Comparing different methods to fix and to dehydrate cells on alginate hydrogel scaffolds using scanning electron microscopy

Scanning electron microscopy (SEM) is commonly used in the analysis of scaffolds morphology, as well as cell attachment, morphology and spreading on to the scaffolds. However, so far a specific methodology to prepare the alginate hydrogel (AH) scaffolds for SEM analysis has not been evaluated. This study compared different methods to fix/dehydrate cells in AH scaffolds for SEM analysis. AH scaffolds were prepared and seeded with NIH/3T3 cell line; fixed with glutaraldehyde, osmium tetroxide, or the freeze drying method and analyzed by SEM. Results demonstrated that the freeze dried method interferes less with cell morphology and density, and preserves the scaffolds structure. The fixation with glutaraldehyde did not affect cells morphology and density; however, the scaffolds morphology was affected in some level. The fixation with osmium tetroxide interfered in the natural structure of cells and scaffold. In conclusion the freeze drying and glutaraldehyde are suitable methods for cell fixation in AH scaffold for SEM, although scaffolds structure seems to be affected by glutaraldehyde. Microsc. Res. Tech. 78:553–561, 2015. © 2015 Wiley Periodicals, Inc.     

放射性骨丢失机制与防护研究进展

放射治疗可引起照射部位骨组织产生放射性骨损伤,发生骨丢失、骨质疏松,甚至病理性骨折。放射性骨丢失的主要原因是电离辐射可促进破骨细胞的骨吸收过程,抑制成骨细胞的骨形成过程。根据电离辐射对骨组织细胞影响的具体机制,以及骨组织细胞对不同剂量的电离辐射的不同响应,可采取相应的治疗和防护措施。本文从动物水平和细胞水平两个方面介绍放射性骨丢失的研究进展,并针对其发生机制提出可能的防护措施。     

Structurally Diverse Histone Deacetylase Photoreactive Probes: Design,Synthesis, and Photolabeling Studies in Live Cells and Tissue

Histone deacetylase (HDAC) activity is modulated in vivo by post-translational modifications and formation of multiprotein complexes. Novel chemical tools to study how these factors affect engagement of HDAC isoforms by HDAC inhibitors (HDACi) in cells and tissues are needed. In this study, a synthetic strategy to access chemically diverse photoreactive probes (PRPs) was developed and used to prepare seven novel HDAC PRPs 9 – 15 . The class I HDAC isoform engagement by PRPs was determined in biochemical assays and photolabeling experiments in live SET-2, HepG2, HuH7, and HEK293T cell lines and in mouse liver tissue. Unlike the HDAC protein abundance and biochemical activity against recombinant HDACs, the chemotype of the PRPs and the type of cells were key in defining the engagement of HDAC isoforms in live cells. Our findings suggest that engagement of HDAC isoforms by HDACi in vivo may be substantially modulated in a cell- and tissue-type-dependent manner.     

One-Step Rapid Fabrication of Cell-Only Living Fibers

Cellular aggregates are used as relevant regenerative building blocks, tissue models, and cell delivery platforms. Biomaterial-free structures are often assembled either as 2D cell sheets or spherical microaggregates, both incompatible with free-form deposition, and dependent on challenging processes for macroscale 3D upscaling. The continuous and elongated nature of fiber-shaped materials enables their deposition in unrestricted multiple directions. Cellular fiber fabrication has often required exogenously provided support proteins and/or the use of biomaterial-based sacrificial templates. Here, the rapid (<24 h) assembly of fiberoids is reported: living centimeter-long scaffold-free fibers of cells produced in the absence of exogenous materials or supplements. Adipose-derived mesenchymal stem cell fiberoids can be easily modulated into complex multidimensional geometries and show tissue-invasive properties while keeping the secretion of trophic factors. Proangiogenic properties studied on a chick chorioallantoic membrane in an ovo model are observed for heterotypic fiberoids containing endothelial cells. These micro-to-macrotissues may find application as morphogenic therapeutic and tissue-mimetic building blocks, with the ability to integrate 3D and 4D full biological materials.     

Rapid Biofabrication of Printable Dense Collagen Bioinks of Tunable Properties

Recent convergence of the 3D printing of tissue‐like bioinks and regenerative medicine offers promise in the high‐throughput engineering of in vitro tissue models and organoids for drug screening and discovery research, and of potentially implantable neo‐tissues with tailored structural, biological, and mechanical properties. However, the current printing approaches are not compatible with collagen, the native scaffolding material. Herein, a unique biofabrication approach that uses automated gel aspiration‐ejection (GAE) is reported to potentially overcome these challenges. Automated‐GAE generates highly defined, aligned, dense collagen gel bioinks of various geometries (i.e., cylindrical, quadrangular, and tubular), dimensions, as well as tunable microstructural and mechanical properties that modulate seeded cellular responses. By densifying initial naturally derived reconstituted collagen hydrogels incorporating cells, automated‐GAE generates mini‐tissue building blocks with tailored protein fibril density and alignment, as well as cell loading, density and orientation according to the intended use. Surprisingly, a simple mathematical relationship defining the bioink compaction factor is found to be highly effective in predicting the initial and temporal properties of the bioinks in culture. Therefore, automated‐GAE will potentially also enable a fourth dimension to biofabrication, where cell–cell communications and cell‐extracellular matrix interactions as a function of time in culture can be predicted and modeled.     

