Back scattered electron (BSE) images of glasses and glass-ceramics with different degrees of bone-bonding behaviour have been investigated. After the implantation of probes in the femur of rats changes of the surface material, induced by leaching and corrosion (material response) and of the tissue in the drill hole (host response), were studied. The results demonstrate advantages of using BSE images and give further information for understanding the sequential changes in the implant material which occur mainly in bone-bonding materials. The density of the leached zone was not homogeneous and it therefore might be possible that preferential leaching of the glass matrix contributes to the mineralization of extracellular matrix. These findings could be of use for further development of surface reactive materials.

This study explored the microstructure of human cranial bone at different ages, and the survival, remodelling and modelling of cranial bone grafts. A combination of reflection and fluorescence confocal optical microscopy and scanning electron microscopy in the backscattered electron imaging mode was employed to examine highly polished block faces of plastic-embedded bone fragments as harvested for grafting, or recovered after a period in situ as a graft. The methods enabled remarkably detailed information on bone content, maturation and turnover to be gleaned from tiny scraps of bone. Microfractures in the harvested bone were repaired at the graft site, with welding of old and new bone indicating revascularization. Human cranial bone grafts successfully stimulated bone cell differentiation, supported new bone formation on resorbed and unresorbed surfaces, and underwent bone turnover. The type and organization of new bone reflected the growth rate and maturation of the graft rather than the age of the patient.

Octopus vulgaris drills holes in the shells of a variety of molluscs. The walls of the cavities drilled exhibit dissolution of mineral and organic material. The features which characterize the cavities have been described. The composition and structure of the shell itself is important in determining the size, shape and form of the cavity drilled, and not the size of the octopus. Capture, drilling the shell, and eating the occupant may take less than one hour.

The use of calcium phosphate biomaterials as a bone substitute necessitates the use of normative biocompatibility and biodegradation techniques which must be fast, simple and reproducible. In the present study, we have developed an in vitro model to study and to compare different calcium phosphate ceramics. After activation with 1,25-dihydroxy-vitamin D3 and phorbol 12,13-dibutyrate, the monoblastic U937 cells became multinucleated, expressed tartrate-resistant acid phosphatase and several markers of monocyte/macrophage differentiation. Activated U937 cells did not express the vitronectin receptor (VNR) (as revealed using monoclonal antibodies 23C6 or 13C2) but around 25% of the cells were strongly reactive with 211D, a novel monoclonal antibody that recognizes an osteoclast-specific membrane antigenic determinant. These cells remain active/viable with hydroxyapatite (HA) or β-tricalcium phosphate (β-TCP) ceramics. In conclusion, activated U937 cells are good candidates to use in a normative in vitro method to evaluate new biomaterials.

The effects of specific inhibitors of cysteine-proteinases [Z-Phe-Ala-CHN2: benzyloxycarbonyl-phenylalanyl alanyl diazomethane and E-64: trans-epoxysuccinyl-L-leucylamido (4-guanidino)-butane) and collagenase [Cl-1: N-(3-N-benzyloxycarbonyl amino-1-R-carboxypropyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide) have been tested on the osteoclastic resorption of dentine. Chick osteoclasts were cultured in the presence or absence of 12.5 μM Z-Phe-Ala-CHN2, 40 or 60 μM E-64, or 40 or 100 μM Cl-1 for 1 or 2 days. In addition, osteoclasts were cultured on oyster shell calcitostracum with or without 12.5 μM Z-Phe-Ala-CHN2. Specimens were studied by light microscopy to count cells and resorption features and by scanning electron microscopy (SEM) stereophotogrammetry for the measurement of the depths, plan-areas and volumes of resorption pits. The numbers, depths and volumes (but not the plan-areas) of the resorption pits in dentine were significantly reduced by Z-Phe-Ala-CHN2 and E-64. Thus, for a given plan-area, the volumes and the depths of resorption pits were smaller in these experimental groups compared with control dentine specimens. The overall inhibition of resorption was at least 75%. Cl-1 did not have this inhibitory effect on the numbers or sizes of resorption pits in dentine. When the oyster calcitostracum was used as a substrate for the osteoclasts, Z-Phe-Ala-CHN2 did not reduce the numbers or volumes of pits, but increased the planareas and prevented the formation of deeper pits. These results indicate that cysteine-proteinases participate in the resorption of adult calcified connective tissues by osteoclasts, but do not provide evidence for an obligatory role for collagenase in this process.

