Atmospheric scanning electron microscopy and its applications for biological specimens.

Scanning electron microscopy in ambient conditions (Air-SEM) was developed recently and has been used mainly for industrial applications. We assessed the potential application of Air-SEM for the analysis of biological tissues by using rat brain, kidney, human tooth, and bone. Hard tissues prepared by grinding and frozen sections were observed. Basic cytoarchitecture of bone and tooth was identified in the without heavy metal staining. Kidney tissue prepared using routine SEM methodology yielded images comparable to those of field emission (FE)-SEM. Sharpness was lower than that of FE-SEM, but foot process of podocytes was observed at high magnification. Air-SEM observation of semithin sections of kidney samples revealed glomerular basement membrane and podocyte processes, as seen using conventional SEM. Neuronal structures of soma, dendrites, axons, and synapses were clearly observed by Air-SEM with STEM detector and were comparable to conventional transmission electron microscopy images. Correlative light and electron microscopy observation of zebrafish embryos based on fluorescence microscopy and Air-SEM indicated the potential for a correlative approach. However, the image quality should be improved before becoming routine use in biomedical research.

90,000 year-old specialised bone technology in the Aterian Middle Stone Age of North Africa.

The question of cognitive complexity in early Homo sapiens in North Africa is intimately tied to the emergence of the Aterian culture (~145 ka). One of the diagnostic indicators of cognitive complexity is the presence of specialised bone tools, however significant uncertainty remains over the manufacture and use of these artefacts within the Aterian techno-complex. In this paper we report on a bone artefact from Aterian Middle Stone Age (MSA) deposits in Dar es-Soltan 1 cave on the Atlantic coast of Morocco. It comes from a layer that can be securely dated to ~90 ka. The typological characteristics of this tool, which suggest its manufacture and use as a bone knife, are comparatively similar to other bone artefacts from dated Aterian levels at the nearby site of El Mnasra and significantly different from any other African MSA bone technology. The new find from Dar es-Soltan 1 cave combined with those from El Mnasra suggest the development of a bone technology unique to the Aterian.

Scanning Electron Microscopy-Energy-Dispersive X-Ray (SEM/EDX): A Rapid Diagnostic Tool to Aid the Identification of Burnt Bone and Contested Cremains.

This study investigates the use of Scanning electron microscopy-energy-dispersive X-ray (SEM-EDX) as a diagnostic tool for the determination of the osseous origin of samples subjected to different temperatures. Sheep (Ovis aries) ribs of two experimental groups (fleshed and defleshed) were burned at temperatures of between 100°C and 1100°C in 100°C increments and subsequently analyzed with the SEM-EDX to determine the atomic percentage of present elements. Three-factor ANOVA analysis showed that neither the exposure temperature, nor whether the burning occurred with or without soft tissue present had any significant influence on the bone's overall elemental makeup (p > 0.05). The Ca/P ratio remained in the osseous typical range of between 1.6 and 2.58 in all analyzed samples. This demonstrates that even faced with high temperatures, the overall gross elemental content and atomic percentage of elements in bone remain stable, creating a unique "fingerprint" for osseous material, even after exposure to extreme conditions.

Detection of glass particles on bone lesions using SEM-EDS.

The problem of identifying the wounding agent in forensic cases is recurrent. Moreover, when several tools are involved, distinguishing the origin of lesions can be difficult. Scanning electron microscopy (SEM)/energy dispersive X-ray analysis (EDS) equipment is increasingly available to the scientific and medical community, and some studies have reported its use in forensic anthropology. However, at our knowledge, no study has reported the use of SEM-EDS in forensic cases involving glass tools, whether in case reports or experiments. We performed an experimental study on human rib fragments, on which we manually created wounds using fragments of window and mirror glass. SEM-EDS was executed on samples without any further preparation on low vacuum mode, then on the same samples after defleshing them completely by boiling them. Window and mirror glass particles were detected on experimental wounds. Both had silica in their spectra, and the opaque side of the mirror contained titanium, allowing for their identification. Boiling and defleshing the bone samples involved a loss of information in terms of the number of wounds detected as positive for glass particles and in the number of glass particles detected, for both window and mirror glass. We suggest the analysis of wounds with suspected glass particles using low vacuum mode and with no defleshment by boiling.

