Calcification Front Research Articles

Localisation of aluminium by histochemical and electron probe x-ray microanalytical techniques in bone tissue of cases of renal osteodystrophy.

Histochemical studies and electron probe x-ray microanalysis for aluminium have been performed on 16 samples of undecalcified bone from cases of renal osteodystrophy associated with a syndrome suggestive of dialysis encephalopathy (five cases), age and sex matched controls for these and a group of patients with chronic renal failure (six cases) who have never been on haemodialysis. Aluminium was detected only in the patients with a dialysis encephalopathy-like syndrome. This group had significant histological bone disease the features of which were broadly consistent with the so-called atypical renal osteomalacia which is thought to be due to a metal toxin. Aluminium was demonstrated at the interface between osteoid and mineralised tissue--that is, at the site of the calcification front, where it could interfere with the mineralisation process. In the group of patients who had never been subjected to haemodialysis there was also significant histological bone disease but no evidence of aluminium accumulation. In this group the bone disease was of a more typical pattern of osteomalacic changes coupled with those of hyperparathyroidism.

Quantitative bone histology in 38 patients with advanced renal failure.

The clinical, biochemical, radiological and histological appearances of the bones of 38 patients with advanced renal failure are presented. Thirty-three patients had histological evidence of hyperparathyroidism and 17 also showed osteomalacia. Of five showing evidence of neither hyperparathyroidism nor osteomalacia, two had borderline osteopenia. There was an inverse correlation between the plasma calcium concentration and trabecular surface covered by osteoid with a tendency for those with the lowest concentrations of plasma calcium to show histological osteomalacia. There was an inverse correlation between extent of calcification front and both volume and surface extent of osteoid. No relation was found between plasma phosphorus concentration and any of the histological measurements made. Patients with radiological hyperparathyroidism had a lower calcium and higher plasma phosphorus than those without. Phalangeal sub-periosteal erosions were as common in those with histological osteomalacia as in those with histological hyperparathyroidism alone. There was no association between plasma alkaline phosphatase activity and type of bone disease. There was no correlation between the radiological second metacarpal index and the histological volume of cancellous iliac bone.

Induced growth of the mandibular condyle in the rat.

Advancement of the calcification front in the cartilage, which is a measure of the growth of the cartilage, can be determined by the administration of two dose bone-markers at specific intervals. It appears that growth still takes place in animals of 12-18 months. This growth is possible by the persistence of chondrogenic cells. The overall growth of the central part of the condyle amounts to about 2 mm over the period from 2 to 18 months. Raising of the bite in the rat has an obvious influence on the temporomandibular joint, and on the condyle in particular. Within a few days an increase of growth-rate of cartilage can be demonstrated. This extra growth is temporary, and the intensity and duration of the response is determined by the age of the animal. This adaptation is related to the presence of chondrogenic cells in the cartilaginous layer in the condyle.

Bone histomorphometric reproducibility in normal patients.

To study bone histomorphometry reproducibility in normal subjects, we performed during orthopedic surgery bone biopsies in 16 postmenopausal women. Each woman had four bone biopsies, two at the usual site in the iliac crest, one on the left and one on the right side, and two other biopsies just behind the usual site, one at each side. We performed measurements of trabecular bone volume, relative osteoid volume, osteoid surfaces, osteoclastic resorption surfaces and calcification front. The average values of the 16 patients were compared, on the one hand, two by two, by a student test, and on the other hand, by a variance analysis. By these two methods the results showed no significant difference between the average values of the 16 patients at each location for any of the histomorphometric parameters studied. However, there was a location variation which was estimated by the intra-individual variation for a given patient. On the other hand, we calculated from the variance analysis the location variance for a group of 10 to 100 patients. In any case all the parameters had a location variation which was high for osteoclastic resorption surfaces and relative osteoid volume when expressed in % of the absolute value of these parameters. The variation of the trabecular bone volume was 0--46. 15% (95% confident limit interval) in a single patient and the hypothetical value of the location variation was 41.6% for a group of 10 patients and 13.0% for a group of 100 patients.

