A technique for the quantification of the 3D connectivity of thin articulations in Bony sutures

The anatomy and development of cranial and facial sutures have been studied in detail using histological sections, 2D radiographs and more recently CT imaging. However, little attention has been paid to evaluating and quantifying the connectivity of these thin cortical bone articulations. More recent technological advances such as micro-CT imaging has the potential to be used to provide quantitative measurements of 3D connectivity in bony articulations. This study presents a new technique for quantifying the connectivity of bony projections inside cranial and facial sutures using a combination of skeletonization, thinning algorithms and 3D intensity mapping. The technique is demonstrated in five sutures through semi-automated analysis and image processing of μCT scans. In the sagittal, coronal and frontozygomatic sutures an average bone connectivity of 6.6–11.6% was found with multiple bony projections providing an interlocking structure between adjacent bones. Much higher bone connectivity was present in the zygomaticotemporal and zygomaticomaxillary sutures (22.7–37.4%) with few bony projections. This method combining μCT scanning and image processing techniques was successfully used to quantify the connectivity of thin bone articulations and allowed detailed assessment of sutural fusion in 3D. The wider application of this technique may allow quantification of connectivity in other structures, in particular fracture healing of long bones.     

Methods of histological treatment of the labyrinth]

The otic labyrinth presents one of the most difficult objects for histological investigations as it is situated in the thick layer of the temporal bone and has a very complex structure. At the same time, a method for treating this object is not given in general hand-books on pathological techniques. The methods for histological treatment of the otic labyrinth elaborated by Wittmaack and later on modified are presented in the article. Methods for fixation, vital fixation including, decalcination, dehydratation and saturation of the objects prepared for histological investigation are described in details. Methods for the temporal bone orientation in order to obtain sections of the internal otic structures in the most advantageous plane depending on the purpose and aim of the investigation are recommended.     

Diagnostic value of micro-CT in comparison with histology in the qualitative assessment of historical human skull bone pathologies

Cases of pathologically changed bone might constitute a diagnostic pitfall and frequently need histological methods to be etiologically properly evaluated. With micro‐computed tomography (μCT), a new epoch of 2D and 3D imaging has been launched. We evaluated the diagnostic investigation of this analytical method versus well established histological investigations of historical human bone. Pathological changes due to various etiologies (infectious, traumatic, endocrinological, neoplasia) observed in autopsy‐based macerated human skulls (Galler Collection, Natural History Museum Basel, Switzerland) were investigated by μCT and compared with histological thin ground sections using polarized light. Micro‐CT images visualize the architecture of the bone with high spatial resolution without preparation or destruction of the sample in the area to be sectioned. Changes in the bone surfaces as well as alterations of the diploë can be assessed. However, morphological patterns caused by reactive response, such as typical arrangements of collagen fibers, can only be visualized by the microscopic investigation of thin ground sections using polarized light. A great advantage of μCT is the high number of slices obtained so that spatial differences within the areas of the specimen become visible. Micro‐CT is a valuable tool for the diagnosis of vestiges of skull bone diseases. Its advantages over histology are the fast, automated image acquisition and the fact that the specimen is not completely destroyed. Only excision of the area to be scanned is necessary, if the specimen is too large to be scanned as a whole. Further, the 3D visualization of the micro‐architecture allows an easy orientation within the sample, for example, for the choice of the location of the histological slices. However, the need to differentiate woven from lamellar bone still makes histology an indispensable method. Am J Phys Anthropol, 2007. © 2007 Wiley‐Liss, Inc.     

Preparation of whole rabbit knee joints for microscopic examination

A paraffin embedding method to prepare whole rabbit knee joints for histological examination is described. This method provides good quality microscopic sections thin enough for the study of cellular detail and does not require prolonged processing. When examining pathologic changes in experimental arthritis, it is advantageous to be able to examine the intact joint with the structural relations of the joint components preserved. Sections of the whole joint provide numerous areas where bone, cartilage and synovium are contiguous for examination. Having obtained poor results using methods recommended for small bony specimens, we modified several existing procedures to obtain a reliable method for preparing excellent microscopic sections of the whole rabbit knee joint.     