This article reviews the applications of SEM methods to human bone pathologies referring to studies made at UCL. We consider the methods which may be most suitable; these prove to be not "routine" in the context of most bio-medical applications of SEM. Valuable information can be obtained from a bone sample if its edges are ground flat, before making either a matrix surface preparation by washing away all the cells or a mineralizing front preparation, by also dissolving the osteoid-for which hydrogen peroxide is recommended to produce a robust specimen. BSE contrast from a cut block surface can be used to measure bone phase volume. SE contrasts from natural surfaces (trabeculae, canals and lacunae) can be used to study forming, resting and resorbing surfaces both qualitatively and quantitatively (except in the case of histological osteomalacia, where the existence of osteoid will go undetected and reversal lines will be difficult to distinguish from recently resorbed surfaces). We also recommend the use of PMMA embedded bone blocks, which can be used as obtained from the pathologist, but are better embedded by a more rigorous procedure. BSE image analysis can be used to quantitate bone density fractions opening up a completely new investigative method for the future. Osteoid can be measured automatically using CL if the bone sample is block stained with brilliant sulphaflavine before embedding or if a scintillant is added to the embeddant. We give examples of observations made from a number of bone diseases: vitamin D resistant rickets, osteogenesis imperfecta; osteomalacia; osteoporosis; hyperparathyroidism; fluorosis; Paget's disease; tumour metastasis to bone.

The assessment of in vitro osteoclastic activity has, until recently, been dependent on the analysis of organ culture experiments. We have developed a single cell resorption assay so that the resorptive function of individual osteoclasts could be studied. This paper examines the biological variation in the sizes of resorption lacunae produced by bone cell cultures derived from neonate rats and rabbits, and prehatch or hatchling chicks. Cultures were run for 24h for all species; and in addition for 48h for rat, 9 or 12 hours for rabbit and 3-7 hours for chick. The numbers of the nuclei of osteoclasts seeded on to plastic were counted for all three species. SEM stereophotogrammetry was used to measure areas, volumes, and maximum and average depths of the lacunae using specially designed instruments and software. Rat osteoclasts were smallest, and more chick osteoclasts were very large. There was a species difference in the onset of resorption and the sizes of pits produced, the chick osteoclasts being more vigorous resorbers than the rabbit ones, and the rat least so. For a given plan area, chick lacunae were deeper. There was a high correlation between area and volume. The range of maximum depths for a given area was high, however. Thus the mean of a few measurements of depths should not be used to calculate volume from area. At 24 hours, 77% of the rat, 47% of the rabbit and 28% of the chick lacunae were less than 1,000 microns 3 in volume; and 11% of the rat, 17% of the rabbit and 22% of the chick lacunae were between 1,000 and 2,000 microns 3 in volume. The mean values at 24 hours were 981, 2796, and 4582 microns 3 for rat, rabbit and chick lacunae respectively.

This article reviews the applications of SEM methods to human bone pathologies referring to studies made at UCL. We consider the methods which may be most suitable; these prove to be not "routine" in the context of most bio-medical applications of SEM. Valuable information can be obtained from a bone sample if its edges are ground flat, before making either a matrix surface preparation by washing away all the cells or a mineralizing front preparation, by also dissolving the osteoid-for which hydrogen peroxide is recommended to produce a robust specimen. BSE contrast from a cut block surface can be used to measure bone phase volume. SE contrasts from natural surfaces (trabeculae, canals and lacunae) can be used to study forming, resting and resorbing surfaces both qualitatively and quantitatively (except in the case of histological osteomalacia, where the existence of osteoid will go undetected and reversal lines will be difficult to distinguish from recently resorbed surfaces). We also recommend the use of PMMA embedded bone blocks, which can be used as obtained from the pathologist, but are better embedded by a more rigorous procedure. BSE image analysis can be used to quantitate bone density fractions opening up a completely new investigative method for the future. Osteoid can be measured automatically using CL if the bone sample is block stained with brilliant sulphaflavine before embedding or if a scintillant is added to the embeddant. We give examples of observations made from a number of bone diseases: vitamin D resistant rickets, osteogenesis imperfecta; osteomalacia; osteoporosis; hyperparathyroidism; fluorosis; Paget's disease; tumour metastasis to bone.