Morphological characteristics of osteotomies using different piezosurgical devices. A scanning electron microscopic evaluation.

PURPOSE: The objective of this study was to compare morphological characteristics of osteotomies performed by 6 Piezosurgical devices. MATERIALS AND METHODS: The 6 Piezosurgical units were: (a) Piezotom, (b) SurgySonic, (c) Piezon Master Surgery, (d) VarioSurg, (e) Surgybone, and (f) Piezosurgery 3. Osteotomies on 9 freshly slaughtered cattle ribs (2 cuts by each unit, per rib) from the cortical (first cut at 5 mm) to the cancellous (second cut at 3 mm) bone layer were performed. The osteotomy margins were compared using scanning electron microscopy analysis. The cutting areas, osteotomy bottoms, and osteotomy margins were analyzed morphologically. Statistical evaluation of the 2 cuts regarding the design of the tips (a-d: tapered tool shanks, f: parallel tool shank) was performed by an unpaired t test. RESULTS: Morphological characteristics were different for each Piezosurgical unit and each examined area. A significant difference (P = 0.0209) of the upper width of the first cut between tapered and parallel tips was shown. CONCLUSIONS: The morphological characteristics of the produced Piezosurgical osteotomies vary and depend on the Piezosurgical unit and tip.

Rib cartilage characterization in patients affected by pectus excavatum.

Pectus excavatum (PE) is the most frequent anterior chest deformity which may be frequently associated with connective tissue disorders. We performed microscopic analyses to better understand cartilage behavior and obtain clues on its pathogenesis. In 37 PE patients, none with Marfan syndrome, we analyzed costal cartilage by light microscopy, immunohistochemistry and transmission electron microscopy. Control tissue specimens were harvested from four patients without any connective tissue disease. In both control and PE patients, chondrocytes were on the average <15 µm in diameter and occupied <10% of tissue volume; in most cases the extracellular matrix was stained by alcian blue, instead of safranin; no difference between PE and control samples was significant. All samples showed an uneven collagen type II immunolabeling both within the cells and pericellular matrix, and occasionally of the territorial matrix. In all cases numerous cells underwent apoptosis accompanied by matrix condensation as shown by electron microscopy. Our results suggest that matrix composition and the cell number and size of costal cartilage are dependent on the subject and not on the disease; the microscopic organization of cartilage is correlated with the stabilization of the defective shape rather than with the onset of the deformity.

Soft sheets of fibrillar bone from a fossil of the supraorbital horn of the dinosaur Triceratops horridus.

Soft fibrillar bone tissues were obtained from a supraorbital horn of Triceratops horridus collected at the Hell Creek Formation in Montana, USA. Soft material was present in pre and post-decalcified bone. Horn material yielded numerous small sheets of lamellar bone matrix. This matrix possessed visible microstructures consistent with lamellar bone osteocytes. Some sheets of soft tissue had multiple layers of intact tissues with osteocyte-like structures featuring filipodial-like interconnections and secondary branching. Both oblate and stellate types of osteocyte-like cells were present in sheets of soft tissues and exhibited organelle-like microstructures. SEM analysis yielded osteocyte-like cells featuring filipodial extensions of 18-20µm in length. Filipodial extensions were delicate and showed no evidence of any permineralization or crystallization artifact and therefore were interpreted to be soft. This is the first report of sheets of soft tissues from Triceratops horn bearing layers of osteocytes, and extends the range and type of dinosaur specimens known to contain non-fossilized material in bone matrix.

Decorin expression, straw-like structure, and differentiation of human costal cartilage.

