Is there a relationship between osteoblasts and collagen orientation in bone?
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Biomedical subjects
Publications and source records attributed to A Boyde.
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Bone was removed from the calvaria of anaesthetized 70 g rats or freshly killed young monkeys and the fibrous periosteum dissected off the inner, formative surface under 0.15 M cacodylate buffer. The bone and undisturbed osteoblasts were fixed in 3% glutaraldehyde in the same buffer for 24 to 48 hours, critical point dried and coated with evaporated carbon and gold for scanning electron microscopy (SEM). Fields of osteoblasts were photographed and chosen cells dissected off the osteoid using a tungsten needle. The control of the dissection was made possible by the use of a system of real-time stereo tv-speed SEM. The fields were rephotographed and the orientations of the osteoblasts were compared with that of the underlying collagen fibres. 62% of all osteoblasts lay with their long axes within 15 degrees of the collagen fiber orientation below and 80% within 30 degrees. Montages of large areas of osteoblasts were also made, and then compared with ones of the same area after the cells had been stripped off on adhesive tape. In general, the orientation of the collagen tended to be the same as the cell that formed it. Collagen fibers below cells at the periphery of a domain sometimes had the orientation of the cells in the adjacent patch. It is not possible to determine whether the cells controlled the orientation of the collagen, or vice versa, from this experiment, but other SEM evidence suggests that the collagen orientation in hard tissue matrices depends on the freedom of cells to move with respect to matrix surface.
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A simple device for holding and moving mechanical tools in the region of a sample being viewed in the scanning electron microscope is described. The unit has a 20:1 mechanical reduction and when fitted with a tungsten carbide dental chisel, it is sufficiently rigid to cut biological hard tissues. Alternately, when fitted with an electro-etched tungsten needle, it can be used, in conjunction with specimen stage controls, to remove individual cells from the surface of soft tissues. Examples of these applications are illustrated.
The principal distance, D, from the centre of perspective in the SEM optical projection to the tilt axis of the specimen stage must be accurately determined before photogrammetric evaluation of stereoscopic pairs of micrographs can proceed. A precise procedure for measuring D is described in which the specimen stage X micrometer is used to measure the width of the field scanned for a particular width of the CRT, when the specimen stage is moved along the electron beam axis by amounts measured with the stage Z micrometer. The Z micrometer is calibrated with an external dial gauge. A plot of field width against Z extrapolated to zero gives the location of the perspective centre. In SEM photogrammetry, it is usual to leave the lens currents unchanged whilst recording the stereo-pairs. The values of D measured with a constant final lens current show that the perspective centre is located close to the final aperture in its conventional position. Previous determinations of D for Stereoscans have used a changing lens current to keep the specimen in focus at varying Z, and found a virtual centre several millimetres above the final aperture. The value of D so obtained should only be used if the micrographs were recorded with dynamic or automatic focusing systems.
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