M. Portigliatti Barbos

Collagen orientation in compact bone: II. Distribution of lamellae in the whole of the human femoral shaft with reference to its mechanical properties

Starting from a previous personal investigation (Portigliatti Barbos et al., 1983) indicating that the distribution of osteons and interstitial bone in the middle of the femoral shaft is related to their structure, a new procedure has been devised to allow information of the same kind to be gathered from the whole of the femoral diaphysis. Twenty-three exactly plane parallel cross-sections, 100 micron thick, each located 1 cm from the next, were prepared using an annular blade microtome. The distribution of longitudinal lamellae (whose fiber bundles and crystallites have longitudinal course and withstand loading by tension) and transverse lamellae (whose fiber bundles and crystallites have a transverse course and withstand loading by compression) was determined for both osteonic and interstitial bone, using circularly polarized light as the illuminating source and a Quantimet 720 image analyzing computer. The results show that along the femoral shaft the transverse and longitudinal lamellae from osteonic and interstitial bone have a characteristic rotational distribution consistent with the distribution of the bending forces normally operative in bone.

Distribution of Osteonic and Interstitial Components in the Human Femoral Shaft with Reference to Structure, Calcification and Mechanical Properties

This paper analyzes the distribution of osteons and interstitial bone in the femoral compacta according to their structure, degree of calcification and mechanical properties. Three cross sections, 100 microns thick, each located 1 cm from the next, were prepared by grinding from the middle third of a human femoral shaft. Starting from the premise that, in lamellar bone, lamellae whose fiber bundles and crystallites have a longitudinal course withstand loading by tension, whereas those whose fiber bundles and crystallites have a transversal course withstand loading by compression, each osteon and fragment of interstitial bone has been given a number recording the percentage of its surface consisting of lamellae with transversally oriented fiber bundles and crystallites (bright under the polarizing microscope). The degree of calcification of the same structures was determined micro-radiographically. The distribution of both osteons and interstitial bone was assessed using a tungsten grid for reference. The total surface of each bone microstructure, and the percentage of that surface consisting of bright lamellae, were all calculated using a Zeiss Video-plan. Our results confirm the view that the distribution of both osteons and interstitial bone is mainly related to their structure--and hence to their mechanical properties. In addition, bone remodeling seems to be most active in areas capable of supporting tensile stress.

Collagen orientation in compact bone: I. A new method for the determination of the proportion of collagen parallel to the plane of compact bone sections

Previous work has shown that different types of osteons that have different mechanical properties with regard to tension and compression can be identified and studied by polarized light microscopy. This study reports the development of an automated method for the analysis of that proportion of collagen in transverse sections of human femurs that lies parallel with the plane of section. The method is based upon the use of circularly polarized light to detect collagen with a major component lying in the plane of the section, correcting the measurement for bright collagen under these circumstances with a corresponding darkfield measurement to determine the area of bone within each field of the section analyzed. The method has been validated in several different ways. The measured value of bright collagen under circularly polarized light depends upon the section thickness, since a linear relationship was found between section thickness and bright areas for a large number of microscopic fields. Plane parallel sections are therefore recommended for this study. The pattern of distribution of bright areas was the same for different observers and different set detection levels in any one section. The pattern found in adjacent sections from the same region of the shaft was identical.

Orientation of collagen in human tibial and fibular shaft and possible correlation with mechanical properties

The pattern of collagen orientation in two human tibiae and fibulae was studied. Serial 100 microns plane parallel cross-sections at 1 cm intervals at the same level in tibia and fibula were cut using an annular blade saw. Distribution of transversely oriented collagen was mapped using circularly polarized light and an image analyzing computer. In both the tibia and fibula the pattern of collagen orientation is characteristic, which may be related to the distribution of the bending forces normally operative in these bones.

Micro-biomechanics vs macro-biomechanics in cortical bone. A micromechanical investigation of femurs deformed by bending

Previous studies have shown that longitudinal and transverse lamellae in compact bone signal the presence of tensile and compressive forces, respectively. On this basis an investigation has been carried out to ascertain the distribution of lamellae in femoral shafts that have been deformed by bending. To do this, a series of undecalcified plane-parallel sections 100 microns thick were prepared, and the proportion of transversely oriented collagen and crystallites in the lamellae was measured, using circularly polarized light as an illuminating source and a Quantimet 720 image-analyzing computer. It has been concluded that the distribution of the two types of lamellae is in accordance with the need to compensate for the deformity produced by the bending of the bones, and that an obvious relationship exists between the macroscopic features of the femurs and their modified microscopic structures.

