

  9. An Acinetobacter trimeric autotransporter adhesin reaped from cells exhibits its nonspecific stickiness via a highly stable 3D structure.

    Science.gov (United States)

    Yoshimoto, Shogo; Nakatani, Hajime; Iwasaki, Keita; Hori, Katsutoshi

    2016-01-01

    Trimeric autotransporter adhesins (TAAs), cell surface proteins of Gram-negative bacteria, mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific, high adhesiveness to abiotic material surfaces as well as to biotic surfaces. AtaA is a homotrimer of polypeptides comprising 3,630 amino acids and forms long nanofibers; therefore, it is too large and structurally complex to be produced as a recombinant protein. In this study, we isolated AtaA's passenger domain (AtaA PSD), which is translocated to the cell surface through the C-terminal transmembrane domain and exhibits biological functions, using a new method. We introduced a protease recognition site and reaped AtaA nanofibers 225 nm in length from the cell surface through proteolytic cleavage with a specific protease. Biochemical and biophysical analyses of the purified native AtaA PSD revealed that it has a stable structure under alkaline and acidic conditions. Temperatures above 80 °C, which disrupted AtaA's higher-order structure but maintained the full-length AtaA polypeptide, inactivated AtaA's nonspecific adhesiveness, suggesting that the stickiness of AtaA requires its 3D structure. This finding refutes the widespread but vague speculation that large unfolded polypeptides readily stick to various surfaces. PMID:27305955

  10. The Effects of Matrix Stiffness and RhoA on the Phenotypic Plasticity of Smooth Muscle Cells in a 3-D Biosynthetic Hydrogel System

    Science.gov (United States)

    Peyton, Shelly R.; Kim, Peter D.; Ghajar, Cyrus M.; Seliktar, Dror; Putnam, Andrew J.

    2008-01-01

    Studies using 2-D cultures have shown that the mechanical properties of the extracellular matrix (ECM) influence cell migration, spreading, proliferation, and differentiation; however, cellular mechanosensing in 3-D remains under-explored. To investigate this topic, a unique biomaterial system based on poly(ethylene glycol)-conjugated fibrinogen was adapted to study phenotypic plasticity in smooth muscle cells (SMCs) as a function of ECM mechanics in 3-D. Tuning compressive modulus between 448–5804 Pa modestly regulated SMC cytoskeletal assembly in 3-D, with spread cells in stiff matrices having a slightly higher degree of F-actin bundling after prolonged culture. However, vinculin expression in all 3-D conditions was qualitatively low and was not assembled into the classic focal adhesions typically seen in 2-D cultures. Given the evidence that RhoA-mediated cytoskeletal contractility represents a critical node in mechanosensing, we molecularly upregulated contractility by inducing SMCs to express constitutively active RhoA. In these cells, F-actin bundling and total vinculin expression increased, and focal adhesion-like structures began to emerge, consistent with RhoA’s mechanism of action cells cultured on 2-D substrates. Furthermore, SMC proliferation in 3-D did not depend significantly on matrix stiffness, and was reduced by constitutive activation of RhoA irrespective of ECM mechanical properties. Conversely, the expression of contractile markers globally increased with constitutive RhoA activation and depended on 3-D matrix stiffness only in cells with heightened RhoA activity. Combined, these data suggest the synergistic effects of ECM mechanics and RhoA activity on SMC phenotype in 3-D are distinct from those in 2-D, and highlight the importance of studying the mechanical role of cell-matrix interactions in tunable 3-D environments. PMID:18342366

  11. Resveratrol, Acetyl-Resveratrol, and Polydatin Exhibit Antigrowth Activity against 3D Cell Aggregates of the SKOV-3 and OVCAR-8 Ovarian Cancer Cell Lines

    OpenAIRE

    Hogg, Simon J.; Kenny Chitcholtan; Wafaa Hassan; Sykes, Peter H.; Ashley Garrill

    2015-01-01

    Resveratrol has aroused significant scientific interest as it has been claimed that it exhibits a spectrum of health benefits. These include effects as an anti-inflammatory and an antitumour compound. The purpose of this study was to investigate and compare any potential antigrowth effects of resveratrol and two of its derivatives, acetyl-resveratrol and polydatin, on 3D cell aggregates of the EGFR/Her-2 positive and negative ovarian cancer cell lines SKOV-3 and OVCAR-8, respectively. Results...

