Glycol methacrylate embedding in histotechnology: factors which influence the evolution of heat during polymerization at room temperature

Benzoyl peroxide is widely applied as an initiator–accelerator system in combination with tertiary amines for the polymerization of water-miscible methacrylates. However, this system is very sensitive to the ambient temperature and the evolution of exothermic heat is, especially at room temperature (293 K), difficult to control. Temperatures involved may increase beyond 373 K. A linear correlation between a graded series of benzoyl peroxide (0.2–1.0 g% benzoyl peroxide/glycol methacrylate) and the maximum temperature within the polymerizing plastic was established using a commercially-available embedding system. Commercially-available glycol methacrylates contain various concentrations of inhibitor in order to prevent spontaneous polymerization of the monomer. We tested a series of 200–300–400–800 ppm of the inhibitor hydroquinone and found a significant influence exerted upon the maximum temperature and the moment at which the maximum temperature was reached. Supplementary addition of inhibitors to the GMA monomer may effect a decrease of the maximum temperature within polymerizing GMA mixtures. Possibilities for standardizing the polymerization of glycol methacrylate by means of the benzoyl peroxide/tertiary amine initiator–accelerator system at room temperature are discussed.     

A new,less toxic polymerization system for the embedding of soft tissues in glycol methacrylate and subsequent preparing of serial sections

The paper describes a new polymerization system for embedding soft tissues in glycol methacrylate (GMA). The polymerization of GMA is initiated by means of a barbituric acid derivative in combination with chloride ions and dibenzoyl peroxide. The catalyst system contains no aromatic amines which constitutes a toxicological advantage over the commonly employed system of peroxide/aromatic amine. Clear blocks are obtained from which 1–2 μm sections are easy to cut. In combination with an appropriate softener, polyethylene glycol 400 , serial sectioning may be practised.     

A simple,practical ‘swiss roll’ method of preparing tissues for paraffin or methacrylate embedding

A simple method of preparing ‘swiss rolls' from strips of tissue up to 500 mm in length is described. This procedure has been applied to a variety of tissues and is suitable for the preparation of both paraffin- and methacrylate-embedded specimens.     

Low‐temperature glycol methacrylate resin embedding method: A protocol suitable for bone marrow immunohistochemistry,PCR, and fish analysis

Molecular analyses such as fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) are demanded to improve diagnostic accuracy in addition to immunohistopathology of bone marrow (BM) trephine specimens. Conventional BM embedding method needs decalcification, and its procedure may impair tissue morphology and DNA quality. Here, we report an undecalcified method by which glycol methacrylate resin is polymerized at low temperature (4°C). Using this method, BM enzyme activity and antigenic determinants are well preserved, and moreover, DNA extracted from plastic embedding sections is suitable for PCR amplification and sequencing, FISH analysis can be well done because of the DNA integrity of BM sections. If working with BM trephine specimen, our protocol offers the possibility to combine superior morphology with modern molecular analysis. Microsc. Res. Tech. 73:1067–1071, 2010. © 2010 Wiley‐Liss, Inc.     

Spatial and temporal changes of subchondral bone proceed to articular cartilage degeneration in rats subjected to knee immobilization

This study was aimed to investigate the spatial and temporal changes of subchondral bone and its overlying articular cartilage in rats following knee immobilization. A total of 36 male Wistar rats (11–13 months old) were assigned randomly and evenly into 3 groups. For each group, knee joints in 6 rats were immobilized unilaterally for 1, 4, or 8 weeks, respectively, while the remaining rats were allowed free activity and served as external control groups. For each animal, femurs at both sides were dissected after sacrificed. The distal part of femur was examined by micro‐CT. Subsequently, femoral condyles were collected for further histological observation and analysis. For articular cartilage, significant changes were observed only at 4 and 8 weeks of immobilization. The thickness of articular cartilage and chondrocytes numbers decreased with time. However, significant changes in subchondral bone were defined by micro‐CT following immobilization in a time‐dependent manner. Immobilization led to a thinner and more porous subchondral bone plate, as well as a reduction in trabecular thickness and separation with a more rod‐like architecture. Changes in subchondral bone occurred earlier than in articular cartilage. More importantly, immobilization‐induced changes in subchondral bone may contribute, at least partially, to changes in its overlying articular cartilage. Microsc. Res. Tech. 79:209–218, 2016. © 2016 Wiley Periodicals, Inc.     

Friable rock embedding in methyl methacrylate for thin-sectioning in geological preparations

In most cases, technical problems prevent the production of good geological thin-slabs from friable rocks such as muds, sands or decomposed granites. Usual impregnation methods using Araldite or epoxies lead to incompletely impregnated blocks. Furthermore the excessive heat occurring during polymerization alters the micromorphology of heat sensitive minerals. Methyl methacrylate (MMA) is a widely used plastic for bone embedding. We have developed a new procedure using vacuum infiltration with MMA in a cold environment. Wafers from friable rocks are easily obtained and can be processed in the same way as those of firm and coherent rocks, without grain loss nor mineral heat alterations.     

