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Tissue Engineering. Part C, Methods

Zhuoming Liu, Rudell Screven, Lynne Boxer, Michael Myers, Lax Devireddy
In this manuscript, we report on the development of a defined serum-free medium capable of supporting the culture expansion of mesenchymal stromal/stem cells (MSCs) from canine adipose tissue (canine Ad-MSCs). The potential benefits of serum-free media can only be utilized if cells cultured in serum-free media maintain the same functional characteristics as cells cultured in serum-containing media. Therefore, we analyze the characteristics of canine Ad-MSCs cultured in this serum-free medium or in serum-containing medium through evaluation of growth kinetics, clonogenic capacity, senescence, and differentiation capacity...
May 17, 2018: Tissue Engineering. Part C, Methods
Maurits Geert Laurent Olthof, Marianna A Tryfonidou, Mahrokh Dadsetan, Wouter Dhert, Michael J Yaszemski, Diederik H R Kempen, Lichun Lu
Local sustained delivery of bioactive molecules from biomaterials is a promising strategy to enhance bone regeneration. To optimize delivery vehicles for bone formation, the design characteristics are tailored with consequential effect on BMP-2 release and bone regeneration. Complying with the 3R principles, the growth factor release is often investigated in vitro using several buffers to mimic the in vivo physiological environment. However, this remains an unmet need. Therefore, this study investigates the correlation between the in vitro and in vivo (IVIVC) BMP-2 release from complex delivery vehicles in several commonly used in vitro buffers: cell culture model, phosphate buffered saline, and a strong desorption buffer...
May 14, 2018: Tissue Engineering. Part C, Methods
Thomas Dixon, Eliad Cohen, Dana Meredith Cairns, Maria Rodriguez, Juanita Mathews, Rod Jose, David L Kaplan
The physical connection between motoneurons and skeletal muscle targets is responsible for the creation of neuromuscular junctions (NMJs), which allow electrical signals to be translated to mechanical work. NMJ pathology contributes to the spectrum of neuromuscular, motoneuron, and dystrophic disease. Improving in vitro tools that allow for recapitulation of the physiology of the neuromuscular connection will enable researchers to better understand the development and maturation of NMJs, and will help to decipher mechanisms leading to NMJ degeneration...
May 9, 2018: Tissue Engineering. Part C, Methods
Hanis Nazihah Hasmad, Mohd Reusmaazran Yusof, Zainul Rashid Mohd Razi, Ruszymah Binti Haji Idrus, Shiplu Roy Chowdhury
Fabrication of composite scaffolds is one of the strategies proposed to enhance the functionality of tissue engineered scaffolds for improved tissue regeneration. By combining multiple elements together, unique biomimetic scaffolds with desirable physical and mechanical properties can be tailored for tissue-specific applications. Despite having a highly porous structure, the utility of electrospun fibers (EF) as scaffold is usually hampered by their insufficient mechanical strength. In this study, we attempted to produce a mechanically competent scaffold with cell-guiding ability by fabricating aligned PLGA fibers on decellularized human amniotic membrane (HAM), known to possess favorable tensile and wound healing properties...
April 25, 2018: Tissue Engineering. Part C, Methods
Marco Viganò, Carlotta Perucca Orfei, Laura de Girolamo, John Pearson, Enrico Ragni, Paola De Luca, Alessandra Colombini
The use of biochemical inducers of mesenchymal stem cells (MSCs) differentiation into tenogenic lineage represents an investigated aspect of tendon disorder treatment. Bone morphogenetic protein 12 (BMP-12) is a widely studied factor, representing along with ascorbic acid (AA) and basic fibroblast growth factor (bFGF) one of the most promising stimulus in this context so far. Quantitative gene expression of specific tenogenic marker is commonly used to assess the efficacy of these supplements. Nevertheless, the reliability of these data is strongly associated with the choice of stable housekeeping genes...
April 20, 2018: Tissue Engineering. Part C, Methods
Mitchell R Ladd, Laura Y Martin, Adam Werts, Cait Costello, Chhinder Sodhi, William Fulton, John C March, David Hackam
Short bowel syndrome is a major cause of morbidity and mortality in the pediatric population, for which treatment options are limited. To develop novel approaches for the treatment of short bowel syndrome, we now focus on the development of a tissue-engineered intestine (also known as an "artificial intestine") in which intestinal stem cells are cultured onto an absorbable bioscaffold followed by implantation into the host. In order to enhance the translational potential of these pre-clinical studies, we have developed three clinically relevant models in neonatal piglets, which approximate the size of the human infant, and which were evaluated after implantation and establishment of intestinal continuity over the long-term: The models included 1) a staged, multi-operation approach; 2) a single operation with a de-functionalized loop of small intestine; and 3) a single operation with an intestinal bypass...