  12. A computer-assisted 3D model for analyzing the aggregation of tumorigenic cells reveals specialized behaviors and unique cell types that facilitate aggregate coalescence.

    Directory of Open Access Journals (Sweden)

    Amanda Scherer

    Full Text Available We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity, and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 μm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. Images are converted to mathematical representations from which quantitative parameters can be derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named "facilitators" and "probes." A third cell type, the "dervish", is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.

  13. Stem Cell Bioprinting: Functional 3D Neural Mini-Tissues from Printed Gel-Based Bioink and Human Neural Stem Cells (Adv. Healthcare Mater. 12/2016).

    Science.gov (United States)

    Gu, Qi; Tomaskovic-Crook, Eva; Lozano, Rodrigo; Chen, Yu; Kapsa, Robert M; Zhou, Qi; Wallace, Gordon G; Crook, Jeremy M

    2016-06-01

    On page 1429 G. G. Wallace, J. M. Crook, and co-workers report the first example of fabricating neural tissue by 3D bioprinting human neural stem cells. A novel polysaccharide based bioink preserves stem cell viability and function within the printed construct, enabling self-renewal and differentiation to neurons and supporting neuroglia. Neurons are predominantly GABAergic, establish networks, are spontaneously active, and show a bicuculline induced increased calcium response. PMID:27333401

  14. Effect of novel chitosan-fluoroaluminosilicate resin modified glass ionomer cement supplemented with translationally controlled tumor protein on pulp cells.

    Science.gov (United States)

    Wanachottrakul, Nattaporn; Chotigeat, Wilaiwan; Kedjarune-Leggat, Ureporn

    2014-04-01

    Dental materials that can promote cell proliferation and function is required for regenerative pulp therapy. Resin modified glass ionomer cement (RMGIC), a broadly used liner or restorative material, can cause apoptosis to pulp cells mainly due to HEMA (2-hydroxyethyl methacrylate), the released residual monomer. Recent studies found that chitosan and albumin could promote release of protein in GIC while translationally controlled tumor protein (TCTP) has an anti-apoptotic activity against HEMA. The aim of this study was to examine the effect of chitosan and albumin modified RMGIC (Exp-RMGIC) supplemented with TCTP on pulp cell viability and mineralization. Exp-RMGIC+TCTP was composed of RMGIC powder incorporated with 15 % of chitosan, 5 % albumin and supplemented with TCTP mixed with the same liquid components of RMGIC. The effect of each specimen on pulp cells was examined using the Transwell plate. From the MTT assay, Exp-RMGIC+TCTP had the highest percentages of viable cells (P supplemented with TCTP had less cytotoxicity than RMGIC and can protect cells from apoptosis better than RMGIC supplemented with TCTP.

  15. The relevance of using 3D cell cultures, in addition to 2D monolayer cultures, when evaluating breast cancer drug sensitivity and resistance.

    OpenAIRE

    O'Driscoll, Lorraine

    2016-01-01

    PUBLISHED 2016 Jun 10. doi: 10.18632/oncotarget.9935. [Epub ahead of print] Solid tumours naturally grow in 3D wherein the spatial arrangement of cells affects how they interact with each other. This suggests that 3D cell culture may mimic the natural in vivo setting better than traditional monolayer (2D) cell culture, where cells are grown attached to plastic. Here, using HER2-positive breast cancer cell lines as models (BT474, HCC1954, EFM192A), the effects of culturing c...

  16. Effects of epicatechin, a crosslinking agent, on human dental pulp cells cultured in collagen scaffolds

    Directory of Open Access Journals (Sweden)