Preparation of Whole Rabbit Knee Joints for Microscopic Examination

A paraffin embedding method to prepare whole rabbit knee joints for histological examination is described. This method provides good quality microscopic sections thin enough for the study of cellular detail and does not require prolonged processing. When examining pathologic changes in experimental arthritis, it is advantageous to be able to examine the intact joint with the structural relations of the joint components preserved. Sections of the whole joint provide numerous areas where bone, cartilage and synovium are contiguous for examination. Having obtained poor results using methods recommended for small bony specimens, we modified several existing procedures to obtain a reliable method for preparing excellent microscopic sections of the whole rabbit knee joint.     

A new method for identification of cement lines in undecalcified, plastic embedded sections of bone

A gallocyanin method for demonstrating cement lines in thin, undecalcified sections of bone has been developed that is compatible with prestaining with osteochrome before plastic embedding. After sectioning at 5 microns on the Jung K heavy duty microtome, the sections are attached to a microslide using Haupt's adhesive mounting medium, placed on a slide warmer at 37 C until completely dry, and deplasticized in xylene at 45 C for 16-24 hr. Sections are stained with 0.15% gallocyanin-5% chrome alum solution for 30 min, followed by staining in buffered Villanueva blood stain for 1-1 1/2 hr, quickly dehydrated, differentiated in equal parts xylene and 100% ethanol, cleared, and mounted in Eukitt's medium. Reversal lines appear as thin, scalloped, blue or purple lines approximately 0.3 micron wide, and arrest lines as thick, homogeneous, straight or evenly curved, dark blue or purple lines approximately 2 microns wide. The method also demonstrates abnormal halo volumes around osteocytes, old and new bone matrix, osteoid seams, and the granular mineralization front at the osteoid-bone interface. It promises to be valuable in the study of age-related bone loss, osteoporosis, and metabolic bone disease.     

A New Method for Identification of Cement Lines in Undecalcified, Plastic Embedded Sections of Bone

A gallocyanin method for demonstrating cement lines in thin, undecalcified sections of bone has been developed that is compatible with prestaining with ostcochrome before plastic embedding. After sectioning at 5 pm on the Jung K heavy duty microtome, the sections are attached to a microslide using Haupt's adhesive mounting medium, placed on a slide warmer at 37 C until completely dry, and deplasticized in xylene at 45 C for 16-44 hr. Sections are stained with 0.15% gallocyanin-5% chrome alum solution for 30 min, followed by staining in buffered Villanueva blood stain for 1-1 1/2 hr, quickly dehydrated, differentiated in equal parts xylene and 100% ethanol, cleared, and mounted in Eukitt's medium. Reversal lines appear as thin, scalloped, blue or purple lines approximately 0.3 pm wide, and arrest lines as thick, homogeneous, straight or evenly curved, dark blue or purple lines approximately 2 pm wide. The method also demonstrates abnormal halo volumes around ostcocytes, old and new bone matrix, osteoid seams, and the granular mineralization front at the osteoid-bone interface. It promises to be valuable in the study of age-related bone loss, osteoporosis, and metabolic bone disease.     

A simple method for preparing thin (10 microM) histological sections of undecalcified plastic embedded bone with implants

A simple method for preparing undecalcified thin sections of bone with implants has been developed. After exposing a surface of bone and implant in a plastic block by sawing thick sections, the surface is stained prior to making a thin section. A glass coverslip is affixed with a thin layer of cement to the stained surface to stabilize the tissue and implant during sectioning. A mixture of glycerine and water is used as a coolant and lubricant. The orientation in situ is preserved allowing demonstration of bone architecture and cells, and the tissue-implant interface.     

