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Tissue Engineering. Part C, Methods

Natalia Becerra, Claudia Marcela Arenas Gómez, Maria Isabel Patiño, Jean Paul Delgado, Carlos Enrique Muskus, Luz Marina Restrepo
Non-viral transfection has important implications on gene therapy, because of its safety. In particular, polyfection and nucleofection are two widely used systems for non-viral gene delivery. Their potential depends on the transfection efficiency achieved, which is influenced in turn by the type of cells transfected and by the plasmid which carries the gene of interest. The efficiency of transfection by polyfection or nucleofection in human fibroblast and keratinocytes was evaluated in this study. Transfections were performed with plasmids containing a gene of interest (Human Cathelicidin Antimicrobial Peptide) and two reporter genes (Red or Green Fluorescent Protein) that included or not an Internal Ribosome Entry Site (IRES)...
February 28, 2018: Tissue Engineering. Part C, Methods
Grace Limandjaja, Lenie J van den Broek, Melanie Breetveld, Taco Waaijman, Stan Monstrey, Edith de Boer, Rik Scheper, Frank Niessen, Susan Gibbs
To understand scar pathology, develop new drugs, and provide a platform for personalized medicine, physiologically relevant human scar models are required which are characteristic of different scar pathologies. Hypertrophic scars and keloids are two types of abnormal scar resulting from unknown abnormalities in the wound healing process. While they display different clinical behavior, differentiation between the two can be difficult - which in turn means that it is difficult to develop optimal therapeutic strategies...
February 28, 2018: Tissue Engineering. Part C, Methods
Brian C Syverud, John P Gumucio, Brittany L Rodriguez, Olga M Wroblewski, Shelby E Florida, Chris Mendias, Lisa Marie Larkin
The growing deficit in suitable tissues for patients awaiting organ transplants demonstrates the clinical need for engineered tissues as alternative graft sources. Demonstrating safety and efficacy by tracking the migration and fate of implanted cells is a key consideration required for approval of promising engineered tissues. Cells from transgenic animals that express green fluorescent protein (GFP) are commonly used for this purpose. However, GFP can create difficulties in practice due to high levels of green autofluorescence in many musculoskeletal tissues...
February 28, 2018: Tissue Engineering. Part C, Methods
Rui Li, Guanglin Yu, Samira Azarin, Allison Hubel
Inadequate preservation methods of human induced pluripotent stem cells (hiPSCs) have impeded efficient reestablishment of cell culture after freeze-thaw. In this study, we examined the roles of cooling rate, seeding temperature and the difference between cell aggregates (3-50 cells) and single cells in controlled rate freezing of hiPSCs. Intracellular ice formation (IIF), post-thaw membrane integrity, cell attachment, apoptosis and cytoskeleton organization were evaluated to understand the different freezing responses between hiPSC single cells and aggregates, among cooling rates of 1, 3, 10˚C/min, and between seeding temperatures of -4, -8˚C...
February 25, 2018: Tissue Engineering. Part C, Methods
Aila Daugs, Nadine Lehmann, Dilem Eroglu, Martina Meinke, Annina Markhoff, Oliver Bloch
Tissue antigenicity represents the main limitation for the use of xenografts in clinical practice. To eliminate xenoantigens and avoid graft rejection in human, decellularization is often used to remove all immunoreactive components from the extracellular matrix (ECM). After decellularization, acellular scaffolds are required to be investigated regarding the presence of antigens, but commonly used detection methods solely focus on known xenoantigens such as alpha Gal (Galα1,3-Galβ1-4GlcNAc-R) or major histocompatibility complex-I (MHC-I) However, there are unknown xenoantigens, which escape the standard methods...
February 24, 2018: Tissue Engineering. Part C, Methods
Zhihua Han, Mit Bhavsar, Liudmila Leppik, Karla M C Oliveira, John H Barker
Large bone defects are a major problem in trauma and orthopedic surgery. Tissue engineering based treatments have emerged as promising alternatives of traditional bone grafting techniques. Critical size bone defect animal models had been developed and widely used to evaluate and compare therapeutic effectiveness in bone tissue engineering treatments. To measure healing in a given defect, after treatment histological assessment methods are commonly used. These histological methods are typically either qualitative and/or measure the amount of newly formed bone...
February 21, 2018: Tissue Engineering. Part C, Methods
Kang-Gon Lee, Kang-Sik Lee, Yu-Jeoung Kang, Jong-Hyun Hwang, Se-Hwan Lee, Sang-Hyug Park, Yongdoo Park, Young-Sam Cho, Bu-Kyu Lee
Bone graft materials are commonly used to regenerate various bone defects, but their application is often limited because of the complex defect shape in various clinical conditions. Hence, customized bone grafts using three-dimensional (3D) printing techniques have been developed. However, conventional simple bone defect models are limited for evaluating the benefits and manufacturing accuracy of 3D-printed customized bone grafts. Thus, the aim of the present study was to develop a complex-shaped bone defect model...