Costal cartilage is much understudied compared with the load-bearing cartilages. Abnormally grown costal cartilages are associated with the inherited chest wall deformities pectus excavatum and pectus carinatum resulting in sunken and pigeon chests, respectively. A lack of understanding of the ultrastructural and molecular biology of costal cartilage is a major confounder in predicting causes and outcomes of these disorders. This study analyzed the structure of marginal human costal cartilage (ribs 6-10) through scanning electron and atomic force microscopes and identified the presence of straw-like structures running longitudinally. We also demonstrated that chondrocytes tend to occur singly or as doublets and that centrally located cells produce high levels of aggrecan compared with more peripherally located cells measured using immunohistochemistry. Gene expression from mRNA extracted from cartilage showed high levels of decorin expression, likely associated with the large, complex tubular structures running through this cartilage type. COL2A1, ACAN, and TIMP1 also showed higher levels of expression compared with ACTB. Analysis of gene expression ratios demonstrate that costal cartilage is under differentiated compared with published ratios for articular cartilage, likely due to the vastly different biomechanical environments of each cartilage type. Further studies need to establish whether findings described here from the costal margins are significantly different than the cartilage of the "true ribs" and how these values change with age.

The temperature of cremation and its effect on the microstructure of the human rib compact bone.

The presented study deals with the effect of the cremation temperature on the microstructure and morphology of the human compact bone. The biological material consisted of samples from ribs of recent Central European origin belonging to individuals of known age, sex and cause of death. Each bone sample was divided into several sections. One section remained unburned and the rest were burned at 700, 800 and 1000 degrees C. A few samples were burned also at the temperature of 600 degrees C. The undecalcified unstained ground cross-sections were made from burned and unburned bones; photographed and analysed using the SigmaScan Pro 5 programme. During burning, both the macroscopic and microscopic dimensions of the bone shrink, including the measures of the individual microstructures. The percentual representation of the area of individual microstructures on the area of the cross-section decreases. The number of individual microstructures per mm2 of the compact bone cross-section increases. Most microstructural variables demonstrated statistically significant differences at the individual temperatures of cremation. The burned bones showed a large scale of the colours, especially at 700 degrees C.

A four-stage method of age at death estimation for use in the subadult rib cortex.

Age estimation in the subadult skeleton can be rather precise when the epiphyses and dentition are present, but incomplete or commingled remains still present a challenge. Histomorphometric age-at-death estimation methods developed for use on adults are based on the age-associated accumulation of osteons. In the growing skeleton, there is a poor correlation between osteon numbers and age until the latter half of the second decade. As a result, there has been no histological aging method for use in subadults. The analysis of the rib cortex of 72 subadults ranging in age from 2 to 21 years has identified a series of developmental changes in the bone microstructure that can be used to estimate age. This qualitative method utilizes the systematic changes in rib cortical morphology to classify ribs into one of four age phases. This method can be applied to immature skeletons in forensic, archaeological, and paleontological contexts.

Application of fracture mechanics to failure in manatee rib bone.

BACKGROUND: The Florida manatee (Trichechus manatus latirostris) is listed as endangered by the U.S. Department of the Interior. Manatee ribs have different microstructure from the compact bone of other mammals. Biomechanical properties of the manatee ribs need to be better understood. Fracture toughness (K(C)) has been shown to be a good index to assess the mechanical performance of bone. Quantitative fractography can be used in concert with fracture mechanics equations to identify fracture initiating defects/cracks and to calculate the fracture toughness of bone materials. METHOD OF APPROACH: Fractography is a standard technique for analyzing fracture behavior of brittle and quasi-brittle materials. Manatee ribs are highly mineralized and fracture in a manner similar to quasi-brittle materials. Therefore, quantitative fractography was applied to determine the fracture toughness of manatee ribs. RESULTS: Average fracture toughness values of small flexure specimens from six different sizes of manatees ranged from 1.3 to 2.6 MPa(m)(12). Scanning electron microscope (SEM) images show most of the fracture origins were at openings for blood vessels and interlayer spaces. CONCLUSIONS: Quantitative fractography and fracture mechanics can be combined to estimate the fracture toughness of the material in manatee rib bone. Fracture toughness of subadult and calf manatees appears to increase as the size of the manatee increases. Average fracture toughness of the manatee rib bone materials is less than the transverse fracture toughness of human and bovine tibia and femur.

Haematopoiesis and a new mechanism for the release of mature blood cells from the bone marrow into the circulation in snakes (Ophidia).