Temperature control in Lowicryl K4M and glycol methacrylate during polymerization: is there a low-temperature embedding method?

An apparatus for embedding tissues at resin temperatures down to 228 K is described. By placing thermocouples in the resin the temperature has been monitored during embedding at low temperature with glycol methacrylate (GMA) and Lowicryl K4M. Even in this apparatus with a liquid cooling bath the heat of polymerization is not dissipated and the resin temperature rises. This rise is directly proportional to the resin temperature at the onset of polymerization and is higher in Lowicryl K4M than GMA. The initial resin temperature also affects the time taken for polymerization. The time to the onset of the peak and its duration are both increased as the temperature is lowered. This effect is more pronounced with GMA than Lowicryl K4M and polymerization of GMA is inhibited at the lowest temperature used. When Lowicryl K4M, polymerized at low temperature, is warmed up to ambient a further exothermic reaction occurs, which causes the resin temperature to rise well above ambient. Both this temperature peak and that during polymerization are reduced, but not totally eliminated, by reducing the resin volume. Aircooled systems are inefficient compared with the low-temperature apparatus used here and the resin temperature rise is consequently greater and, even with small resin volumes, it can be very high. It is therefore unlikely for published methods that the temperature specified has been maintained in the resin during polymerization. The implications of these findings are discussed in relation to enzyme and antigen survival. Recommendations include use of very small volumes of resin, refrigerated liquid-bath rather than air-cooled systems and contact with a heat sink when specimens are warmed up to ambient temperature. Examples of enzyme reaction, antigen survival and structural preservation obtained with the method are presented.     

Immunohistochemical processing of tissue which allows for embedding in Araldite®, and orientation and correlation between light and electron microscopic staining

This paper describes a simple and efficient method for the immmunohistochemical processing of tissue sections on glass slides and the subsequent transfer of the entire section to an Araldite® medium. These sections present in one plane can then be orientated and when cut provide the user with whole tissue, free from gaps due to folding. This procedure therefore enables the changes in protein concentration and distribution to be assessed and correlated within and between different cells at the light and electron microscopic level. This procedure may be of use in other microscopic techniques studies.     

A comparative study of softeners and catalyst systems upon dimensional changes and sectioning quality of glycol methacrylate sections

It is shown that softeners and temperature have an important influence upon the stretching parameters of glycol methacrylate sections. Also it is demonstrated that the catalyst system used may significantly interfere with section stretching.     

Preparation of rodent kidney for light microscopy by freeze-drying and embedding in water-soluble methacrylate

Methods for the elimination of five main artifacts common to frozen-dried specimens embedded in methacrylate are described in detail. Two of these are associated with the drying technique and three to the methacrylate embedding process. One of the “drying” artifacts, contamination of the tissues by pump oil, can be overcome by inserting an oil trap between the vacuum pump and the freeze-dryer. The other, “zoning” in the specimens, is shown to be due to incomplete drying. Using an Edwards TD2 or TD4 freeze dryer a point in the drying process is reached when the pressure exerted by the water extracted from the specimens equals that exerted by the residual water within the specimens. Drying then ceases and cannot be completed merely by prolonging the procedure. To complete drying, the tissues are transferred to a second clean drying head and the process is repeated. Good impregnation of tissues is achieved by evacuating them in a chamber before adding the methacrylate, which is then allowed to penetrate the tissues at atmospheric pressure. This calls for a viscous methacrylate such as the glycol compound. The rate of penetration varies greatly for different tissues and methacrylates and a glycol/butyl methacrylate mixture penetrates frozen-dried kidney very slowly at a rate of about 1 mm in twelve hours. The remaining artifacts are ballooning of the tissues, leaching out of material from them, and bubbling of the methacrylate. The properties of glycol, butyl, and methyl methacrylates, alone and in mixtures, have been studied in association with different catalyst concentrations. A mixture of 85 p.c. distilled glycol and 15 p.c. distilled butyl methacrylate with 0.5 p.c. benzoyl peroxide catalyst added, satisfies the vacuum embedding requirements, obviates ballooning of tissues and leaching out of material from them, does not bubble and produces a block with an acceptable cutting texture. It is necessary to polymerize some of the mixture to within ½ hour of completion for casting the final block.     