Ultrastructural localization of complex carbohydrates in odontoblasts, predentin, and dentin.

Glycosaminoglycans (GAGs) and glycoproteins (GPs) are essential components for dentinogenesis. We have examined rat odontoblasts, predentin, and dentin decalcified with EDTA and stained with: 1) Spicer's hig-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycoconjugates, and 2) Thiéry's periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HIS-TCH-SP stained distended portions of Golgi saccules and secretory granules. The predentin contained three times the number of HID-TCH-SP stain precipitates when compared to the mineralization front of the dentin matrix. PA-TCH-SP weakly stained membranes of Golgi saccules and cisternae of rough endoplasmic reticulum (RER), whereas stronger staining was observed in secretory granules, lysosomes, and multivesicular bodies (MVBs). Collagen fibrils in predentin demonstrated moderate PA-TCH-SP staining. In contrast, strong PA-TCH-SP staining was observed on and between collagen fibrils in the mineralization front of the dentin matrix. TCH-SP controls of unosmicated specimens lacked significant staining, however, osmicated control specimens did contain some TCH-SP stain deposits in the mineralization front. These results indicate that sulfated and vicinal glycol-containing glycoconjugates are packaged in the same type of secretory granule and released into the extracellular matrix; subsequently vicinal glycol-containing glycoconjugates concentrate in the calcification front, whereas sulfated glycoconjugates accumulate in the predentin and are either removed or masked to staining in the dentin.

Heterogeneity of Adult Hypophosphatasia Report of Severe and Mild Cases

Two cases of women with adult hypophosphatasia illustrate the clinical spectrum and potential difficulties in the diagnosis of this condition. Both patients had subnormal serum alkaline phosphatase activity, absence of leukocyte alkaline phosphatase, increased amounts of urinary phosphoethanolamine, and normal levels of immunoreactive calcitonin and parathyroid hormone. In undecalcified bone biopsy specimens, the number of osteoblasts and the tetracycline-labeled calcification front were similar in the two patients, although the percentage of unmineralized bone matrix and the extent of osteoid-covered bone surface were different. Twenty years of bone pain, severe skeletal deformities, and a generalized increase of osteoid in one patient contrasted with an 18-month history of bone pain and patchy osteoid in the other. These cases suggest that adult hypophosphatasia is a heterogeneous disorder and may be more common than previously realized.

Scanning electron microscopy of teeth in dominant osteogenesis imperfecta: support for genetic heterogeneity.

Scanning electron microscopic studies were performed on 25 deciduous and permanent teeth from members of 7 kindreds with dominant nonlethal osteogenesis imperfects (OI). Two families had normal teeth on clinical and radiological examination; five families had blue or brown opalescent teeth with specific radiologic findings. Enamel surfaces and prism organization were normal on all teeth. On fractured surfaces, the dentin tubules of normal teeth from patients with OI were evenly distributed and coursed regularly to the dentin-enamel junction. Opalescent teeth had few tubules and those present were short, narrow, and tortuous. Dentin calcification fronts of normal teeth were composed of many nodules with regularly spaced openings on their surfaces. Calcification fronts of opalescent teeth were composed of irregularly spaced, small nodules, which varied greatly in size and the nodules lacked tubule openings on their surfaces. The results of this study support the concept that at least two dominant forms of OI exist--one in which all individuals with IO have normal teeth, and the other in which all with OI have blue or brown opalescent teeth with characteristic changes on SEM.

The mineralization processes in teleost fish scales.

