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.     

The effects of osmium tetroxide post‐fixation and drying steps on leafy liverwort ultrastructure study by scanning electron microscopy

Leafy liverwort is one of the most abundant and diverse plants in Indonesia. Their high variation and beneficial secondary metabolites contained in the oil bodies have attracted researchers' attention. The ultrastructural analysis of leafy liverworts is important as a means of species identification and also for further exploration of their oil bodies. However, the optimization of the preparation steps for observing leafy liverworts by SEM is necessary to avoid sample destruction. Fixation and drying play important roles in maintaining a sample's structure as close to its natural state as possible. Thus, in this study, we evaluated the effect of 4% Osmium tetroxide (OsO₄) and drying on leafy liverworts ultrastructure. Microlejeunea, Acrolejeunea, and Frullania were fixed with 2.5% glutaraldehyde. Some samples were then post‐fixed with 4% OsO₄, while the rest were directly dehydrated with an ethanol series and then subjected to different drying methods, i.e. air drying, freeze drying, and drying with hexamethyldisilazane (HMDS). According to the data obtained, post‐fixation with 4% OsO₄ could better maintain the integrity of the samples and enhance the contrast of leafy liverwort SEM images. In addition, samples dried with HMDS showed more detailed structures compared to those that were air dried. Different ultrastructure were found among the different leafy liverworts observed by SEM. Our data suggested the advantages of SEM in providing ultrastructure information on leafy liverworts as well as the optimum conditions to observe them with less deformation. OsO₄ post‐fixation could enhance the contrast of leafy liverwort SEM images and maintain the structure of the samples. Drying with HMDS provided a convenient way for rapid SEM preparation with less structural distortion.     

Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells

Critical point drying (CPD) is a common method of drying biological specimens for scanning electron microscopy (SEM). Drying by evaporation of hexamethyldisilazane (HMDS) has been described as a good alternative. This method, however, is infrequently used. Therefore, we reassessed HMDS drying. Cultured rat hepatic sinusoidal endothelial cells (LEC), possessing fragile fenestrae and sieve plates, were subjected to CPD and HMDS drying and evaluated in the scanning electron microscope, atomic force microscope (AFM) and transmission electron microscope (TEM). We observed no differences between the two methods regarding cellular ultrastructure. In contrast with CPD, HMDS drying takes only a few minutes, less effort, low costs for chemicals and requires no equipment. We conclude that HMDS-dried specimens have equal quality to CPD ones. Furthermore, the method also proved useful for drying whole-mount cells for TEM and AFM.     

Design of multi-scaffold fabrication system for various 3D scaffolds

Three-dimensional (3D) porous scaffolds have been fabricated recently for tissue engineering applications through solid free-form fabrication (SFF) technologies. A multi-scaffold fabrication system for the fabrication of scaffolds, such as polymer, polymer/ceramic, ceramic, and nanofiber, was designed in this study. The various components, including a dispenser with a maximum pressure of 750 kPa, a thermostat with a maximum temperature of 250°C, a high-voltage power supply with a maximum output of 60 kV, and a syringe pump with small flow control, play important roles in determining the process characteristics of scaffolds. The system can process applicable biomaterials with extremely high accuracy with a precision nozzle. Several 3D scaffolds, including PCL, PCL/PLGA/β-TCP, β-TCP, and PCL nanofibers, were fabricated. The morphology and pore size of fabricated scaffolds were observed through scanning electron microscopy. Results show that the scaffolds manufactured in this study can be effectively utilized as bone regeneration scaffolds.     

New application of air-drying techniques for studying Ephemeroptera and Plecoptera eggs by scanning electron microscopy

Hexamethyldisilizane (HMDS) and tetramethylsilane are organic compounds that are volatile at ambient temperature and which can therefore be used for air-drying biological samples for SEM studies. The techniques using these compounds provide results that are comparable with those obtained by critical point drying, but which involve a very simple process that saves time and money. Both techniques were applied to SEM studies of Ephemeroptera and Plecoptera eggs in order to assess their suitability as alternative methods to critical point drying for these kinds of biological material. The results show no morphological differences between eggs HMDS air-dried and critical point-dried.     