    Eun-su Lim

    2016-02-01

    Full Text Available ABSTRACT Objective The purpose of this study was to investigate the biological effects of epicatechin (ECN, a crosslinking agent, on human dental pulp cells (hDPCs cultured in collagen scaffolds. Material and Method To evaluate the effects of ECN on the proliferation of hDPCs, cell counting was performed using optical and fluorescent microscopy. Measurements of alkaline phosphatase (ALP activity, alizarin red staining, and real-time polymerase chain reactions were performed to assess odontogenic differentiation. The compressive strength and setting time of collagen scaffolds containing ECN were measured. Differential scanning calorimetry was performed to analyze the thermal behavior of collagen in the presence of ECN. Results Epicatechin increased ALP activity, mineralized nodule formation, and the mRNA expression of dentin sialophosphoprotein (DSPP, a specific odontogenic-related marker. Furthermore, ECN upregulated the expression of DSPP in hDPCs cultured in collagen scaffolds. Epicatechin activated the extracellular signal-regulated kinase (ERK and the treatment with an ERK inhibitor (U0126 blocked the expression of DSPP. The compressive strength was increased and the setting time was shortened in a dose-dependent manner. The number of cells cultured in the ECN-treated collagen scaffolds was significantly increased compared to the cells in the untreated control group. Conclusions Our results revealed that ECN promoted the proliferation and differentiation of hDPCs. Furthermore, the differentiation was regulated by the ERK signaling pathway. Changes in mechanical properties are related to cell fate, including proliferation and differentiation. Therefore, our study suggests the ECN treatment might be desirable for dentin-pulp complex regeneration.

  17. 3D Animation Essentials

    CERN Document Server

    Beane, Andy

    2012-01-01

    The essential fundamentals of 3D animation for aspiring 3D artists 3D is everywhere--video games, movie and television special effects, mobile devices, etc. Many aspiring artists and animators have grown up with 3D and computers, and naturally gravitate to this field as their area of interest. Bringing a blend of studio and classroom experience to offer you thorough coverage of the 3D animation industry, this must-have book shows you what it takes to create compelling and realistic 3D imagery. Serves as the first step to understanding the language of 3D and computer graphics (CG)Covers 3D anim

  18. 3D video

    CERN Document Server

    Lucas, Laurent; Loscos, Céline

    2013-01-01

    While 3D vision has existed for many years, the use of 3D cameras and video-based modeling by the film industry has induced an explosion of interest for 3D acquisition technology, 3D content and 3D displays. As such, 3D video has become one of the new technology trends of this century.The chapters in this book cover a large spectrum of areas connected to 3D video, which are presented both theoretically and technologically, while taking into account both physiological and perceptual aspects. Stepping away from traditional 3D vision, the authors, all currently involved in these areas, provide th

  19. Characterization and mesenteric lymph node cells-mediated immunomodulatory activity of litchi pulp polysaccharide fractions.

    Science.gov (United States)

    Huang, Fei; Zhang, Ruifen; Liu, Yang; Xiao, Juan; Su, Dongxiao; Yi, Yang; Wang, Guangjin; Wei, Zhencheng; Zhang, Mingwei

    2016-11-01

    Three water-soluble hetero-polysaccharides, designated LP1-3, were isolated from litchi pulp. Their structures, solution properties and immunomodulatory activities were evaluated. LP1 contained (1→4,6)-β-d-Glc and (1→4)-α-l-Gal, while LP2 contained (1→3)-α-l-Ara and (l→2)-β-d-Gal, and LP3 contained α-l-Ara and (l→4)-β-Rha. Their molecular weights ranged from 105,880 to 986,470g/mol. LP1 had a spherical conformation with hyper-branched structure and LP2 was semi-flexible chain, while the polysaccharide chains of LP3 were cross linked to form network-like conformation in solution. In addition, all fractions strongly stimulated mesenteric lymph node cell proliferation, IFN-γ and IL-6 secretion in the dose range of 25-100μg/mL compared with untreated control group (pcell proliferation and cytokine secretion, which may be attributed to its unique chemical structure and chain conformation. This is the first report on the solution properties and intestinal immunity activities of polysaccharides from litchi pulp. PMID:27516297

  20. Effects of two fast-setting calcium-silicate cements on cell viability and angiogenic factor release in human pulp-derived cells.

    Science.gov (United States)

    Chung, Chooryung J; Kim, Euiseong; Song, Minju; Park, Jeong-Won; Shin, Su-Jung

    2016-05-01

    Mineral trioxide aggregate (MTA) is considered a pulp-capping agent of choice, but has the drawback of a long setting time. This study aimed to assess two different types of calcium-silicate cements as pulp-capping agents, by investigating their in vitro cytotoxicity and angiogenic effects in human pulp cells. ProRoot MTA, Endocem Zr, and Retro MTA were prepared as set or freshly mixed pellets. Human pulp-derived cells were grown in direct contact with these three cements, Dycal, or no cement, for 7 days. Initial cell attachment, viability, calcium release, and the levels of vascular endothelial growth factor (VEGF), angiogenin, and basic fibroblast growth factor (FGF-2) were evaluated statistically using a linear mixed model (P calcium concentration compared with the control group (P  0.05). We demonstrate that Retro MTA, which has a short setting time, has similar biocompatibility and angiogenic effects on human pulp cells, and can therefore potentially be as effective in pulp capping as ProRoot MTA. Endocem Zr showed intermittent cytotoxicity and elicited lower levels of VEGF and angiogenin expression. PMID:25596932

