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Tissue Engineering. Part C, Methods

Samuel A Einstein, Bradley P Weegman, Meri T Firpo, Klearchos Papas, Michael Garwood
Techniques to monitor the oxygen partial pressure (pO<sub>2</sub>) within implanted tissue-engineered grafts (TEGs) are critically necessary for TEG development, but current methods are invasive and inaccurate. In this study, we investigated an accurate and noninvasive technique to monitor TEG pO<sub>2</sub> utilizing proton (<sup>1</sup>H) or fluorine (<sup>19</sup>F) magnetic resonance spectroscopy (MRS) relaxometry. The value of the spin-lattice relaxation rate constant (R<sub>1</sub>) of some biocompatible compounds is sensitive to dissolved oxygen (and temperature), while insensitive to other external factors...
October 19, 2016: Tissue Engineering. Part C, Methods
Lauren Sfakis, Tim Kamaldinov, Melinda Larsen, James Castracane, Alexander Khmaladze
Quantifying confocal images to enable location of specific proteins of interest in 3D is important for many tissue engineering applications. Quantification of protein localization is essential for evaluation of specific scaffold constructs for cell growth and differentiation for application in tissue engineering and tissue regeneration strategies. Although obtaining information regarding protein expression levels is important, the location of proteins within cells grown on scaffolds is often key to evaluating scaffold efficacy...
October 19, 2016: Tissue Engineering. Part C, Methods
Katie Pollock, Guanglin Yu, Ralph Moller-Trane, Marissa Koran, Peter Dosa, David McKenna, Allison Hubel
There is demand for non-DMSO cryoprotective agents that maintain cell viability without causing poor post thaw function or systemic toxicity. The focus of this investigation involves expanding our understanding of multicomponent osmolyte solutions and their ability to preserve cell viability during freezing. Controlled cooling rate freezing, Raman microscopy, and differential scanning calorimetry (DSC) were utilized to evaluate the differences in recovery and ice crystal formation behavior for solutions containing multiple cryoprotectants including sugars, sugar alcohols, and small molecule additives...
October 19, 2016: Tissue Engineering. Part C, Methods
Wenlong Liu, Xiuli Dan, Ting Wang, William Weijia Lu, Haobo Pan
The development of an optimal animal model which could provide fast assessments of the interaction between bone and orthopaedic implants is essential for both pre-clinical and theoretical researches in the design of novel biomaterials. Compared with other animal models, mice have superiority in accessing the well-developed transgenic modification techniques (e.g. cell tracing, knock-off, knock-in ect.) which serve as powerful tools in studying molecular mechanisms. In this study, we introduced the establishment of a mouse model which is specifically tailored for the assessment of bone-implant interaction in a load-bearing bone marrow microenvironment and could potentially allow the molecular mechanism study of biomaterials by using transgenic technologies...
October 12, 2016: Tissue Engineering. Part C, Methods
Timothy J Ewald, Justin A Walker, Eric Alexander Lewallen, William H Trousdale, Michael J Yaszemski, Arlen D Hanssen, Bernard F Morrey, Andre J van Wijnen, Joaquin Sanchez-Sotelo, Mark E Morrey, Matthew P Abdel
Implantable biomaterials supporting extended release of pharmacologic agents may permit localized intra-articular delivery of drugs that modulate the fibrotic response to injuries and surgery. Oligo[Poly (ethylene glycol)] Fumarate (OPF) is an attractive organic carrier, but its safety profile within synovial joints remains unclear. Here, we assessed the safety of OPF sponges using a validated in vivo model of knee arthrofibrosis. A cohort of 102 rabbits was divided into 5 groups: arthrotomy only (24), arthrotomy with OPF scaffold placement (24), surgically induced contracture (24), surgically induced contracture with OPF scaffold placement (24), and control without any surgical intervention (6)...
September 27, 2016: Tissue Engineering. Part C, Methods
Yuanyuan Zheng, Gang Zhao, Fazil Fazil Panhwar, Xiaoming He
Cryopreservation of human umbilical vein endothelial cells (HUVECs) is important to tissue engineering applications and the study of the role of endothelial cells in cardiovascular and cerebrovascular diseases. The traditional methods for cryopreservation by vitrification (cooling samples to a cryogenic temperature without apparent freezing) using high concentration of cryoprotective agents (CPAs) and slow-freezing are suboptimal due to the severe toxicity of high concentration of CPAs and ice formation induced cryoinjuries, respectively...
September 27, 2016: Tissue Engineering. Part C, Methods
Luis G Hurtado-Aguilar, Shane Mulderrig, Ricardo Moreira, Nima Hatam, Jan Spillner, Thomas Schmitz-Rode, Stefan Jockenhoevel, Petra Mela
Tissue-engineered heart valves are developed in bioreactors where biochemical and mechanical stimuli are provided for extracellular matrix formation. During this phase, the monitoring possibilities are limited by the need to maintain the sterility and the integrity of the valve. Therefore, noninvasive and nondestructive techniques are required. As such, optical imaging is commonly used to verify valve's functionality in vitro. It provides important information (i.e. leaflet symmetry, geometric orifice area, closing and opening times) which is, however, usually limited to a singular view along the central axis from the outflow side...