This is the first description of haematopoiesis in snakes. Studies were carried out on the following species belonging to Ophidia: Bothrops jararaca, Bothrops jararacusu, Waglerophis merremii, Elaphe taeniura taeniura, Boa constrictor,and Python reticulatus. Smears of the peripheral blood and histological preparations from the vertebrae, ribs, liver, and spleen were studied under a light and electron microscope. Myeloid cells were present in the following locations in the vertebrae: the neural spine, zygoapophysial processes, floor of the neural canal, lacunae in the bodies of vertebrae and also inside the ribs. Although the vascular system was well developed, especially around the ribs, vessels inside the marrow cavities were scarce, both in the ribs and elsewhere where haematopoiesis was found. Venous sinuses were well developed in the vertebrae and in the rib regions from their costal head towards the middle area. They consisted of one layer of fine endothelial cells. Mature cells in the process of migration into the general circulation were only sporadically encountered when venous sinuses were studied on perfusion-fixed specimens. In contrast, almost every sinus venosus contained protrusions directed towards the lumen, filled mostly with mature and immature blood cells. Various stages of their formation were seen in the cross sections of venous sinuses ranging from small, newly formed to large, elongated ones, filled with many fully developed and some maturing blood cells. In many cases the apices of the protrusions were ruptured, and mature blood cells, as well as a few immature ones, were seen in their vicinity. This observation led us to a new hypothesis that blood cells are released from the extravascular space into the lumen of venous sinuses. In snakes, these cells are released into the systemic circulation mainly via the rupture of protrusions filled with mature blood cells and, to a lesser degree, by transcytosis as known in mammals. In the spleens from young specimens, 1-2 foci of haematopoiesis were encountered where lymphopoiesis predominated. Haematopoiesis was not detected in the liver.

[Ultrastructural changes of child rib cartilage in different deformations of chest cells]. / Osobennosti ul'trastrukturnykh izmenennii rebernogo khriashcha detei pri razlichnykh deformatsiiakh grudnoi kletki.

Different chest deformations are accompanied by appearance of asbestos-like fibrills in pericellular matrix, development of significant lipid and carbohydrate chondrocyte dystrophy, irreversible changes in their majority. Anomalous accumulation of intermediate filaments, lipid and glycogen, may substitute the cytoplasm organells in Marfan's syndrome. Pathologic disturbances of costal cartilage, connected with ultrastructural changes of chondrocytes, intercellular substance and extracellular matrix are morphologic substrate of different chest deformations.

[Structural organization of compact and cancellous tissue of human rib in aging]. / Strukturnaia organizatsiia kompaktnogo i gubchatogo veshchestva rebra cheloveka pri starenii.

Using the scanning electron microscopy the thickness of external and internal layers of compact bone of the rib as well as the structure of its cancellous bone was investigated in humans aged from 19 to 89 years. The differences of bone remodeling mechanism were found in men and women of the older age groups. In men, the gradual attenuation of osseous structures was taking place. In women, bone loss was found to be rapidly progressing due to the elimination of its structural elements (transformation of compact layer into cancellous bone and disappearance of some part of trabeculae from the latter).

The biomechanical, morphologic, and histochemical properties of the costal cartilages in children with pectus excavatum.