February 21, 2018: Tissue Engineering. Part C, Methods
Anita Krouwels, Ferry Melchels, Mattie H P Van Rijen, F Cumhur Oner, Wouter Dhert, M Tryfonidou, Laura Creemers
Hydrogels can facilitate nucleus pulposus regeneration, either for clinical application or research into mechanisms of regeneration. However, many different hydrogels and culture conditions for human degenerated nucleus pulposus (NP) have been employed, making literature data difficult to compare. Therefore, we compared six different hydrogels of natural polymers and investigated the role of serum in the medium and of osmolarity during expansion or redifferentation in an attempt to provide comparators for future studies...
February 17, 2018: Tissue Engineering. Part C, Methods
Sakib F Elahi, Seung Yup Lee, William R Lloyd, Leng-Chun Chen, Shiuhyang Kuo, Ying Zhou, Hyungjin Myra Kim, Robert Kennedy, Cynthia Marcelo, Stephen E Feinberg, Mary-Ann Mycek
Fluorescence lifetime sensing has been shown to noninvasively characterize the pre-implantation health and viability of engineered tissue constructs. However, current practices to monitor post-implantation construct integration are either qualitative (visual assessment) or destructive (tissue histology). We employed label-free fluorescence lifetime spectroscopy for quantitative, noninvasive optical assessment of engineered tissue constructs that were implanted in a murine model. The portable system was designed to be suitable for intravital measurements and included a handheld probe to precisely and rapidly acquire data at multiple sites per construct...
February 16, 2018: Tissue Engineering. Part C, Methods
Yonggang Pang, Jing Yang, Zhixin Hui, Brian Grottkau
Collective cell migration, in which cells migrate as a group, is fundamental in many biological and pathological processes. There is increasing interest in studying the collective cell migration in high throughput. Cell-scratching, insertion blocker and gel-dissolving techniques are some methodologies used previously. However, these methods have the drawbacks of cell damage, substrate surface alteration, limitation in media exchange and solvent interference. The superhydrophobic surface, on which the water contact angle is greater than 150 degrees, has been recently utilized to generate patterned arrays...
February 3, 2018: Tissue Engineering. Part C, Methods
Rebeka Dalma Major, Martin Kluge, Maximilian Jara, Maximilian Nösser, Rosa Horner, Joseph Gassner, Benjamin Struecker, Peter Tang, Steffen Lippert, Anja Reutzel-Selke, Dominik Geisel, Timm Denecke, Martin Stockmann, Johann Pratschke, Igor M Sauer, Nathanael Raschzok
The need for primary human hepatocytes is constantly growing, for basic research as well as for therapeutic applications. However, the isolation outcome strongly depends on the quality of liver tissue, and we are still lacking a preoperative test that allows the prediction of the hepatocyte isolation outcome. Here we evaluated the "maximal liver function capacity test" (LiMAx) as predictive test for the quantitative and qualitative outcome of hepatocyte isolation. This test is already used in clinical routine to measure preoperative and to predict postoperative liver function...
January 30, 2018: Tissue Engineering. Part C, Methods
Laura Macri-Pellizzeri, Nigel De Melo, Ifty Ahmed, David M Grant, Brigitte Scammell, Virginie Sottile
The final stage of in vitro osteogenic differentiation is characterized by the production of mineral deposits containing calcium cations and inorganic phosphates, which populate the extracellular matrix surrounding the cell monolayer. Conventional histological techniques for the assessment of mineralization, such as Von Kossa and Alizarin Red S staining, are end-point techniques requiring cell fixation. Moreover, in both cases staining quantitation requires dye extraction which irreversibly alters the ECM conformation and structure, therefore preventing the use of the sample for further analysis...
January 21, 2018: Tissue Engineering. Part C, Methods
Seyed Hossein Mahfouzi, Ghassem Amoabediny, Ali Doryab, Seyed Hamid Safiabadi-Tali, Mostafa Ghanei
Maintaining cell viability within 3D tissue engineering scaffolds is an essential step toward a functional tissue or organ. Assessment of cell viability in 3D scaffolds is necessary to control and optimize tissue culture process. Monitoring systems based on respiration activity of cells (e.g., oxygen consumption) have been used in various cell cultures. In this research, an online monitoring system based on respiration activity was developed to monitor cell viability within acellular lung scaffolds. First, acellular lung scaffolds were recellularized with human umbilical cord vein endothelial cells, and then, cell viability was monitored during a 5-day period...
