S. Yousuf Ali

Electron probe analysis of cryosections of epiphyseal cartilage

Abstract Conventional methods for preparing specimens for analysis of the chemical composition of mineralizing tissues at the electron microscopic level can produce artefacts during fixation, dehydration, embedding and wet-sectioning. Cryoultramicrotomy provides a more reliable approach to electron probe analysis of initial apatite formation in calcifying cartilage. Two procedures are described (a) for a micro-analytical approach and (b) for a morphological study. By using a negative staining procedure (b) cytoplasmic details in terms of subcellular organelles and extracellular, membrane-bound, matrix vesicles could easily be identified in relatively large sections of epiphyseal cartilage. Unfixed, dry, unstained cryosections obtained by procedure (a) showed dense mineral granules (50–120 nm in diameter) in the mitochondria of chondrocytes and electron probe analysis of these gave a mean Ca/P mass ratio of 1.20. Adjacent cytoplasmic areas showed negligible levels of calcium. In the longitudinal septa matrix, vesicle-like particles could be identified with characteristic crystal needle clusters. Microanalysis of these structures showed them to have a mean Ca/P mass ratio of 2.15. Non-vesicular matrix appeared to be very low in calcium and phosphorus. The combination of cryoultramicrotomy and electron probe analysis of different areas in the same cryosection has provided some information on the coexistence of amorphous calcium phosphate in the mitochondria and of crystalline apatite in matrix vesicles a few micrometers away. These findings may thus explain the sequence of hydroxyapatite formation in epiphyseal cartilage.

Calcium phosphate microcrystal deposition in the human intervertebral disc

A variety of crystals have been identified in both normal and pathological connective tissues. Calcium phosphate ‘cuboidal’ microcrystal deposition has been found, using transmission electron microscopy (TEM), in femoral articular cartilage, where they are distributed as a band 5–50 µm beneath the articular surface. These cuboid microcrystals have been identified as magnesium whitlockite. Our objective was to investigate their presence in the human intervertebral disc. In this study, two degenerate and 15 scoliotic discs were examined using light microscopy and TEM techniques to determine the presence of calcium phosphate crystals. Calcium pyrophosphate dihydrate (CPPD) deposition was identified in one degenerate disc specimen. Using TEM and electron probe analysis, cuboid microcrystals were identified in the annulus fibrosus and nucleus pulposus of both degenerate specimens, but not in the discs from young scoliotic patients. Cuboid microcrystal deposition was found predominantly around cells, which were mainly necrotic, with some association with extracellular lipidic/membranous debris. This is the first TEM report of whitlockite in the intervertebral disc. In one specimen coexistence of cuboid and CPPD crystal deposition was found.

A Morphometric analysis of osteoid collagen fibril diameter in osteogenesis imperfecta

Osteogenesis imperfecta is a genetic disorder of connective tissue characterised by frequent bone fracture following minimal trauma. Mutations of type I procollagen genes have been widely reported as the cause of OI and such mutations have been shown to introduce kinks into the collagen molecule. A study was performed to examine type I collagen fibrils at the ultrastructural level in the transmission electron microscope (TEM). Type I collagen fibrils from the bone osteoid of OI patients and age- and site-matched normal control bone were photographed in the electron microscope. A histomorphometric analysis of the diameters of collagen fibrils photographed in the TEM indicated that type I collagen in OI bone was larger in diameter compared with normal bone. This increase in diameter of type I collagen fibrils may represent an alteration in the quaternary structure of the collagen fibril as a consequence of kinked, poorly packed collagen molecules. Such alteration in the collagen fibrils may affect the formation and stability of bone mineral associated with it.

An ultrastructural and immunogold localization study of proteoglycans associated with the osteocytes of fetal bone in osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a rare, heterogeneous, inherited connective tissue disorder frequently caused by abnormalities of type I collagen. It is characterized by bone fragility, osteopenia, and progressive skeletal deformities. Electron microscopy of three OI type II fetal bone samples revealed numerous large osteocyte lacunae. In addition, there was a perilacunar osteoid-like band of collagen surrounding the osteocytes, which was unmineralized and morphologically unusual. Furthermore, large osteocyte lacunae contained fine particles and filamentous material similar to the expected ultrastructural appearance of proteoglycans. More detailed examination was carried out using histochemical and immunogold localization of proteoglycans at light and ultrastructural levels. These tests and the use of electron probe X-ray microanalysis confirmed that the material in the osteocyte lacunae was proteoglycan. In contrast, in the age- and site-matched normal fetal bone, all the osteocyte lacunae appeared negative for proteoglycan. Proteoglycans are regarded as inhibitors of calcification. Our observation of substantial amounts of proteoglycan in abnormally enlarged osteocytic lacunae of some OI fetal bone suggests association with the abnormal bone of this particular subtype of OI type II.

A microanalytical study of bone mineral in a transgenic mouse model for brittle bone disease

No single analytical technique will provide all the information necessary for the interpretation of a complex biological system. The mineralisation of bone is no exception, especially in a pathological situation. Brittle Bone disease, a genetically and biochemically heterogeneous group of disorders is characterised by the ease and frequency of bone fracture. Although numerous type I procollagen gene mutations have been detected, few reports exist of the mineral and its relationship with the altered collagen. In this study the calcium to phosphorus ratio in normal, human and or cortical bone has been compared with bone from normal mouse and a transgenic mouse model containing a shortened human procollagen gene. This mouse has exhibited many phenotypic features of human or (Cassella et ai, 1992).