A structural study of the bovine vaginal fluid at estrus.

The present study describes the structural components of the bovine vaginal fluid at estrus by scanning electron microscopy (SEM) following critical point- and freeze-drying preparation procedures. Confocal scanning laser microscopy (CLSM) was also used to evaluate the structural integrity of samples, and a control sample was assessed by adding sperm to the vaginal fluid. Samples were collected from 10 cows at the time of artificial insemination, prepared for SEM by using critical point- and freeze-drying procedures, gold coated, and observed by SEM. Mesh size and filament thickness were measured with an image analyzer. Of the 10 samples processed, 4 were considered altered following critical point drying. Compaction and lack of filaments were observed in these samples. A small area of one sample showed a honey comb-like structure when freeze drying was used. Nonoriented filaments with different thicknesses and with a network-like structure were observed throughout the remainder of the samples. Filaments throughout all samples were also observed by CSLM. After critical point drying, the mesh area ranged from 0.8 to 101.4 microns 2; the minor axis from 0.7 to 10.8 microns; and filament thickness from 40 to 442 nm. Using freeze drying, the mesh area ranged from 0.9 to 493.8 microns 2; the minor axis from 0.7 to 27.5 microns; and filament thickness from 40 to 800 nm. When samples were freeze dried, mesh values were similar to the interstrand channels observed by CSLM. In sperm-vaginal fluid samples, following critical point- or freeze-drying procedures, spermatozoa were oriented randomly in the vaginal fluid and did not seem to alter filamentous structure. Our data suggest that the freeze-drying procedure better preserves the true structural dimensions of the vaginal fluid. Furthermore, the filamentous structure of the vaginal fluid does not appear to impede sperm transport.     

Scanning electron microscopy of piliated Neisseria gonorrhoeae processed with hexamethyldisilazane

Piliated Neisseria gonorrhoeae are virulent and attach readily to some human mucosal cells. The study of interactions between piliated Neisseria gonorrhoeae and surface structures of eukaryotic cells in tissue culture requires consistent high resolution imaging in scanning electron microscopy (SEM). The combination of the fixatives glutaraldehyde, osmium, tannic acid, and uranyl acetate improves preservation of pili and other delicate structures. Following the critical point drying (CPD) process, pili bundles remained intact, but charging produced image distortion in most of the specimens. The use of hexamethyldisilazane (HMDS) with air drying substantially reduced charging and image distortion. Less contrast and greater resolution of pili bundles and surface structures of bacteria or tissue culture cells were obtained at magnifications of 10,000 or higher. As an alternative to CPD, HMDS processing of cell culture monolayers was simple and was more efficient when a large number of samples was processed.     

Cell and organelle shrinkage during preparation for scanning electron microscopy: effects of fixation, dehydration and critical point drying.

The critical point drying method of preparing samples for scanning electron microscopy is associated with a variable amount of specimen shrinkage. We studied the causes of this phenomenon is isolated mouse hepatocyte nuclei and in human erythrocytes and found that the critical point drying process itself caused most of the shrinkage that we observed (a 25-30% reduction in diameter in both specimens). Glutaraldehyde fixation and ethanol dehydration caused only minimal size reduction, prior to critical point drying. Substitution of an inert (ethylene glycol-ethylene glycol monethyl ether) dehydration technique did not alter the final result. Previous studies in our laboratory using high resolution SEM and correlative transmission microscopy of isolated nuclei have demonstrated that the shrinkage represents a miniaturization of the organelles in which all structural components retain their usual relationships.     

