METODOLOGIA EXPERIMENTAL

Vittelaro-Zuccarello et al (1984) described the morphology of connective tissue cells in 01 and reported on mitochondria but made no mention of inclusion bodies. Kjaer et al (1975) reported mitochondrial granules in the osteoblasts of a child with 01 (type unspecified). No explanation as to their presence in the disease was made.

In the mitochondria of most cells the matrix contains electron-dense granules 300- 500A in diameter (Ghadially, 1977). These have been referred to as intermitochondrial

granules, or matricial granules. These granules are more prominent and encountered more

frequently in the mitochondria of tissues which transport large quantities of ions and water. Previous studies on isolated dense granules indicated that they contain calcium, magnesium, phosphorus and inorganic material (Ali et al. 1977). Peachey (1964) proposed that if modest amounts of calcium ions were presented to the mitochodria, they would be deposited on the dense granules, but if large amounts of calcium ions, preferably with inorganic phosphate were offered, they would be likely to form de novo

precipitates in the mitochondrial matrix. In some pathological states the mitochondria may be filled with calcium deposits. It is important to note that this is not calcification of necrotic tissue, for intermitochondrial calcification precedes the necrosis and appears to be a factor in its production; an example being the kidney tubules of mice after the administration of parathyroid extract (Cauflield and Schray, 1964).

Osteoclasts and osteoblasts have been found to contain mitochondrial granules after administration of parathormone (Matthew et al. 1970). Matthews et al. (1970) demonstrated that one specific modification induced in the mitochondria of chondrocytes by a low phosphate, vitamin Dz-deficient diet was the reduced ability to form mineral- coating mitochondrial granules. It may be possible that vitamin D is related to the production of ûie organic component essential for stabilising mitochondrial mineral. Matthews et al (1970) noted the disappearance of mitochondrial granules in the chondrocyte strata immediately adjacent to the site of onset of mineralisation, further suggesting a relationship to the mineralisation process. It has yet to be demonstrated whether these granules play an active role in the mineralisation process or whether they simply represent an intracellular mechanism for storing calcium and phosphorus in cells

Cameron et al (1967) observed spherical electron dense particles in mitochondria in osteoblasts of rats following parathormone treatment. They interpreted the increase in number and density of mitochondrial granules to be a function of calcium increase. Ali et al (1977) reported dense granules in unstained sections of rabbit calcifying cartilage. Ali and Wisby (1975) suggested that a compound like calcium B-glycerophosphate might be present in mitochondrial granules found in chondrocytes. These granules were 50- 120nm in diameter and electron probe micoanalysis indicated a Ca/P mass ratio of 1.14. These authors were the first to report the occurrence of calcium-deficient, amorphous calcium phosphate in mitochondrial granules in the cytoplasm of chondrocytes. There were approximately six to ten granules in a group. Under some intense beam conditions, the granules were reported to sublimate and assume a ’frothy’ appearance. This was not a feature seen in the mitochondrial granules observed in the 01 patients.

Ozawa and Yamamoto (1983) demonstrated numerous mitochondrial granules in unstained sections of various calcifying tissues prepared by anhydrous methods (Ozawa et al. 1979; Landis et al. 1977). Elemental analysis of these mitochondrial granules revealed distinct peaks for cal^m and phosphorus, while electron diffraction patterns of these granules demonstrated only ’hazy rings’ indicating that they consisted of non- ^stalline calcium phosphate. Landis (1979) reported single mitochondrial granules within chondrocytes of growth plate cartilage. Calcium phosphate was also deposited in other intracellular organelles. Correlative electron microscopy and electron diffraction established that no patterns of a specific calcium phosphate solid phase were generated from the granules.

Lehninger (1970) reported that under certain conditions the mitochondria of many types of cells from a variety of animal species can accumulate large deposits of stable amorphous calcium phosphate. Lehninger (1974) reported the presence of significant quantities of Mg and ATP in stable amorphous granules and concluded that Mg and ATP were required for the formation of mitochondrial calcium phosphate.

Krane and Glimcher (1978) concluded that ATP molecules which are present in high concentrations in the mitochondria, might be bound to the mitochondrial granules where they susequently donate their terminal phosphate groups by a phosphoryl transfer to the solid phase granule surface, forming pyrophosphate groups as an intimate part of the solid phase surface structure.

The effect of the presence of these additional phosphate groups not only stabilises the growth of the solid phase and prevents further phase changes characteristic of maturation, but in part explains the low Ca/P ratios of solid phase of Ca-P in mitochondria.

Anderson (1976) considered it likely that calcium and phosphate released from mitochondria were in a soluble form as they diffuse into the bone matrix, where they can be utilised by vesicles during calcification. As such the calcified mitochondria were regarded to represent a mechanism of active Ca transport into the calcifying area (Brighton et al. 1976; Matthews et al. 1971; 1973), whereby the high Mg and ATP content guaranteed a stabilisation of amorphous calcium phosphate (Posner et al. 1978).

The presence of mitochondrial granules in OI indicates that some metabolic imbalance in these cells has occurred. Two forms of these mitochondrial inclusions were found. One form, spherules has been reported before but needles which contained calcium and phosphorus as determined by X-ray microanalysis were also detected. However, these inclusions demonstrated a low molar calcium-phosphate ratio (Ca/P= l.KX)) compared to normal hydroxyapatite (Ca/P =1.602, see Chapter 4). Electron diffraction of these inclusions revealed a ’hazy’ set of diffraction rings indicating a poorly crystalline material made up of small crystallites.