  1. A nano-microstructured artificial-hair-cell-type sensor based on topologically graded 3D carbon nanotube bundles.

    Science.gov (United States)

    Yilmazoglu, O; Yadav, S; Cicek, D; Schneider, J J

    2016-09-01

    A design for a unique artificial-hair-cell-type sensor (AHCTS) based entirely on 3D-structured, vertically aligned carbon nanotube (CNT) bundles is introduced. Standard microfabrication techniques were used for the straightforward micro-nano integration of vertically aligned carbon nanotube arrays composed of low-layer multi-walled CNTs (two to six layers). The mechanical properties of the carbon nanotube bundles were intensively characterized with regard to various substrates and CNT morphology, e.g. bundle height. The CNT bundles display excellent flexibility and mechanical stability for lateral bending, showing high tear resistance. The integrated 3D CNT sensor can detect three-dimensional forces using the deflection or compression of a central CNT bundle which changes the contact resistance to the shorter neighboring bundles. The complete sensor system can be fabricated using a single chemical vapor deposition (CVD) process step. Moreover, sophisticated external contacts to the surroundings are not necessary for signal detection. No additional sensors or external bias for signal detection are required. This simplifies the miniaturization and the integration of these nanostructures for future microsystem set-ups. The new nanostructured sensor system exhibits an average sensitivity of 2100 ppm in the linear regime with the relative resistance change per micron (ppm μm(-1)) of the individual CNT bundle tip deflection. Furthermore, experiments have shown highly sensitive piezoresistive behavior with an electrical resistance decrease of up to ∼11% at 50 μm mechanical deflection. The detection sensitivity is as low as 1 μm of deflection, and thus highly comparable with the tactile hair sensors of insects, having typical thresholds on the order of 30-50 μm. The AHCTS can easily be adapted and applied as a flow, tactile or acceleration sensor as well as a vibration sensor. Potential applications of the latter might come up in artificial cochlear systems. In

  2. A nano-microstructured artificial-hair-cell-type sensor based on topologically graded 3D carbon nanotube bundles

    Science.gov (United States)

    Yilmazoglu, O.; Yadav, S.; Cicek, D.; Schneider, J. J.

    2016-09-01

    A design for a unique artificial-hair-cell-type sensor (AHCTS) based entirely on 3D-structured, vertically aligned carbon nanotube (CNT) bundles is introduced. Standard microfabrication techniques were used for the straightforward micro-nano integration of vertically aligned carbon nanotube arrays composed of low-layer multi-walled CNTs (two to six layers). The mechanical properties of the carbon nanotube bundles were intensively characterized with regard to various substrates and CNT morphology, e.g. bundle height. The CNT bundles display excellent flexibility and mechanical stability for lateral bending, showing high tear resistance. The integrated 3D CNT sensor can detect three-dimensional forces using the deflection or compression of a central CNT bundle which changes the contact resistance to the shorter neighboring bundles. The complete sensor system can be fabricated using a single chemical vapor deposition (CVD) process step. Moreover, sophisticated external contacts to the surroundings are not necessary for signal detection. No additional sensors or external bias for signal detection are required. This simplifies the miniaturization and the integration of these nanostructures for future microsystem set-ups. The new nanostructured sensor system exhibits an average sensitivity of 2100 ppm in the linear regime with the relative resistance change per micron (ppm μm‑1) of the individual CNT bundle tip deflection. Furthermore, experiments have shown highly sensitive piezoresistive behavior with an electrical resistance decrease of up to ∼11% at 50 μm mechanical deflection. The detection sensitivity is as low as 1 μm of deflection, and thus highly comparable with the tactile hair sensors of insects, having typical thresholds on the order of 30–50 μm. The AHCTS can easily be adapted and applied as a flow, tactile or acceleration sensor as well as a vibration sensor. Potential applications of the latter might come up in artificial cochlear systems. In

  3. Bioconductive 3D nano-composite constructs with tunable elasticity to initiate stem cell growth and induce bone mineralization.