Fabrication and characterization of three-dimensional poly(ether- ether- ketone)/-hydroxyapatite biocomposite scaffolds using laser sintering

The ability to have precise control over porosity, scaffold shape, and internal pore architecture is critical in tissue engineering. For anchorage-dependent cells, the presence of three-dimensional scaffolds with interconnected pore networks is crucial to aid in the proliferation and reorganization of cells. This research explored the potential of rapid prototyping techniques such as selective laser sintering to fabricate solvent-free porous composite polymeric scaffolds comprising of different blends of poly(ether-ether-ketone) (PEEK) and hydroxyapatite (HA). The architecture of the scaffolds was created with a scaffold library of cellular units and a corresponding algorithm to generate the structure. Test specimens were produced and characterized by varying the weight percentage, starting with 10 wt% HA to 40 wt% HA, of physically mixed PEEK-HA powder blends. Characterization analyses including porosity, microstructure, composition of the scaffolds, bioactivity, and in vitro cell viability of the scaffolds were conducted. The results obtained showed a promising approach in fabricating scaffolds which can produce controlled microarchitecture and higher consistency.     

RGD功能化HA/PLLA多孔复合材料的制备和表征

通过碳二亚胺法将精氨酸-甘氨酸-天冬氨酸(RGD)接枝到羟基磷灰石(HA)颗粒表面,增强HA颗粒识别细胞的功能,然后将其均匀地分布于左旋聚乳酸(PLLA)中制备多孔复合材料,分别用XPS和SEM等对HA颗粒和多孔复合材料进行了表征。结果表明:RGD成功地接枝到了HA颗粒表面,多孔复合材料中RGD功能化的HA颗粒是纳米尺寸的,且分布均匀;RGD功能化HA/PLLA多孔复合材料的细胞粘附率从普通HA/PLLA多孔复合材料的37.21%提高到了69.11%。     

A simple and rapid scanning electron microscope preparative technique for delicate "gymnodinioid" dinoflagellates

Light microscopy (LM) is routinely used to investigate delicate (unarmoured and lightly armoured) "gymnodinioid" dinoflagellate species but at this level of resolution, morphological features such as apical grooves, apical pores, thin thecal plates, and scales are often difficult to observe, thereby necessitating the use of scanning electron microscopy (SEM). Good results were obtained when harvested cells were fixed with osmium tetroxide (OsO(4)) as the primary fixative, adhered with poly-L-lysine to round glass coverslips, dehydrated in an ethanol series, and dried with hexamethyldisilazane (HMDS). Poly-L-lysine has in the past effectively been used to adhere biological material such as human red blood cells, mouse leukemic cells, and marine dinoflagellates to glass coverslips. HMDS has been used to substitute critical point drying (CPD) to dry soft insect tissues, rat hepatic endothelial cells, and the cilia of rat trachea. By combining and fine-tuning these two protocols in SEM studies of delicate "gymnodinioid" dinoflagellates, it is possible to overcome cell distortion such as shrinking and collapsing that result from centrifuging, filtering, and CPD. The combination of poly-L-lysine and HMDS not only produces good results but also requires limited expertise and equipment, is inexpensive, and is less time-consuming.     

Electron microscopy of interfaces in a wollastonite — tricalcium phosphate bioeutectic®

The study investigated the morphology and structure of interfaces between a pseudowollastonite (psW) and α-tricalcium phosphate (α-TCP) eutectic ceramic material (Bioeutectic®) obtained by slow solidification (0.5 °C h⁻¹) through the eutectic temperature region (1402 ± 3 °C) of the binary system. The eutectic material contained 60 wt% psW and 40 wt% α-TCP and was composed of spherical colonies formed by alternating radial lamellae of psW and α-TCP phases. Small quantities of impurities were found to affect the microstructure and ultrastructure of adjacent colonies. The observations showed the presence of dislocation loops and dislocation pairs in the psW phase away from the interfaces with α-TCP. The α-TCP phase remained defect-free. Two types of interface were identified between the triclinic high-temperature form of wollastonite (psW) and orthorhombic α-TCP. In single colonies, the phases were aligned with [011¯]psW and [001¯] α-TCP. High-resolution electron microscopy revealed differences in morphologies between the two interfaces, and showed that the lattice planes in the interfaces were defect-free.     