BACKGROUND/PURPOSE: The cause of the pectus excavatum (PE) remains unclear, although some results of research have indicated that the disturbance of the sternum or costal cartilage might be responsible for this deformity. But no decisive evidence has been gained. The authors have analyzed the biomechanical, morphologic, and histochemical properties of the cartilage in PE and intend to support the belief that the disturbance of the cartilage might contribute to the development of PE. METHODS: Thirty-eight specimens of the sixth cartilage were obtained at operation for the PE group (aged from 3 to 6 years; mean, 4.2 years). And 28 specimens of the control group (aged from 3 to 6 years; mean, 4.4 years) were gained from routine postmortem examinations in which the cause of death was unlikely to have affected the cartilage. The biomechanical test was carried out in a material testing machine (Shimadzu AG-10TA, Tokyo, Japan). The relation curve of load-deformation in tensile and compressive tests and the curve of load-time in the flexuous test were recorded automatically. The values of the ultimate strength and strain were calculated from this relation curve. The specimens also underwent H&E staining. The values of the area, circumference, mean diameter, maximal diameter, and morphologic factor of the cell and the nucleus of the cartilage in superficial and deep area were determined with the help of image analysis software (GT-2 model, China). The superficial zone (SZ) and deep zone (DZ) of the cartilage were examinated with electron microscopy (JEM-100SX, Japan). The distribution and intensity of type II collagen was shown by immunohistochemistry staining and analyzed with the image analysis software (GT-2 model, Huakang Co, Chengdu, China). The extent and distribution of proteoglycan were analyzed after Safranin-O and periodic acid shiff (PAS) staining. RESULTS: The mean strength of the costal cartilage in the experimental group was less than that in the control group in terms of tension, compression, and flexure (P <.05). The shape of the stress-strain curve for tension and compression in the experimental group was different from the control group. The fracture load in the experimental group was less than in the control group in tension (1.5 MPa versus 2.8 MPa) and in compression (.2 MPa versus 8.3 MPa). The time of fracture in experimental group was 30 seconds compared with 38 seconds in control group. No denaturation or necrosis could be found in light microscopical examination. There was no manifestation of hyperplasia or hypoplasia in the costal cartilage of the PE group. In SZ and DZ areas, the pattern and the number of mitochondria, endoplasmic reticulum, and Golgi in the experimental group were the same as the control group in transmission electron microscopy. Furthermore, the distribution and the number of proteoglycan in the 2 groups did not show a significant difference both in SZ and DZ areas. Although the distribution of the collagen in SZ areas was normal, this pattern was disturbed in DZ areas in the experiment group. The results of type II collagen immunohistochemistry examination was concordant with that change. No significant difference between control and experimental group could be seen in Safranin-O and PAS staining for proteoglycan. CONCLUSIONS: The biomechanical stability of the cartilage was decreased in the PE group. This might be caused by the disorderly arrangement and distribution of the collagen in the cartilage of PE patients. J Pediatr Surg 36:1770-1776.

Visualisation of three-dimensional microcracks in compact bone.

Microdamage in bone contributes to the loss of bone quality in osteoporosis and is thought to play a major role in both fragility and stress fractures (Schaffler et al. 1995). In this study, in vivo microcracks in human ribs were bulk-stained in basic fuchsin and viewed in longitudinal section and in 3 dimensions using 2 different computer-based methods of reconstruction: (1) serial sectioning of methylmethacrylate embedded sections using a sledge macrotome and identification of microcracks using UV epifluorescence followed by computerised reconstruction of microcracks using software and (2) laser scanning confocal microscopy of thick sections followed by reconstruction of microcracks into a 3-D image. The size and shape of microcracks were found to be similar using both techniques. Both techniques of reconstruction showed microcracks to be approximately elliptical in shape. From the serial sectioning reconstructions (n = 9), microcracks were found to have a mean length of 404 +/- 145 microm (mean +/- S.D.) (in the longitudinal direction) and mean width of 97 +/- 38 microm (in the transverse direction). Using epifluorescence microscopy, 92 microcracks were identified; mean microcrack length was 349 +/- 100 microm in the longitudinal direction. This was consistent with other results (Burr & Martin, 1993) and with the theoretical prediction of an elliptical crack shape with aspect ratio (longitudinal: transverse) of 5:1 deduced from analysis of random 2-D sections (Taylor & Lee, 1998). The results obtained provide new data on the nature of microcracks in bone and the method has the potential to become a useful tool in the calculation of stress intensity values which indicate the probability of an individual microcrack propagating to cause a stress or fragility fracture.

Bone turnover associated with antler growth in red deer (Cervus elaphus).

Although it is known that skeletal bone depletion occurs during antler growth in deer, it is not clear whether repletion of the skeleton takes place before or after completion of antler development. This study attempted to correlate repeated scanning electron microscopic measures of ilium and rib bone porosity from six approximately 2-monthly biopsy samples (using back-scattered imaging) and biochemical markers of bone turnover (serum hydroxyproline and osteocalcin concentrations) taken for 11 months with antler growth in six red deer stags. No changes were detected in ilium samples but changes in porosity of rib bones and an elevation of the biochemical markers indicated that skeletal depletion occurred during the antler growth period. However, the decrease in rib bone porosity and decline in markers of bone turnover took place before completion of antler growth, indicating that a considerable amount of skeletal repletion could have occurred whilst antlers were also undergoing bone accretion. This latter finding extends the current view of antler growth being accompanied by a form of reversible osteoporosis in the skeleton by showing that there is a period when the antlers and skeleton are both undergoing net bone formation.