植入了骨修复材料的小型猪腰椎椎体不脱钙病理组织切片制备方法

摘要 目的：建立植入了骨修复材料小型猪腰椎椎体骨组织标本的不脱钙病理组织切片制备方法。方法：将含骨修复材料的腰椎椎体骨组织标本进行分割暴露组织切面,梯度浓度乙醇脱水后经Technovit 7200 VLC光聚树脂浸润,经黄蓝光共同辐照进行光聚合包埋,借助硬组织病理切磨系统制备含骨修复材料不脱钙病理组织切片。结果：结果显示通过上述方法制备的病理组织切片,经苏木精-伊红（HE）染色及甲苯胺蓝染色后光学显微镜下观察能较好地显示骨的各种组织细胞结构,可清晰的观察到骨小梁的走向及连接情况。结论：研究建立了含骨修复材料骨组织标本病理组织切片制备方法,实现了含骨修复材料不脱钙骨组织病理切片的制备,经病理染色后实现了带植入物的组织学观察,为生物材料及医疗器械动物试验研究提供了新的病理检测手段及组织学评价途径。     

A Simple Method for Preparing Thin (10 μm) Histological Sections of Undecalcified Plastic Embedded Bone with Implants

A simple method for preparing undecalcified thin sections of bone with implants has been developed. After exposing a surface of bone and implant in a plastic block by sawing thick sections, the surface is stained prior to making a thin section. A glass coverslip is affixed with a thin layer of cement to the stained surface to stabilize the tissue and implant during sectioning. A mixture of glycerine and water is used as a coolant and lubricant. The orientation in situ is preserved allowing demonstration of bone architecture and cells, and the tissue-implant interface.     

A Three-Dimensional Histological Method for Direct Determination of the Number of Trabecular Termini in Cancellous Bone

Osteoporotic fractures occur frequently in aging populations. Established methods for analyzing microarchitecture indicate that cancellous bone loss in the elderly is associated with progressive reduction in the connectivity of the trabecular network. This disconnection may explain the increased skeletal fragility that is sometimes out of proportion to the amount of bone lost. Connectivity, however, is difficult to measure and usually requires indirect methods. We describe development of a simple, inexpensive and direct procedure for counting sites of trabecular disconnection. The method is based upon preparation of 300-500 fjim thick slices of methylmethacrylate embedded material rather than the more usual thin 8 μm. histological sections. The marrow tissue is retained within the thick slice; this is essential for conservation of any detached bone fragments. In such preparations differential superficial staining of the upper and lower surfaces with alizarin red and light green, respectively, allows the two-dimensional image to be viewed at the same time as its three-dimensional counterpart. In this way, “real” (i. e., unstained) trabecular termini can be distinguished from “apparent” (i. e., stained red or green) termini that are artifacts of the plane of section. Partly polarized light enhances the microscope image. The method does not destroy the material for subsequent bone histomorphom-etry and, therefore, may be a useful adjunct to iliac bone biopsy analysis in studies of metabolic bone disease.     

Dry,High Resolution Autoradiography

Microtomed sections of freeze-dried, paraffin-embedded tissues are placed on pieces of thin sheet-Teflon backed by a felt pad. The sections are then pressure-mounted on dry photographic emulsion. After suitable exposure, the sections are firmly cemented to the emulsion with 0.45% cellulose acetate in a 10:1 mixture of 2-butanone and acetone. This prevents the specimens from falling off or moving during photographic processing, though the tissue can be stained through the cellulose acetate binder. The method has been tested with tissues containing tritium-labelled DNA, and it produced resolution comparable to that obtained with standard liquid emulsion or stripping film techniques.     

Dry, High Resolution Autoradiography

Microtomed sections of freeze-dried, paraffin-embedded tissues are placed on pieces of thin sheet-Teflon backed by a felt pad. The sections are then pressure-mounted on dry photographic emulsion. After suitable exposure, the sections are firmly cemented to the emulsion with 0.45% cellulose acetate in a 10:1 mixture of 2-butanone and acetone. This prevents the specimens from falling off or moving during photographic processing, though the tissue can be stained through the cellulose acetate binder. The method has been tested with tissues containing tritium-labelled DNA, and it produced resolution comparable to that obtained with standard liquid emulsion or stripping film techniques.     