    Science.gov (United States)

    Sagar, Nitin; Khanna, Kunal; Sardesai, Varda S; Singh, Atul K; Temgire, Mayur; Kalita, Mridula Phukan; Kadam, Sachin S; Soni, Vivek P; Bhartiya, Deepa; Bellare, Jayesh R

    2016-12-01

    Bioactive 3D composites play an important role in advanced biomaterial design to provide molecular coupling and improve integrity with the cellular environment of the native bone. In the present study, a hybrid lyophilized polymer composite blend of anionic charged sodium salt of carboxymethyl chitin and gelatin (CMChNa-GEL) reinforced with nano-rod agglomerated hydroxyapatite (nHA) has been developed with enhanced biocompatibility and tunable elasticity. The scaffolds have an open, uniform and interconnected porous structure with an average pore diameter of 157±30μm and 89.47+0.03% with four dimensional X-ray. The aspect ratio of ellipsoidal pores decrease from 4.4 to 1.2 with increase in gelatin concentration; and from 2.14 to 1.93 with decrease in gelling temperature. The samples were resilient with elastic stain at 1.2MPa of stress also decreased from 0.33 to 0.23 with increase in gelatin concentration. The crosslinker HMDI (hexamethylene diisocyanate) yielded more resilient samples at 1.2MPa in comparison to glutaraldehyde. Increased crosslinking time from 2 to 4h in continuous compression cycle show no improvement in maximum elastic stain of 1.2MPa stress. This surface elasticity of the scaffold enables the capacity of these materials for adherent self renewal and cultivation of the NTERA-2 cL.D1 (NT2/D1), pluripotent embryonal carcinoma cell with biomechanical surface, as is shown here. Proliferation with MG-63, ALP activity and Alizarin red mineralization assay on optimized scaffold demonstrated ***psize defect. Therefore, this nHA-CMChNa-GEL scaffold composite exhibits inherent and efficient physicochemical, mechanical and biological characteristics based on gel concentrations, gelatin mixing and gelling temperature thus points to creating bioactive 3D scaffolds with tunable elasticity for orthopedic applications. PMID:27612764

  4. A nano-microstructured artificial-hair-cell-type sensor based on topologically graded 3D carbon nanotube bundles

    Science.gov (United States)

    Yilmazoglu, O.; Yadav, S.; Cicek, D.; Schneider, J. J.

    2016-09-01

    A design for a unique artificial-hair-cell-type sensor (AHCTS) based entirely on 3D-structured, vertically aligned carbon nanotube (CNT) bundles is introduced. Standard microfabrication techniques were used for the straightforward micro-nano integration of vertically aligned carbon nanotube arrays composed of low-layer multi-walled CNTs (two to six layers). The mechanical properties of the carbon nanotube bundles were intensively characterized with regard to various substrates and CNT morphology, e.g. bundle height. The CNT bundles display excellent flexibility and mechanical stability for lateral bending, showing high tear resistance. The integrated 3D CNT sensor can detect three-dimensional forces using the deflection or compression of a central CNT bundle which changes the contact resistance to the shorter neighboring bundles. The complete sensor system can be fabricated using a single chemical vapor deposition (CVD) process step. Moreover, sophisticated external contacts to the surroundings are not necessary for signal detection. No additional sensors or external bias for signal detection are required. This simplifies the miniaturization and the integration of these nanostructures for future microsystem set-ups. The new nanostructured sensor system exhibits an average sensitivity of 2100 ppm in the linear regime with the relative resistance change per micron (ppm μm-1) of the individual CNT bundle tip deflection. Furthermore, experiments have shown highly sensitive piezoresistive behavior with an electrical resistance decrease of up to ˜11% at 50 μm mechanical deflection. The detection sensitivity is as low as 1 μm of deflection, and thus highly comparable with the tactile hair sensors of insects, having typical thresholds on the order of 30-50 μm. The AHCTS can easily be adapted and applied as a flow, tactile or acceleration sensor as well as a vibration sensor. Potential applications of the latter might come up in artificial cochlear systems. In

  5. Metabonomic study on the antitumor effect of flavonoid derivative 3d in HepG2 cells and its action mechanism.

    Science.gov (United States)