Mechanical and tribological properties of poly(hydroxyethyl methacrylate) hydrogels as articular cartilage substitutes

Poly(hydroxyethyl methacrylate) (p(HEMA)) hydrogels have been proposed as promising biomaterials to replace damaged articular cartilage. A major obstacle to their use as replacement bearing tissue is their poor mechanical properties in comparison with healthy articular cartilage. The purpose of this study was to obtain p(HEMA) hydrogels with physicochemical and mechanical properties close to healthy articular cartilage, by introducing a hydrophilic monomer, namely acrylic acid (AA). Formulations of hydrogels with different amounts of hydrophilic monomer (acrylic acid, AA) were synthesized and tested: p(HEMA), p(HEMA-co-5%AA), p(HEMA-co-25%AA). The macro-mechanical tests were reproduced at nanoscale in order to verify if the superficial properties of the hydrogels are similar to the bulk ones.     

Quantitative zonal differentiation of articular cartilage by microscopic magnetic resonance imaging,polarized light microscopy,and Fourier‐transform infrared imaging

This study aimed to synchronize the zonal differentiation of the full‐thickness articular cartilage by three micro‐imaging techniques, namely microscopic magnetic resonance imaging (µMRI), polarized light microscopy (PLM), and Fourier‐transform infrared imaging (FTIRI). Eighteen cartilage‐bone blocks from three canine humeral joints were imaged by: (a) µMRI T₂ relaxation at 0° and 55° orientations in a 7 T magnetic field, (b) PLM optical retardation and azimuthal angle, and (c) FTIRI amide I and amide II anisotropies at 0° and 90° polarizations relative to the articular surface. In addition, µMRI T₁ relaxation was imaged before and after the tissue being immersed in gadolinium (contrast agent) solution, to calculate the proteoglycan concentration. A set of previously established criteria in cartilage imaging was revised. The new criteria could simultaneously correlate the thicknesses of the three consecutive subtissue zones in articular cartilage among these imaging techniques. Microsc. Res. Tech. 76:625–632, 2013. © 2013 Wiley Periodicals, Inc.     

The effects of glycol methacrylate as a dehydrating agent on the dimensional changes of liver tissue

The dimensional changes of liver sections during the course of processing with glycol methacrylate (GMA) or with ethanol are described. Tissue processing with ethanol served as a control. During prolonged processing steps (24 h each), linear shrinkage of tissue specimens dehydrated with GMA at room temperature was 13.2%. Subsequent infiltration with GMA resulted in trivial swelling, and polymerization in slight shrinkage (2.3%). In comparison, processing with cold GMA resulted in shrinkage during dehydration (about 10.8%), a slight swelling in pure GMA, followed by shrinkage during polymerization (2.2%). Short routine processing schedules resulted in similar shrinkage/swelling patterns, although precise values differed slightly. In all experiments, ethanolic dehydration resulted in smaller dimensional tissue changes than did GMA dehydration. The dimensional changes of tissue sections during stretching on water, mounting and drying compensated for the major part of the shrinkage manifested during processing.     

Long bone human anlage longitudinal and circumferential growth in the fetal period and comparison with the growth plate cartilage of the postnatal age

The patterns of longitudinal and peripheral growth were analyzed in human autopod cartilage anlagen (fetal developmental stage 20th–22nd week) through morphometric assessment of chondrocyte parameter size, shape, alignment and orientation between peripheral and central sectors of the anlage transition zone defined by primary ossification center and the epiphyseal basis. The aim was to correlate the chondrocyte dynamics with the longitudinal and peripheral growth. A further comparison was carried out between the corresponding sectors of the postnatal (3–5 months old) growth plate cartilage documenting: (1) the different chondrocyte framework and the new peripheral mechanism; (2) the opposite direction of fetal periosteal ossification versus the Lacroix bone bark. Measurement of multiple parameters (% lac area, % total matrix area, total lac density and mean single lac area), which characterize the cartilage Anlage growth, suggested the following correlations with chondrocyte duplication rate: (a) slow duplication rate ≈ coupled, intralacunar chondrocytes (in central epiphysis); (b) repeated/frequent cell duplications ≈ clusters (in the basal epiphyseal layer); (c) clusters of chondrocytes before becoming hypertrophic were stacked up on the top of each other (both in the Anlage transition zone or in the columns of metaphyseal growth plate); (d) enhanced osteoclastic resorption of the Lacroix bone bark lower end, extended to the more external metaphyseal trabeculae counterbalancing the discrepancy between the epiphyseal and the diaphyseal circumferential growth.     