September 27, 2016: Tissue Engineering. Part C, Methods
Jung-Seok Lee, Seul Ki Kim, Jae-Kook Cha, Byung-Joo Jung, Seong-Bok Choi, Eun Young Choi, Chang Sung Kim
Centrifugation based on density gradients is a general methodology for isolating of human bone marrow (hBM)-derived mesenchymal stem cells (hBMSCs). The mononuclear cell (MNC) layer can be obtained using a density-gradient solution in the conventional protocol. But, this is not suitable for direct transplantation due to the possible toxicity of this solution. The results obtained are also influenced by skill level when applying the technique, which involves time-consuming processes. We have developed a novel protocol for isolating hBMSCs using hyaluronic acid (HA), which is the most widely used injectable biomaterial in the clinical setting and a major component of the extracellular matrix...
September 8, 2016: Tissue Engineering. Part C, Methods
Tonia C Rothuizen, Raymond Kemp, Jacques M G J Duijs, Hetty C De Boer, Roel Bijkerk, Eric P Van der Veer, Lorenzo Moroni, Anton Jan Van Zonneveld, Anthony S Weiss, Ton J Rabelink, Joris I Rotmans
BACKGROUND: Elastin, critical for its structural and regulatory functions, is a missing link in vascular tissue engineering. Several elastin-inducting compounds have previously been reported, but their relative efficiency in promoting elastogenesis by adult arterial and venous vascular smooth muscle cells (VSMCs) and fibroblasts, four main vascular and elastogenic cells, has not been described. In addition to elasto-inductive substances, microRNA-29a was recently established as a potent post-transcriptional inhibitor of elastogenesis...
September 7, 2016: Tissue Engineering. Part C, Methods
Kevin Stanley Bielawski, Andrea Leonard, Shiv Bhandari, Charles E Murry, Nathan Sniadecki
Engineered heart tissues made from human pluripotent stem cell derived cardiomyocytes have been used for modeling cardiac pathologies, screening new therapeutics, and providing replacement cardiac tissue. Current methods measure the functional performance of engineered heart tissue by their twitch force and beating frequency, typically obtained by optical measurements. In this paper, we describe a novel method for assessing twitch force and beating frequency of engineered heart tissue using magnetic field sensing, which enables multiple tissues to be measured simultaneously...
September 6, 2016: Tissue Engineering. Part C, Methods
Ruchi Mishra, Ryan S Sefcik, Tyler J Bishop, Stefani M Montelone, Nisha Crouser, Jean F Welter, Arnold I Caplan, David Dean
One approach to the development of an artificial graft material could rely on uniform coverage of a resorbable biomaterial with bone extracellular matrix (ECM). To achieve this on the surface of poly(propylene fumarate) (PPF) scaffolds, we selected a growth factor regime of basic fibroblast growth factor (FGF-2) (5 ng/mL), platelet-derived growth factor (PDGF-BB) (40 ng/mL), and epidermal growth factor (EGF) (20 ng/mL) to stimulate proliferation of bone marrow-derived human mesenchymal stem cells (BM-hMSCs)...
September 2016: Tissue Engineering. Part C, Methods
Pedro S Babo, Alexey Klymov, Joost teRiet, Rui L Reis, John A Jansen, Manuela E Gomes, X Frank Walboomers
Micro- and nanotextured biomaterial surfaces have been widely studied for their capacity to drive the regeneration of organized tissues. Nanotopographical features in the shape of groove-ridge patterns aim at mimicking the extracellular matrix organization. However, to date, a wide array of groove and ridge sizes has been described. In this work, we therefore tested a device composed of a multipatterned array consisting of six patterns of radially arranged parallel nanogrooves, with a pitch ranging from 0 to 1000 nm and a depth ranging from 0 to 170 nm, to be used as a tool for the expeditious and simultaneous screening of surface topographies aiming the regeneration of anisotropically organized tissues such as ligament...
September 2016: Tissue Engineering. Part C, Methods
Wendy E Brown, Jerry C Hu, Kyriacos A Athanasiou
Juvenile and fetal, primary, fully differentiated cells are widely considered to be ideal cell types for tissue engineering applications. However, their use in tissue engineering may be hindered through contamination by undesirable cell types. These include blood-associated cells as well as unwanted resident cell types found both in healthy and pathologic donor tissues. Ammonium-chloride-potassium (ACK) lysing buffer is used to lyse red blood cells (RBCs) during the isolation of stem cell populations, but has not been explored for the purification of fully differentiated cells...
September 2016: Tissue Engineering. Part C, Methods
Neda Latifi, Hossein K Heris, Scott L Thomson, Rani Taher, Siavash Kazemirad, Sara Sheibani, Nicole Y K Li-Jessen, Hojatollah Vali, Luc Mongeau
The human vocal folds (VFs) undergo complex biomechanical stimulation during phonation. The aim of the present study was to develop and validate a phono-mimetic VF flow perfusion bioreactor, which mimics the mechanical microenvironment of the human VFs in vitro. The bioreactor uses airflow-induced self-oscillations, which have been shown to produce mechanical loading and contact forces that are representative of human phonation. The bioreactor consisted of two synthetic VF replicas within a silicone body. A cell-scaffold mixture (CSM) consisting of human VF fibroblasts, hyaluronic acid, gelatin, and a polyethylene glycol cross-linker was injected into cavities within the replicas...