    Gao, Dan; Jin, Feng; Liu, Hongxia; Wang, Yini; Jiang, Yuyang

    2014-01-01

    A novel flavonid derivate, 1-(3-chloro-4-(6-ethyl-4-oxo-4H-chromen-2-yl)phenyl)-3-(4-chlorophenyl)urea (3d) synthesized in our lab possesses potent antitumor activity against HepG2 cells. Our previous studies on pharmacological mechanism of 3d mostly focused on cell and gene levels, little is about its metabolomics study. Herein, an ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) based metabolomics approach was established to investigate the antitumor effect of 3d on HepG2 cells and its action mechanism. Q-TOF MS was used to identify metabolites, and tandem mass spectrometry was used to confirm their identity. Comparing 3d-treated HepG2 cells with vehicle control (dimethyl sulfoxide), 32 distinct metabolites involved in glutathione metabolism, glycerophospholipid metabolism, cysteine and methionine metabolism, fatty acid metabolism, and phenylalanine metabolism. The reduced level of glutathione (GSH) and decreased ratio of reduced/oxidized glutathione (GSH/GSSG) in 3d-treated cells indicated the increased oxidative stress after 3d treatment. The significant decrease of phosphatidylcholine (PC) levels and increase of lysophosphatidylcholine (LPC) levels suggested alterations in lipid composition which were causally related to decline in mitochondrial function. Depletion of carnitine and increase of long chain carnitines and fatty acids reflected decline in fatty acid metabolism. The further biological experiments including ROS and MMP measurements confirmed the above probabilities presumed from metabolomic results. Our findings suggested that 3d caused the perturbation of multiple cellular pathways. The increased oxidative stress and the resulting mitochondrial dysfunction resulted in the antiproliferative effect of 3d. The UPLC/Q-TOF MS based metabolomics approach provides new insights into the mechanistic studies of new compounds that distinct from traditional biological studies.

  6. Scalable, incremental learning with MapReduce parallelization for cell detection in high-resolution 3D microscopy data

    KAUST Repository

    Sung, Chul

    2013-08-01

    Accurate estimation of neuronal count and distribution is central to the understanding of the organization and layout of cortical maps in the brain, and changes in the cell population induced by brain disorders. High-throughput 3D microscopy techniques such as Knife-Edge Scanning Microscopy (KESM) are enabling whole-brain survey of neuronal distributions. Data from such techniques pose serious challenges to quantitative analysis due to the massive, growing, and sparsely labeled nature of the data. In this paper, we present a scalable, incremental learning algorithm for cell body detection that can address these issues. Our algorithm is computationally efficient (linear mapping, non-iterative) and does not require retraining (unlike gradient-based approaches) or retention of old raw data (unlike instance-based learning). We tested our algorithm on our rat brain Nissl data set, showing superior performance compared to an artificial neural network-based benchmark, and also demonstrated robust performance in a scenario where the data set is rapidly growing in size. Our algorithm is also highly parallelizable due to its incremental nature, and we demonstrated this empirically using a MapReduce-based implementation of the algorithm. We expect our scalable, incremental learning approach to be widely applicable to medical imaging domains where there is a constant flux of new data. © 2013 IEEE.

  7. COUPLED SIMULATION OF 3D ELECTRO-MAGNETO-FLOW FIELD IN HALL-HEROULT CELLS USING FINITE ELEMENT METHOD

    Institute of Scientific and Technical Information of China (English)

    J. Li; W. Liu; Y.Q. Lai; Q.Y. Li; Y.X. Liu

    2006-01-01

    Two full 3D steady mathematical models are developed by finite element method (FEM) to calculate coupled physics fields: the electro-magnetic model is built and solved first and so is the fluid motion model with the acquired electromagnetic force as source body forces in Navier-Stokes equations. Effects caused by the ferromagnetic shell, busbar system around, and open boundary problem as well as inside induced current were considered in terms of the magnetic field. Furthermore, a new modeling method is found to set up solid models and then mesh them entirely with so-called structuralized grids, namely hex-mesh. Examples of 75kA prebaked cell with two kinds of busbar arrangements are presented. Results agree with those disclosed in the literature and confirm that the coupled simulation is valid. It is also concluded that the usage of these models facilitates the consistent analysis of the electric field to magnetic field and then flow motion to the greater extent, local distributions of current density and magnetic flux density are very much dependent on the cell structure, the steel shell is a shield to reduce the magnetic field and flow pattern is two dimensional in the main body of the metal pad.