Distribution of lamellae in human femoral shafts deformed by bending with inferences on mechanical properties

On the basis of previous investigations indicating that the distribution of osteonic and interstitial lamellae in a normal femur depends on its mechanical properties, a procedure has been devised to provide information on the distribution of lamellae in human femora diaphyses deformed by bending. To achieve this, exactly plane parallel cross sections, 100 micron thick, were prepared from the portion of maximum bending, using an annular blade microtome. An index of the distribution of longitudinal lamellae (whose fiber bundles and crystallites have a longitudinal course and withstand loading by tension) and transverse lamellae (whose fiber bundles and crystallites have a transverse course and withstand loading by compression) was determined using circularly polarized light as illuminating source and a Quantimet 720 image analyzing computer. The results show that, even in a pathologically deformed bone, both the microscopic structure of sections at the level of osteonic and interstitial lamellae, and their macroscopic shape may be governed by the distribution of the forces active in bone.

Recurrent Secondary Hyperparathyroidism Due to Parathyroid Carcinoma: Usefulness of Ki-67 Immunostaining in the Diagnosis of a Malignant Parathyroid Tumor

Parathyroid carcinoma is a very rare disease occurring in less than 2-3% of all the cases showing clinical features of primary hyperparathyroidism. Several histological markers have been used for distinguishing between benign and malignant tumors of the parathyroid glands. However, most of these markers are not easily applicable and clinical prognosis cannot be predicted by histopathological criteria alone. A recent study has drawn attention to the role of the cell cycle associated antigen Ki-67 detected by MIB-1 monoclonal immunocytochemistry in parathyroid tumors: in fact, Ki-67 seems to be a valuable marker of malignancy in such tumors since it permits an easy detection of proliferating and dividing cells. Here we report in detail a case of severe recurrent hyperparathyroidism in a 51-year-old female patient undergoing regular hemodialysis treatment. In the surgical specimens of the parathyroid glands, the tumor proliferative fraction of 56, expressed as the number of Ki-67-positive nuclei per thousand cells, and the mean mitosis count of 0.5, expressed as the percentage of the total amount of Ki-67 positive nuclei, support the diagnosis of parathyroid carcinoma despite the scanty amount of microscopical signs considered characteristic of malignancy, i.e. extensive thick fibrous bands or prominent nucleoli. To our knowledge this paper is the first clinical report that supports the diagnostic role of the cell cycle associated antigen Ki-67 in parathyroid carcinoma in a case of secondary hyperparathyroidism in a patient undergoing hemodialysis.

Relationship between mechanical properties and distribution of lamellae in the human femoral shaft

The correlation between microstructure and stress distribution along a bone has always been one of the main concerns of skeletal biomechanics. By applying an original technique for isolating osteon samples and loading them under tension or compression along their axis, Ascenzi and co-workers (1, 2, 3, 4) stated that the lamellae best able to support tensile stress are those with longitudinally oriented fibre bundles (dark in cross sections observed under the polarizing microscope), while the lamellae best able to support compressive stress are those with transversely oriented fibre bundles (bright in cross sections observed under the polarizing microscope). From this result it appears clear that an analysis of the distribution and thickness of the two types of lamellae would make it possible to provide informations about the distribution of tensile vs. compressive strenght within compact bone. Such a type of analysis can be carried out using either a semiautomatic (9). or an automatic apparatus for acquisition and computation of geometric data. According to our experience the two methods give comparable results. However, an automatic apparatus is most convenient because it allows to reduce dramatically the v/orking time. In fact, the time required for analysing a cross section of a femoral diaphysis drops from two months to two hours when a semi-automatic apparatus is replaced by an automatic apparatus. So the proposal of one of us, Prof. A. Boyde, to further develop this research in his laboratory, using a Quantimet 720 image analysing computer, was accepted with enthusiasm (6, 10). The material used in this investigation was a serie. of undecalcified cross sections, 100 micra thick, each located 1 cm. from the next, and prepared using a Leitz annular blade microtome. The distribution of osteon. es and interstitial bone in terms of the proportion of their cross-sectional area taken up by bright lamellae as assessed by polarized light microscopy was done by performing a series of three operations. The first was the calculation of the surface area taken up by bright lamellae with respect to the dark background. The purpose of the second operation was to calculate the proportion of the surface actually occupied by the bone section, that is, excluding the discontinuities in the section. The aim of the third operation was to calculate what proportion of the surface of the bone section was occupied by bright lamellae through computer processing of the results of the first two operations.