September 2016: Tissue Engineering. Part C, Methods
Meng Fatt Leong, Hong Fang Lu, Tze Chiun Lim, Karthikeyan Narayanan, Shujun Gao, Luna Yue Wang, Rebecca P K Toh, Henrik Funke, Muhammad Hamizan Abdul Samad, Andrew C A Wan, Jackie Y Ying
Pluripotent human embryonic stem cells (hESCs) are a potential renewable cell source for regenerative medicine and drug testing. To obtain adequate cell numbers for these applications, there is a need to develop scalable cell culture platforms to propagate hESCs. In this study, we encapsulated hESCs in calcium alginate microfibers as single cells, for expansion and differentiation under chemically defined conditions. hESCs were suspended in 1% (w/v) alginate solution at high cell density (>10(7) cells/mL) and extruded at 5 m/min into a low calcium concentration bath (10 mM) for gelation...
September 2016: Tissue Engineering. Part C, Methods
Rosa Horner, Martin Kluge, Joseph Gassner, Maximilian Nösser, Rebeka Dalma Major, Anja Reutzel-Selke, Annekatrin K Leder, Benjamin Struecker, Mehmet H Morgul, Johann Pratschke, Igor M Sauer, Nathanael Raschzok
Liver tissue obtained from partial hepatectomy is a common source for isolation of primary human hepatocytes. Until now, liver resections were most commonly performed by conventional open surgery. Although the laparoscopic approach is currently emerging in liver surgery, data on the outcome of hepatocyte isolation from laparoscopically resected liver tissue are not available. A total of 22 hepatocyte isolations were performed using the two-step collagenase perfusion technique from October 2015 to March 2016...
September 2016: Tissue Engineering. Part C, Methods
Matthias Schweinlin, Sabine Wilhelm, Ivo Schwedhelm, Jan Hansmann, Rene Rietscher, Christian Jurowich, Heike Walles, Marco Metzger
Intestinal in vitro models are valuable tools in drug discovery and infection research. Despite several advantages, the standard cell line-based Transwell(®) models based for example on colonic epithelial Caco-2 cells, lack the cellular complexity and transport activity associated with native small intestinal tissue. An additional experimental set-back arises from the most commonly used synthetic membranes, on which the cells are routinely cultured. These can lead to an additional barrier activity during in vitro testing...
September 2016: Tissue Engineering. Part C, Methods
Jee-Wook Lee, Hui-Suk Yun, Takayoshi Nakano
Changes in the biological apatite (BAp) c-axis orientation were investigated as a bone quality parameter in bone regeneration using hydroxyapatite/poly ɛ-caprolactone (HA/PCL) composite scaffolds. Three-dimensional (3D) HA/PCL composite scaffolds were fabricated using a layer manufacturing process in three grid sizes (200-, 600-, and 1000 μm) and grafted into the forearm ulna of New Zealand white rabbits. The cross-sectional areas of the bones regenerated from the scaffolds with 600- and 1000-μm grid sizes were significantly larger than those from the scaffold with 200-μm grid sizes, whereas bone mineral density in the regenerated regions did not differ between the three grid sizes...
September 2016: Tissue Engineering. Part C, Methods
Su-Eon Jin, Hyo-Sun Ahn, Ji Hye Kim, Yoshie Arai, Soo-Hong Lee, Tae-Jong Yoon, Sung-Joo Hwang, Jong-Hyuk Sung
Adipose-derived stem cells (ASCs) are typically expanded to acquire large numbers of cells for therapeutic applications. Diverse stimuli such as sphingosylphosphocholine and vitamin C have been used to increase the production yield and regenerative potential of ASCs. In the present study, we hypothesized that ZnO nanorods have promising potential for the enhancement of ASC proliferation. ZnO nanorods were prepared using three different methods: grinding and boiling at low temperature with and without surfactant...
September 2016: Tissue Engineering. Part C, Methods
Olaia F Vila, Cristina Garrido, Irene Cano, Marta Guerra-Rebollo, Melba Navarro, Oscar Meca-Cortés, Stephen P Ma, Elisabeth Engel, Nuria Rubio, Jerónimo Blanco
Bioreactor systems allow safe and reproducible production of tissue constructs and functional analysis of cell behavior in biomaterials. However, current procedures for the analysis of tissue generated in biomaterials are destructive. We describe a transparent perfusion system that allows real-time bioluminescence imaging of luciferase expressing cells seeded in scaffolds for the study of cell-biomaterial interactions and bioreactor performance. A prototype provided with a poly(lactic) acid scaffold was used for "proof of principle" studies to monitor cell survival in the scaffold (up to 22 days)...
September 2016: Tissue Engineering. Part C, Methods
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