  8. Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells

    Science.gov (United States)

    Rietdorf, Jens; Stephens, David J.; Squire, Anthony; Simpson, Jeremy; Shima, David T.; Paccaud, Jean-Pierre; Bastiaens, Philippe I.; Pepperkok, Rainer

    2000-04-01

    Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in selective packaging of anterograde cargo into coated transport vesicles budding from the ER. COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER. We have used multi spectral 3D imaging to visualize COPI and COPII behavior simultaneously with various GFP-tagged secretory markers in living cells. This shows that COPII and COPI act sequentially whereby COPI association with anterograde transport complexes is involved in microtubule-based transport and the en route segregation of ER recycling molecules from secretory cargo within TCS in transit to the Golgi complex. We have also investigated the possibility to discriminate spectrally GFP fusion proteins by fluorescence lifetime imaging. This shows that at least two, and possibly up to three GFP fusion proteins can be discriminated and localized in living cells using a single excitation wavelength and a single broad band emission filter.

  9. A New Crank Arm-Based Load Cell for the 3D Analysis of the Force Applied by a Cyclist

    Directory of Open Access Journals (Sweden)

    Alexandre Balbinot

    2014-12-01

    Full Text Available This report describes a new crank arm-based force platform designed to evaluate the three-dimensional force applied to the pedals by cyclists in real conditions. The force platform was designed to be fitted on a conventional competition bicycle crankset while data is transmitted wirelessly through a BluetoothTM module and also stored on a SD card. A 3D solid model is created in the SolidWorks (Dassault Systèmes SOLIDWORKS Corp. to analyze the static and dynamic characteristics of the crank arm by using the finite elements technique. Each crankset arm is used as a load cell based on strain gauges configured as three Wheatstone bridges. The signals are conditioned on a printed circuit board attached directly to the structure. The load cell showed a maximum nonlinearity error between 0.36% and 0.61% and a maximum uncertainty of 2.3% referred to the sensitivity of each channel. A roller trainer equipped with an optical encoder was also developed, allowing the measurement of the wheel’s instantaneous velocity.

  10. Increased extracellular matrix density decreases MCF10A breast cell acinus formation in 3D culture conditions.

    Science.gov (United States)

    Lance, Amanda; Yang, Chih-Chao; Swamydas, Muthulekha; Dean, Delphine; Deitch, Sandy; Burg, Karen J L; Dréau, Didier

    2016-01-01

    The extracellular matrix (ECM) contributes to the generation and dynamic of normal breast tissue, in particular to the generation of polarized acinar and ductal structures. In vitro 3D culture conditions, including variations in the composition of the ECM, have been shown to directly influence the formation and organization of acinus-like and duct-like structures. Furthermore, the density of the ECM appears to also play a role in the normal mammary tissue and tumour formation. Here we show that the density of the ECM directly influences the number, organization and function of breast acini. Briefly, non-malignant human breast MCF10A cells were incubated in increasing densities of a Matrigel®-collagen I matrix. Elastic moduli near and distant to the acinus structures were measured by atomic force microscopy, and the number of acinus structures was determined. Immunochemistry was used to investigate the expression levels of E-cadherin, laminin, matrix metalloproteinase-14 and ß-casein in MCF10A cells. The modulus of the ECM was significantly increased near the acinus structures and the number of acinus structures decreased with the increase in Matrigel-collagen I density. As evaluated by the expression of laminin, the organization of the acinus structures present was altered as the density of the ECM increased. Increases in both E-cadherin and MMP14 expression by MCF10A cells as ECM density increased were also observed. In contrast, MCF10A cells expressed lower ß-casein levels as the ECM density increased. Taken together, these observations highlight the key role of ECM density in modulating the number, organization and function of breast acini.

  11. HER2 signaling pathway activation and response of breast cancer cells to HER2-targeting agents is dependent strongly on the 3D microenvironment

    Energy Technology Data Exchange (ETDEWEB)