April 11, 2018: Tissue Engineering. Part C, Methods
Callie An Knuth, Caoimhe H Kiernan, Virginia Palomares Cabeza, Johannes Lehmann, Janneke Witte-Buoma, Derk Ten Berge, Pieter A J Brama, Eppo B Wolvius, Elske M Strabbing, Martin Koudstaal, Roberto Narcisi, Eric Farrell
Mesenchymal stem cells/marrow stromal cells (MSCs) are attractive for applications ranging from research and development to use in clinical therapeutics. However, the most commonly studied MSCs, adult bone marrow MSCs (A-MSCs), are limited by significant donor variation resulting in inconsistent expansion rates and multilineage differentiation capabilities. We have recently obtained permission to isolate paediatric MSCs (P-MSCs) from surplus iliac crest bone chips. Here, we developed a simple and easily replicable isolation protocol yielding P-MSCs which adhere to MSC defining guidelines...
April 10, 2018: Tissue Engineering. Part C, Methods
Vahid Mirshafiee, Brendan A C Harley, Mary L Kraft
Characterization of the heterogeneity within stem cell populations, which affects their differentiation potential, is necessary for the design of artificial cultures for stem cell expansion. In this study, we assessed whether self-organizing maps (SOMs) of single cell time-of-flight secondary ion mass spectrometry (TOF-SIMS) data provide insight into the spectral, and thus the related functional heterogeneity between and within three hematopoietic cell populations. SOMs were created of TOF-SIMS data from individual hematopoietic stem and progenitor cells (HSPCs), lineage-committed common lymphoid progenitors (CLPs), and fully differentiated B cells that had been isolated from murine bone marrow via conventional flow cytometry...
March 16, 2018: Tissue Engineering. Part C, Methods
Maaike Vera Jasmijn Braham, Tilman Ahlfeld, Ashwini Rahul Akkineni, Monique C Minnema, Wouter Dhert, F Cumhur Oner, Catherine Robin, Anja Lode, Michael Gelinsky, Jacqueline Alblas
The bone marrow microenvironment is the preferred location of multiple myeloma, supporting tumor growth and development. It is composed of a collection of interacting subniches, including the endosteal and perivascular niche. Current in vitro models mimic either of these subniches. By developing a model combining both niches, this study aims to further enhance the ability to culture primary myeloma cells in vitro. Also the dependency of myeloma cells on each niche was studied. A 3D bone marrow model containing two subniches was created using 3D bioprinting technology...
March 8, 2018: Tissue Engineering. Part C, Methods
Brian C Syverud, Jonathan P Gumucio, Brittany L Rodriguez, Olga M Wroblewski, Shelby E Florida, Christopher L Mendias, Lisa M 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...
May 2018: Tissue Engineering. Part C, Methods
Rui Li, Guanglin Yu, Samira M Azarin, Allison Hubel
Inadequate preservation methods of human induced pluripotent stem cells (hiPSCs) have impeded efficient reestablishment of cell culture after the freeze-thaw process. In this study, we examined roles of the cooling rate, seeding temperature, and 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, and 10°C/min, and between seeding temperatures of -4°C and -8°C...
May 2018: Tissue Engineering. Part C, Methods
Aila Daugs, Nadine Lehmann, Dilem Eroglu, Martina C 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)...
May 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 to traditional bone grafting techniques. Critical size bone defect animal models have 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 qualitative and only measure the amount of newly formed bone...
May 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...
May 2018: Tissue Engineering. Part C, Methods
Natalia Yiset Becerra Colorado, Claudia Marcela Arenas Gómez, Maria Isabel Patiño Vargas, Jean Paul Delgado Charris, Carlos Enrique Muskus López, Luz Marina Restrepo Múnera
Nonviral transfection has important implications on gene therapy because of its safety. In particular, polyfection and nucleofection are two widely used systems for nonviral 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 that carries the gene of interest. The efficiency of transfection by polyfection or nucleofection in human fibroblasts 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)...
April 2018: Tissue Engineering. Part C, Methods
Grace C Limandjaja, Lenie J van den Broek, Melanie Breetveld, Taco Waaijman, Stan Monstrey, Edith M de Boer, Rik J Scheper, Frank B 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...
April 2018: Tissue Engineering. Part C, Methods
Anita Krouwels, Ferry P W Melchels, Mattie H P van Rijen, F Cumhur Öner, Wouter J A Dhert, Marianna A Tryfonidou, Laura B Creemers
Hydrogels can facilitate nucleus pulposus (NP) regeneration, either for clinical application or research into mechanisms of regeneration. However, many different hydrogels and culture conditions for human degenerated 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 redifferentiation in an attempt to provide comparators for future studies...
April 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 preimplantation health and viability of engineered tissue constructs. However, current practices to monitor postimplantation 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 into 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...
April 2018: Tissue Engineering. Part C, Methods
Yonggang Pang, Jing Yang, Zhixin Hui, Brian E 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 medium 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...
April 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...
April 2018: Tissue Engineering. Part C, Methods
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