Rib lesions and tuberculosis: the palaeopathological evidence.

SETTING: Tuberculosis diagnosis in past populations relies on lesions in the spine and major weight bearing joints of the body. Bone formation on visceral surfaces of ribs has also been suggested to be the result of chronic pulmonary disease. OBJECTIVE: To test whether these lesions are the result of pulmonary infection (most likely tuberculosis), by reviewing past work, and to discuss whether these lesions could be considered another diagnostic criterion for pulmonary tuberculosis. DESIGN: A review of the literature on new bone formation on ribs, and consideration of further evidence from archaeological skeletal material from the UK. RESULTS: Results from modern studies suggest that bone formation on ribs is often the result of pulmonary tuberculosis, that lesions are relatively common in archaeological skeletal material, and that some skeletons have rib lesions plus pathognomonic changes of tuberculosis. CONCLUSION: Evidence suggests that new bone formation on visceral surfaces of ribs should be considered a possible indicator of tuberculosis. If accepted, historical evidence, when correlated with rib data, produces closer approximations to the frequency of the disease in the past. This study indicates the importance of palaeopathology in identifying sometimes subtle lesions that may not be noted by clinicians because of their non-visibility on radiographs.

A comparative study of perichondrial tissue in mammalian cartilages.

The general organization, cellular and extracellular components, and structural variation of perichondrium have been studied in different mammalian cartilages by polarized light and transmission electron microscopy. The overall structure is that of a dense connective tissue composed of variable numbers of thin, stratified, closely-packed lamellae, themselves composed of closely-matted collagen fibres running in the plane of the cartilage surface, but oriented at various angles to each other. Variations mainly concern the arrangement of the fibre bundles in the transition zones between perichondrial and cartilage matrices, and between perichondrium and surrounding tissues. Perichondrial cells have the characteristics of fibrocytes. A cambial layer of undifferentiated stem cells was never observed. A layer of 'perichondrial lining cells' with distinctive ultrastructural characteristics was observed in some cartilage units, which separates the perichondrium from the surrounding loose connective tissue. The ultrastructural results demonstrate that the cartilage and perichondrial extracellular matrices are distinct, and what have been designated perichondrial 'transition' and 'proliferative' zones are in fact parts of the most superficial cartilage layer. Variations in perichondrial structure appear to correlate with diversity of cartilage function and we conclude that each cartilage unit plus perichondrium forms a tightly-integrated entity, best regarded as a unitary organ within the skeletal system.

Localization of collagens and alkaline phosphatase activity during mineralization and ossification of human first rib cartilage.

The localization of type X collagen and alkaline phosphatase activity was examined in order to gain a better understanding of tissue remodelling during development of human first rib cartilage. First rib cartilages from children and adolescents showed no staining for type X collagen and alkaline phosphatase activity. After onset of mineralization in the late second decade, a peripheral ossification process preceded by mineralized fibrocartilage could be distinguished from a more central one preceded by mineralized hyaline cartilage. No immunostaining for type X collagen was found in either type of cartilage. However, strong staining for alkaline phosphatase activity was detected around chondrocyte-like cells within fibrocartilage adjacent to the peripheral mineralization front, while a weaker staining pattern was observed around chondrocytes of hyaline cartilage near the central mineralization front. In addition, the territorial matrix of some chondrocytes within the hyaline cartilage revealed staining for type I collagen, suggesting that these cells undergo a dedifferentiation process, which leads to a switch from type II to type I collagen synthesis. The study provides evidence that mineralization of the hyaline cartilage areas in human first rib cartilage occurs in the absence of type X collagen synthesis but in the presence of alkaline phosphatase. Thus, mineralization of first rib cartilage seems to follow a different pattern from endochondral ossification in epiphyseal discs.