    Weigelt, Britta; Lo, Alvin T; Park, Catherine C; Gray, Joe W; Bissell, Mina J

    2009-07-27

    Development of effective and durable breast cancer treatment strategies requires a mechanistic understanding of the influence of the microenvironment on response. Previous work has shown that cellular signaling pathways and cell morphology are dramatically influenced by three-dimensional (3D) cultures as opposed to traditional two-dimensional (2D) monolayers. Here, we compared 2D and 3D culture models to determine the impact of 3D architecture and extracellular matrix (ECM) on HER2 signaling and on the response of HER2-amplified breast cancer cell lines to the HER2-targeting agents Trastuzumab, Pertuzumab and Lapatinib. We show that the response of the HER2-amplified AU565, SKBR3 and HCC1569 cells to these anti-HER2 agents was highly dependent on whether the cells were cultured in 2D monolayer or 3D laminin-rich ECM gels. Inhibition of {beta}1 integrin, a major cell-ECM receptor subunit, significantly increased the sensitivity of the HER2-amplified breast cancer cell lines to the humanized monoclonal antibodies Trastuzumab and Pertuzumab when grown in a 3D environment. Finally, in the absence of inhibitors, 3D cultures had substantial impact on HER2 downstream signaling and induced a switch between PI3K-AKT- and RAS-MAPKpathway activation in all cell lines studied, including cells lacking HER2 amplification and overexpression. Our data provide direct evidence that breast cancer cells are able to rapidly adapt to different environments and signaling cues by activating alternative pathways that regulate proliferation and cell survival, events that may play a significant role in the acquisition of resistance to targeted therapies.

  12. Human Dental Pulp-Derived Cells Produce Bone-Like Tissue and Exhibit Bone Cell-Like Responsiveness to Mechanical Loading

    DEFF Research Database (Denmark)

    Kraft, David Christian Evar; Melsen, Birte; Bindslev, Dorthe Arenholt

    2010-01-01

    architecture to the prevailing mechanical load and should be able to conduct bone cell-specific functions, such as bone remodeling. In vitro investigation of the responsiveness of different cell types to mechanical loading is so far a relative new research field. The aim of this study was to establish...... and characterize cell lines from human 3rd molar dental pulp tissue to determine whether human dental pulp-derived cells (DPCs) are osteogenic and responsive to mechanical loading by pulsating fluid flow (PFF) in vitro. Methods: Human DPCs used for this study were characterized by measuring proliferation......Recent studies have shown that dental pulp cells possess stem cell like potential and thus may be potential candidates for tissue engineering purposes particularly in the oro-facial region. Successful tissue engineering ideally requires that newly formed bone adapts its mass, shape, and trabecular...

  13. Dental Pulp Stem Cells as a multifaceted tool for bioengineering and the regeneration of craniomaxillofacial tissues

    Directory of Open Access Journals (Sweden)

    Maitane eAurrekoetxea

    2015-10-01

    Full Text Available Dental pulp stem cells, or DPSC, are neural crest-derived cells with an outstanding capacity to differentiate along multiple cell lineages of interest for cell therapy. In particular, highly efficient osteo/dentinogenic differentiation of DPSC can be achieved using simple in vitro protocols, making these cells a very attractive and promising tool for the future treatment of dental and periodontal diseases. Among craniomaxillofacial organs, the tooth and salivary gland are two such cases in which complete regeneration by tissue engineering using DPSC appears to be possible, as research over the last decade has made substantial progress in experimental models of partial or total regeneration of both organs, by cell recombination technology. Moreover, DPSC seem to be a particularly good choice for the regeneration of nerve tissues, including injured or transected cranial nerves. In this context, the oral cavity appears to be an excellent testing ground for new regenerative therapies using DPSC. However, many issues and challenges need yet to be addressed before these cells can be employed in clinical therapy. In this review, we point out some important aspects on the biology of DPSC with regard to their use for the reconstruction of different craniomaxillofacial tissues and organs, with special emphasis on cranial bones, nerves, teeth, and salivary glands. We suggest new ideas and strategies to fully exploit the capacities of DPSC for bioengineering of the aforementioned tissues.

  14. Dental pulp stem cells as a multifaceted tool for bioengineering and the regeneration of craniomaxillofacial tissues.

    Science.gov (United States)

    Aurrekoetxea, Maitane; Garcia-Gallastegui, Patricia; Irastorza, Igor; Luzuriaga, Jon; Uribe-Etxebarria, Verónica; Unda, Fernando; Ibarretxe, Gaskon

    2015-01-01

    Dental pulp stem cells, or DPSC, are neural crest-derived cells with an outstanding capacity to differentiate along multiple cell lineages of interest for cell therapy. In particular, highly efficient osteo/dentinogenic differentiation of DPSC can be achieved using simple in vitro protocols, making these cells a very attractive and promising tool for the future treat