The first experimental studies concerning observations of changes in bone cell functional morphology were made using the SEM, and SEM has remained paramount in this field. Bone forming and resorbing cells only exist on surfaces – which are available for study after removal of adjacent tissue layers: The underlying matrix surface can then be studied after removal of the cells, and the mineral front examined after removing the matrix (with an appropriate solvent or by plasma ashing). In this review, we analyse the main findings which we have made in this laboratory concerning the biological activities of osteoblasts (bone forming cells) and osteoclasts (bone resorbing cells). The technical problems of specimen preparation of cells which shrink more than the substrate to which they are attached have been convered previously (Boyde et al. 1977). Such problems obviously affect the lateral, cell to cell inter-relationships more than the cell to substrate effects which we cover here. At present, we can conclude that SEM has made a major contribution to bone biology by permitting observation of normal cells and natural and surrogate substrates. We confidently predict that it will continue to play a pivotal role in the closer observation of cell-cell-substrate interactions particularly in respect of local hormonal effects, as well as in bone pathology and implantology.

A description is given of an approach to the objective qualification and quantification of information obtained by secondary electron imaging of the three-dimensional shape and surface ultrastructure of cells grown attached to the flat substratum in vitro . Objective cell assessment in the SEM (OCAS) is based on the evaluation of cell phenotype in standard defined culture conditions according to a fixed protocol. The human capacity to analyse complex images is used for the elementary collection of data and this is analysed by computerised mathematical processing. The OCAS method described was tested on a training set of populations of mostly rat mesenchymal normal and neoplastic cells. The results show a significant correlation between OCAS data structures describing a cell population and the following biological properties: malignancy, tumorigenicity, virogenicity and growth properties in vitro . These findings correspond to those of others and to an intuitive evaluation of the secondary electron SEM images of cells. In a detailed analysis the OCAS differences were screened for the indicative power to show the particular biological properties of the investigated cells. This was achieved by varying the representation of the OCAS variables in all three groups used: microecology, cell shape, and surface ultrastructure. It was found that the variable which describes the cell surface features points out tumorigenicity and malignancy while the width/height ratio alone indicates tumorigenicity. When these two criteria are combined, then all important biological properties coming into consideration (malignancy, tumorigenicity, virogenicity and growth properties in vitro ) are shown. If only microecological criteria are used, then anchorage independence alone is revealed. This led to the conclusion that, for the prediction of malignancy, just the surface ultrastructure, especially the presence of various microvilli, and the three-dimensional shape of the mesenchymal cell in vitro are decisive. Microecological criteria can be omitted unless the adaptation to the growth under in vitro conditions is investigated. Moreover, from our results it follows that the expression of the influences of neoplastic transformation (tumorigenicity) and the adaption to growth in vitro (anchorage independent growth) on the cell morpho-type are to some extent at variance, which deserves further study.

The dimensional changes of small cubes of glutaraldehyde fixed mouse liver tissue were measured using a light microscope image projected into the Quantimet 720 Image Analysing computer system. The dimensional changes occurring in the critical drying bomb could be followed at all stages when violent turbulence was not occurring. The results show that liver tissue blocks shrink in four stages whilst in the critical point drying bomb: (1) during substitution of the intermediate solvent with the transitional fluid; (2) when the transitional fluid is warmed above the critical temperature; (3) when the transitional fluid, now a gas, is allowed to escape from the CPD bomb – the rate of shrinkage increasing as atmospheric pressure is approached; (4) at atmospheric pressure when all the gas has been allowed to escape from the bomb. Taken together with the authors' previous findings, it would seem that substantial shrinkage of animal soft tissue specimens must occur whilst they are undergoing “critical point drying”. This fact should be taken into account when interpreting SEM images of CPD tissues.