Air-drying of human leucocytes for scanning electron microscopy using the GTGO procedure

The utilization of tannic acid and guanidine hydrochloride as mordants for better osmium binding has been shown to serve as an excellent alternative to metal coating of organ tissue specimens for scanning electron microscopy (SEM). The present report describes the GTGO procedure, a modification of the TAO technique introduced by Murakami et al. (1977, 1978), which we have found successful for the preparation of air dried peripheral blood leucocytes for SEM studies. Air dried, GTGO-treated leucocytes show excellent preservation of surface features with minimal cell shrinkage. When critical point dried, GTGO-treated cells are examined, they also show less shrinkage than cells prepared with standard glutaraldehyde fixation and critical point drying. The potential application of this air drying procedure (GTGO-AD) to other soft biological specimens is currently under investigation. This technique is recommended as a new and effective air drying procedure for the successful preparation of cells for SEM.     

多孔氧化锆基体上涂覆羟基磷灰石涂层材料的制备和性能

用凝胶成型法制备了三维网状开孔结构的多孔氧化锆(ZrO2)支架材料,通过改变浸渍循环次数实现了对材料孔隙率的控制;然后在多孔ZrO2支架的表面涂覆了羟基磷灰石(HA)涂层,中间涂覆了氟磷灰石(FA)层,用XRD对其进行了表征,并进行了体外模拟试验。结果表明:加入FA中间层以后,防止了HA和ZrO2之间的化学反应,提高了涂层性能;涂覆了HA的多孔ZrO2材料的强度是多孔HA材料强度的7倍多;这种有涂层的多孔支架材料很适合细胞的生长,具有良好的生物相容性。     

Fabrication and mechanical properties of PLLA and CPC composite scaffolds

The brittleness and insufficient strength of biomaterials such as calcium phosphate cement (CPC) limit their applications in physiologically non-load-bearing bone lesions. These limitations stimulated the research for developing degradable polymer-ceramic composite materials that can closely match the modulus of bones. In this study, poly (L-lactic acid)/calcium phosphate cement (PLLA/CPC) composite scaffolds were fabricated via a four-step process, namely, measurement, prototyping, compounding, and dissolving. The design and mechanical properties of the PLLA/CPC composite structures were theoretically and experimentally studied. The PLLA/CPC scaffold improved the mechanical properties of the CPC. The CPC??s compressive strength and strengthening percentage increase with higher PLLA volume. Such composites may have a clinical use for load-bearing bone fixation.     

孔隙率可控的多孔羟基磷灰石生物陶瓷的制备

采用凝胶成型法制备用作骨移植和药物传递的多孔羟基磷灰石(HA)生物医用陶瓷,通过改变工艺参数制得了孔隙率和孔径可控、内部连通的三维网状开孔结构的HA,对其孔隙率、孔径及生物相容性进行了分析.结果表明:通过改变循环浸渍次数可以控制调节孔隙率在45%～92%之间;通过改变母体模板和浆料涂层的厚度可以控制孔径;制得的HA具有良好的生物相容性,具有这种特点的孔洞结构有利于骨细胞的生长.     

Ionic liquid enables simple and rapid sample preparation of human culturing cells for scanning electron microscope analysis

Ionic liquid is a kind of salt that stays in a molten state even at room temperature. It does not vaporize at all in vacuum and facilitates electrical conductivity to the sample surfaces for observations with a scanning electron microscope (SEM). In this study, we used an ionic liquid in SEM for the first time to observe fixed human culture cells. The condition for the cell culture using wrapping sheets and SEM settings were varied to elucidate the optimized protocol. Compared to samples prepared by the conventional way, the ionic liquid‐treatment of samples gave SEM images of the cellular ultra structures in more detail, enabling observation of microvilli that made bridges between separated cells. In addition, the ionic liquid treatment is less time consuming as well as less laborious compared with the conventional way that includes dehydration, drying, and conductivity treatments. Totally, we concluded the ionic liquid is a useful reagent for SEM sample preparation. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Early results using high-resolution,low-voltage,low-temperature SEM