January 16, 2018: Tissue Engineering. Part C, Methods
Nathalie Steimberg, Francesca Angiero, Davide Farronato, Angiola Berenzi, Gianguido Cossellu, Andrea Ottonello, Darnell Kaigler, Giovanna Mazzoleni
The human dental follicle (hDF) contains the developing tooth and is involved in regulating tooth maturation and eruption. To investigate the mesenchymal stromal cells of the dental follicle, two 3D culture models were used, based on a dynamic bioreactor: the Rotary Cell Culture System (RCCS) and the 3D culture of precursor cells isolated from follicular tissue (hDFCs). The hDFCs were obtained from impacted third molars of 20 patients. Two 3D culture models were tested. In the first model, intact hDF explants were cultured in 3D conditions, preserving the original tissue architecture; they were studied via histomorphological and molecular analyses...
January 12, 2018: Tissue Engineering. Part C, Methods
Xiaofei Zhu, Jie Liu, Zongdong Yu, Chao-An Chen, Hacer Aksel, Adham A Azim, George T-J Huang
The goal of this study was to establish mini-swine as a large animal model for stem cell-based pulp regeneration studies. Swine dental pulp stem cells (sDPSCs) were isolated from mini-swine and characterized in vitro. For in vivo studies, we first employed both ectopic and semi-orthotopic study models using severe combined immunodeficiency mice. One is hydroxyapatite-tricalcium phosphate (HA/TCP) model for pulp-dentin complex formation, and the other is tooth fragment model for complete pulp regeneration with new dentin depositing along the canal walls...
February 2018: Tissue Engineering. Part C, Methods
Mohammad Izadifar, Dean Chapman, Paul Babyn, Xiongbiao Chen, Michael E Kelly
Biofabrication of cell supportive cardiac patches that can be directly implanted on myocardial infarct is a potential solution for myocardial infarction repair. Ideally, cardiac patches should be able to mimic myocardium extracellular matrix for rapid integration with the host tissue, raising the need to develop cardiac constructs with complex features. In particular, cardiac patches should be electrically conductive, mechanically robust and elastic, biologically active and prevascularized. In this study, we aim to biofabricate a nanoreinforced hybrid cardiac patch laden with human coronary artery endothelial cells (HCAECs) with improved electrical, mechanical, and biological behavior...
February 2018: Tissue Engineering. Part C, Methods
Tohru Takaseya, Hideyuki Fumoto, Junmin Zhu, Akira Shiose, Mariko Kobayashi, Masako Fujiki, Yoko Arakawa, Raymond Dessoffy, Larry D Kramer, Kiyotaka Fukamachi
We established an acute animal model for early, straightforward, and reproducible assessment of a biocompatible material interface. Bilateral femoral artery-to-vein shunts were created in 12 pigs: two tubes per shunt, the left two coated and the right two uncoated. We evaluated two groups: uncontrolled flow (UF; shunt flow unregulated) and controlled flow (CF; shunt flow ∼50 mL/min). For each case on each side, two shunts were evaluated: one for 1 h and the other for 3 h. Arterial blood gas and complete blood count were recorded at baseline, 1, and 3 h...
February 2018: Tissue Engineering. Part C, Methods
Lorena Hidalgo San Jose, Phil Stephens, Bing Song, David Barrow
Stem cell encapsulation technology demonstrates much promise for the replacement of damaged tissue in several diseases, including spinal cord injury (SCI). The use of biocompatible microcapsules permits the control of stem cell fate in situ to facilitate the replacement of damaged/lost tissue. In this work, a novel customized microfluidic device was developed for the reproducible encapsulation of Neural Stem Cells (NSCs) and Dental Pulp Stem Cells (DPSCs) within monodisperse, alginate-collagen microcapsules...
December 19, 2017: Tissue Engineering. Part C, Methods
Mehdi Najar, Laurent Dollé, Emerence Crompot, Stefaan Verhulst, Leo Van Grunsven, Hélène Busser, Laurence Lagneaux
Mesenchymal stromal cells (MSCs) have particular properties that allow their use as therapeutic strategies for several cell-based applications. Historically, bone marrow (BM)-MSCs are isolated by culture adherence since specific cell surface markers are yet to be developed. This original work aimed to identify and characterize isolating expanded BM-MSCs based on their aldehyde dehydrogenase (ALDH) activity known to be a hallmark of stem cells and relevant for their isolation. We thus isolated by FACS technology two functionally different populations of BM-MSCs depending on their ALDH activity (ALDH+ and ALDH-)...
December 14, 2017: Tissue Engineering. Part C, Methods
Xiaoli Chen, Linda Harkness, Zhongfan Jia, Andrew Prowse, Michael Monteiro, Peter P Gray
Human pluripotent stem cells (hPSCs) are viewed as promising candidates for applications in regenerative medicine and therapy due to their proliferative and pluripotent properties. However, obtaining clinically significant numbers of hPSCs remains a limiting factor and impedes their use in therapeutic applications. Conventionally, hPSCs are cultured on 2-dimensional (2D) surfaces coated with a suitable substrate, such as Matrigel™. This method, however, requires a large surface area to generate sufficient cell numbers to meet clinical needs, and is therefore impractical as a manufacturing platform for cell expansion...
December 14, 2017: Tissue Engineering. Part C, Methods
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