少量出血患者使用术中回收式自体输血有效性的随机对照研究

目的　探讨术中回收式自体输血（intraoperative autotransfusion, IAT）能否改善少量出血患者的组织氧合及术后恢复。方法　选择2011年12月至2012年4月在北京协和医院行腰椎后路手术、美国麻醉医师协会（American Society of Anesthesiologists，ASA）分级为Ⅰ~Ⅱ级且预计出血量＜20%血容量的患者，随机分为两组，回输组输注回收自体血，对照组给予等量胶体液。检测两组患者术前、术中及术后血红蛋白（hemoglobin，Hb）、乳酸及脑氧饱和度，随访并比较术后恢复指标，包括体温、不适主诉、切口愈合、下地行走时间及术后住院时间。结果　共38例患者纳入本研究，回输组和对照组各19例。回输组术后0.5 h和术后1 d的Hb均高于术中水平［分别为（116.5±10.7）、（115.4±12.3）、（106.6±12.6）g/L；P=0.001，P=0.004］，而对照组术后Hb与术中比较差异无统计学意义（P＞0.05）。对照组术后0.5 h乳酸高于术前［（2.5±1.0）mmol/L比（1.3±0.6）mmol/L，P=0.016］，而回输组术后乳酸与术前比较差异无统计学意义（P＞0.05）。术后Hb、乳酸及脑氧饱和度两组间差异均无统计学意义（P＞0.05）。两组术后恢复指标差异亦均无统计学意义（P＞0.05）。结论　在健康成人患者少量出血手术中使用IAT，对术后早期组织氧合有一定改善作用，但对术后恢复无显著影响。     

Effect of pH,temperature and storage time on the stability of bovine myelin basic protein

Bovine central nervous system tissue (CNT) harboring highest levels of the infectious agent for prion diseases, has been banned from food and feed supplies. Effective detection of CNT in excessively processed meat and feedstuffs requires the assay based on a stable marker in CNT as the analyte. Myelin basic protein (MBP), the major central nervous system (CNS) myelin protein, was reported to be detectable up to 115 °C in the literature. This study further investigated the effects of pH, temperature and storage time on the stability of purified bovine MBP (18.5 kDa) in solutions. Purified MBP dissolved in 10 mM PBS was adjusted to pH 3.0, 7.2 and 10.5, respectively. Sample at each pH was subjected to three heat treatments, 100 °C, 121 °C, and 133 °C for 30 min. The unheated and 100 °C heated samples at pH 7.2 were stored at 4 °C for 19 days to study the storage stability of bovine MBP. The immunoreactivity of all samples was examined using indirect non-competitive enzyme-linked immunosorbent assay and immunoblotting with an anti-MBP monoclonal antibody. The protein degradation was observed by analyzing samples using SDS-PAGE. Results show that bovine MBP was most stable at pH 7.2 while the protein was least stable at pH 10.5. MBP started to degrade after heating at 121 °C for 30 min at pH 7.2. Storage up to 19 days at 4 °C did not significantly affect the immunoreactivity of unheated and heated samples. However, unheated MBP samples started to show degradation from day 8 of storage; while no degradation was observed in 100 °C heated samples over the storage time. This study demonstrated that bovine MBP can be a suitable marker for the detection of bovine CNT in highly processed food and feed supplies.     

High-frequency dual mode pulsed wave Doppler imaging for monitoring the functional regeneration of adult zebrafish hearts

Adult zebrafish is a well-known small animal model for studying heart regeneration. Although the regeneration of scars made by resecting the ventricular apex has been visualized with histological methods, there is no adequate imaging tool for tracking the functional recovery of the damaged heart. For this reason, high-frequency Doppler echocardiography using dual mode pulsed wave Doppler, which provides both tissue Doppler (TD) and Doppler flow in a same cardiac cycle, is developed with a 30 MHz high-frequency array ultrasound imaging system. Phantom studies show that the Doppler flow mode of the dual mode is capable of measuring the flow velocity from 0.1 to 15 cm s⁻¹ with high accuracy (p-value = 0.974 > 0.05). In the in vivo study of zebrafish, both TD and Doppler flow signals were simultaneously obtained from the zebrafish heart for the first time, and the synchronized valve motions with the blood flow signals were identified. In the longitudinal study on the zebrafish heart regeneration, the parameters for diagnosing the diastolic dysfunction, for example, E/Em < 10, E/A < 0.14 for wild-type zebrafish, were measured, and the type of diastolic dysfunction caused by the amputation was found to be similar to the restrictive filling. The diastolic function was fully recovered within four weeks post-amputation.