Recent advances in the design of the scanning electron microscope (SEM) column, such as the coupling of a field-emission gun to a low-aberration immersion lens and the availability of a high-stability cryo-transfer stage, make low-temperature, low-voltage SEM (LTLVSEM) possible at very high resolution. We have used this combination to obtain results with uncoated biological specimens. The trichocyst from a Paramecium was used as a test specimen to observe the shrinkage of this structure as the temperature is raised from 170 K to room temperature following freeze-drying. High-magnification stereo images were obtained of trichocysts that had been prepared by freezing, freeze-substitution and critical-point drying and which were subsequently viewed by LTLVSEM to reduce beam damage and contamination.     

Ultrastructural evaluation of mesenchymal stem cells from inflamed periodontium in different in vitro conditions

This research aimed to observe the behavior of mesenchymal stem cells (MSCs) isolated from periodontal granulation tissue (gt) when manipulated ex vivo to induce three‐dimensional (3D) spheroid (aggregates) formation as well as when seeded on two bone scaffolds of animal origin. Periodontal gt was chosen as a MSC source because of its availability, considering that it is eliminated as a waste material during conventional surgical therapies. 3D aggregates of cells were generated; they were grown for 3 and 7 days, respectively, and then prepared for transmission electron microscopic analysis. The two biomaterials were seeded for 72 h with gtMSCs and prepared for scanning electronic microscopic observation. The ultrastructural analysis of 3D spheroids remarked some differences between the inner and the outer cell layers, with a certain commitment observed at the inner cells. Both scaffolds showed a relatively smooth surface at low magnification. Macro‐ and micropores having a scarce distribution were observed on both bone substitutes. gtMSCs grew with relative difficulty on the biomaterials. After 72 h of proliferation, gtMSCs scarcely covered the surface of bovine bone scaffolds, demonstrating fibroblast‐like or star‐like shapes with elongated filiform extensions. Our results add other data on the possible usefulness of gtMSC and could question the current paradigm regarding the complete removal of chronically inflamed gts from the defects during periodontal surgeries. Until optimal protocols for ex vivo manipulation of MSCs are available for clinical settings, it is advisable to use biocompatible bone substitutes that allow the development of progenitor cells. Microsc. Res. Tech. 78:792–800, 2015. © 2015 Wiley Periodicals, Inc.     

仿生多材料复合增强骨软骨支架的制造及性能研究

针对关节面上大面积骨软骨缺损修复过程中软骨形态恢复和力学环境恢复困难的问题,设计并制造一种新型聚乙二醇(Polyethylene glycol, PEG)/ 聚乳酸(Polylactide, PLA)/ β-磷酸三钙(β-Tricalcium phosphate, β-TCP)仿生多材料复合增强骨软骨支架。基于CT扫描数据重建的羊膝关节模型上进行仿生多材料骨软骨支架的结构设计,包括多孔定制结构和固定桩及仿生结构;以光固化成形技术与真空灌注工艺相结合制造了的多材料复合增强骨软骨支架,确定灌注温度220℃,真空度–0.08～–0.10 Pa。形貌观测表明真空灌注法能使PLA完全充满整个次级管道,力学试验发现复合材料支架的压缩强度(21.25 MPa ± 1.15 MPa)是单管道多孔生物陶瓷支架(9.76 MPa± 0.64 MPa)的2.17倍, PLA固定桩的剪切强度(16.24 MPa±1.85 MPa)是陶瓷固定桩(0.87 MPa±0.14 MPa)的18.7倍。因此,复合PLA的骨软骨支架具有显著的力学增强和固定能力,有望为大面积骨软骨缺损的修复提供新的治疗手段。