High resolution microscopic survey of third metacarpal articular calcified cartilage and subchondral bone in the juvenile horse: possible implications in chondro-osseous disease

The aim was to survey articular calcified cartilage (ACC) and subchondral bone in the palmar and dorsal regions of the condyles of the third metacarpal bone (Mc3) of young horses with minimal or no signs of musculo-skeletal abnormality. Back-scattered electron scanning electron microscopy (BSE SEM) was conducted on polymethyl methacrylate-embedded mediolateral slices and macerated wedges of the right distal Mc3 from seven each of trained and untrained 2-year-old Thoroughbred horses. Furrows or grooves visible to the naked eye in the mineralizing front (MF) of ACC are the commonest "lesion" and are most common in the palmar portions of the medial and lateral condylar grooves. Cracks running predominantly in the parasagittal plane that infill with hypercalcified matrix are found in the same domain. Common to all these defects are deficiencies or absence of the ACC MF. Other anomalies include local excrescences or depressed areas of the MF. More important condylar lesions show displaced fragmented hypermineralized ACC with underlying excess resorption in the bone domain, leaving a thin ACC layer with cavernous space beneath it. The fragments may dislodge and displace to the joint space. Obvious although small lesions are present in horses that have undertaken little or no training. The nature and sites of the lesions indicate that they are possibly the earliest morphological evidence of changes that may lead to specific joint abnormalities. The lesions appear unlikely to be solely due to functional traumatic forces, and developmental influences are likely to be important in their initiation.     

An easy and versatile embedding method for transverse sections

In several research areas, transverse sections are indispensable for studying structural aspects of specimens. However, the oriented embedding of small cylindrical samples can become problematic, especially when transverse sections at right angles to the main axis of the object are desired. Here, we describe an easy and low‐cost technique for oriented embedding of small (? < 500 µm) as well as of larger specimens (? > 500 µm). The usefulness of the technique is demonstrated for roots and stamens of Arabidopsis thaliana and for adventitious roots of Asplenium demerkense, as examples of small and larger cylindrical samples, respectively. Furthermore, several types of resin (glycol methacrylate, epoxy and acrylic resins) were successfully tested, showing the applicability of the technique for light and electron microscopy and for immunolocalizations. In conclusion, the principle of the technique can be extended to several resins and a wide variety of specimen types, such as stems, leaves and textile fibres. The originality of the technique lies in its simplicity combined with its high efficiency to produce well‐oriented transverse sections.     

A comparison between micro-CT and histology for the evaluation of cortical bone: effect of polymethylmethacrylate embedding on structural parameters

Cortical bone microstructure is an important parameter in the evaluation of bone strength. The aim of this study was to validate the characterization of human cortical bone microarchitecture using microcomputed tomography. In order to do this, microcomputed tomography structural measurements were compared with those obtained through histological examination (the gold standard). Moreover, to calculate structural parameters, microcomputed tomography images have to be binarized with the separation between bone and nonbone structures throughout a global thresholding. As the effect of the surrounding medium on the threshold value is not clear, an easy procedure to find the global uniform threshold for a given acquisition condition is applied. This work also compared the structural parameters of microcomputed tomography cortical sample scan in air or embedded in polymethylmethacrylate; histology was used as a reference. For each acquisition condition, a fixed threshold value was found and was applied on the corresponding microcomputed tomography image for the parameters assessment. Twenty cortical bone samples were collected from human femur and tibia diaphyses. All samples were microcomputed tomography scanned in air, embedded in polymethylmethacrylate, rescanned by microcomputed tomography, examined by histology and finally compared. A good correspondence between the microcomputed tomography images and the histological sections was found. Paired comparisons in cortical porosity, Haversian canal diameter and Haversian canal separation between histological sections and microcomputed tomography cross sections, first in air and then embedded in PolyMethylMethAcrylate, were made: no significant differences were found. None of the comparisons showed significant differences for cortical porosity, Haversian canal diameter and Haversian separation over a three-dimensional volume of interest, between microcomputed tomography scans in air and with samples embedded in PolyMethylMethAcrylate. The very good correlation between bone structural measures obtained from microcomputed tomography datasets and from two-dimensional histological sections confirms that microcomputed tomography may be an efficient tool for the characterization of cortical bone microstructure. Moreover, when the corresponding threshold value for each condition is used, structural parameters determined by microcomputed tomography are not affected by the surrounding medium (PolyMethylMethAcrylate).     

Practical considerations in the use of polarized light microscopy in the analysis of the collagen network in articular cartilage

Polarized light microscopy is a traditional method for visualizing the collagen network architecture of articular cartilage. Articular cartilage repair and tissue engineering studies have raised new demands for techniques capable of quantitative characterization of the scar and repair tissues, including properties of the collagen network. Modern polarized light microscopy can be used to measure collagen fibril orientation, parallelism, and birefringence. New commercial instruments are computer controlled and the measurements are easy to perform. However, often the interpretation of results causes difficulties, even errors, because the theoretical aspects of the technique are demanding. The aim of this study was to describe the instrumentation and properties of a modern polarized light microscope, to point out some sources of error in the interpretation of the results, and to recall the theoretical background of the polarized light microscopy.