A Procedure for Preparing Undecalcified and Unembedded Bone Sections for Light Microscopy

We have developed a procedure for light microscopic investigation of undecalcified and unembeddedbone sections. Biopsy samples of human metatarsus and femur and rat femur were fixed in aldehydes and sectioned with a cutting machine equipped with a diamond saw blade. Free sections 100-150 μm thick, stained with toluidine blue and von Kossa, did not show artifacts following the cutting, and the spatial relations of mineralized and nonmineralized components remained intact. Compact and trabecular bone, bone marrow and all cell types appeared well preserved and easily recognizable. Our procedure provides a simple and rapid method for preparing bone sections which undergo no chemical treatment other than fixation. This method is a useful alternative to standard histological protocols for studying bone specimens.     

Immunohistochemical demonstration of surface antigen of human lymphocytes with monoclonal antibody in acetone-fixed paraffin-embedded sections

Although the majority of reported studies have used fresh-frozen sections in detecting surface antigen of lymphocytes in tissue via monoclonal antibody, detailed histological figures can not be obtained by this method. Nor can the antigenicity be preserved for any length of time. A new method for detecting the surface antigen of lymphocytes using fixed and embedded material is presented. Human spleens were fixed in cold acetone, embedded in low melting point paraffin wax, and the thin sections treated with hyaluronidase. Anti-T lymphocyte monoclonal antibody (anti-Leu-1, anti-Leu-2, anti-Leu-3) and anti-HLA-DR were applied on these sections, and the antigen was detected by the ABC (avidin-biotin-peroxidase complex) method. The results were then compared with those of fresh-frozen sections. There was no great difference in detecting T and B cells or their subsets, but the histological figures were substantially better preserved in sections prepared by the present method. Furthermore, the antigenicity was retained in the materials fixed and embedded for more than two years.     

Cement Line Staining in Undecalcified Thin Sections of Cortical Bone

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 μm, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

Cement line staining in undecalcified thin sections of cortical bone.

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

Cement line staining in undecalcified thin sections of cortical bone

A technique for demonstrating cement lines in thin, undecalcified, transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

Improved Procedure for Histological Identification of Osteoid Matrix in Decalcified Bone

Several improvements on the original method of Yoshiki and coworkers for histological identification of osteoid matrix in decalcified bone are described in this report. The first, fixation of bone with neutral buffered formalin, a popular and stable fixative, should produce better tissue morphology and ensure easy handling in any laboratory. The second is a simple test for aged cyanuric chloride. Aged reagents show poor or no solubility in methanol and have almost no effect on differential staining of osteoid matrix. The third is an application of an organic acid solution in place of neutral EDTA for bone decalcification. Reduced decalcification time with the acid results in rapid preparation of bone sections. Neutral formalin fixation, immersion in the cyanuric chloride solution, decalcification with an organic acid, and hematoxylin and eosin staining, all quite routine laboratory procedures, yield high quality results for identification of osteoid matrix in bone sections.     

Histochemical and immunohistochemical protocols for routine biopsies embedded in Lowicryl resin

We describe a novel method that allows reliable detection of in situ hybridization signals in thin sections of plastic embedded embryos. Sections from plastic embedded embryos are thinner and have superior histological quality compared to paraffin, gelatin, agarose embedded sections or cryosections; however, plastic resin traditionally has not been used as an embedding medium following in situ hybridization because of loss of signal. When signal is detected with alkaline phosphatase and NBT/BCIP, the resulting colored precipitate is subject to fading when samples are exposed to organic compounds. The colored precipitate can be redeposited by repeating the NBT/BCIP reaction following plastic sectioning. This recolorization shows no loss of specificity, because signal is detected only where the anti-digoxigenin/alkaline phosphatase conjugated antibody is bound to the riboprobe. Strong signals can be detected without recolorization; however, weaker signals require the recolorization step. This novel method of re-depositing colored precipitate after processing and sectioning allows accurate determination of the location of gene expression and study of this expression in high quality histological sections of early chick embryos.