In Teleost fish scales, growth and mineralization are continuous. Different mineralization processes can be distinguished. The external layer of the scale is the first to be mineralized and may be classified as the initial calcifying structure of the scale. The initial calcification loci are matrix vesicles of cellular origin always observed during the formation of this layer. This mineralization process takes place progressively, closely following the elaboration of the organic matrix in the scale periphery. The outer limiting and internal layers of the scale are developed after the external layer has been formed. A mineral substance is deposited without the mediation of matrix vesicles, but in contact with the previously mineralized external layer. This type of mineralization is called subsequential. However, the mineralization of the outer limiting layer closely follows the secretion of a collagen-free organic matrix and is thus different from the mineralization of the internal layer in which the calcification front remains remote from the collagen matrix surface and corresponds to a delayed mineralization process. The isolated calcifications (Mandl's corpuscles) which develop in the unmineralized laminae of the internal layer are mineralized in the absence of matrix vesicles and without making contact with a pre-existing calcified tissue, probably by a heterogeneous nucleation of the collagen fibrils.

Ca2+ transport by chondrocyte mitochondria of the epiphyseal growth plate

In a study of the Ca2+ kinetics of mitochondria of chick epiphyseal chondrocytes, the rate of Ca2+ uptake was linear up to a medium Ca2+ concentration of 30 mum. The half maximal transport rate occurred at 34 mum Ca2+. The Ca2+ uptake rate, expressed as a function of time, was 35 nmoles/mg protein/min; the presence of Mg2+ had little effect on Ca2+ accumulation. While these kinetic parameters did not differ significantly from mitochondria of cells of nonmineralizing tissues, the respiratory characteristics of the chondrocyte organelles exhibited functional differences. Thus, up to 350 nmoles Ca2+/mg protein, chondrocyte mitochondria performed coupled oxidative phosphorylation. Calcium uptake was energy supported, while Ca2+ binding was low. Addition of respiratory inhibitors and uncouplers to these mitochondria resulted in a rapid loss of more than 80% of the total Ca2+. The Ca/Pi ratio of the extrudate was very similar to the ratio of the ions in cartilage septum fluid. In the most mineralized zones of the epiphyseal plate, there was little change in the state 4 respiratory rate, but nonspecific Ca2+ binding was elevated and a high percentage of the total Ca2+ was in a nonextrudable form. The results indicate that in cells preparing for mineralization, much of the total mitochondrial Ca2+ is in a form that can be transported to the calcification front. In cells close to the calcification front, nonextrudable Ca2+ may form calcium phosphate granules described by other investigators.

The effect of oxygen, phosphoenolpyruvate and pH on the release of calcium from chondrocyte mitochondria

Chondrocyte mitochondria were isolated from the epiphyseal growth plate of the chick and calcium release was measured at an oxygen tension corresponding to the pO 2 of the most hypoxic region of the plate. At this oxygen tension no significant release of calcium was observed. Since this result suggested that low oxygen tension had no direct effect on mitochondria calcium efflux, an indirect effect via glycolytic intermediates was explored. There was no release of calcium when mitochondria were incubated with 1,2-diphosphoglycerate, 3-phosphor glycerate, or lactate. However, in the absence of added ATP, phosphoenolpyruvate (PEP) caused inhibition of calcium uptake and release of more than 50% of endogeneous calcium; this effect was maximal in the presence of a NAD-linked substrate. Analysis of PEP levels in the growth plate showed that the highest quantity was present in the most hypoxic zone. As a low oxygen tension would stimulate glycolysis, the effects of pH on calcium transport were also studied. It was found that at a low pH (4.0–6.5) there was release of mitochondrial calcium. The findings from this investigation suggest that the hypoxic conditions at the calcification front stimulate glycolysis. This results in an increase in PEP, a decrease in ATP and a drop in cytosolic pH. In concert, these factors induce calcium release from chondrocyte mitochondria.

The effect of 1α(OH)D 3 and 1α,25(OH) 2D 3 on the bone in patients with renal osteodystrophy

Six patients with chronic renal disease and variable degrees of renal osteodystrophy were treated for three weeks with either 1α,25-dihydroxyvitamin D 3 (1α25(OH)D 3) or 1α,hydroxyvitamin D 3 (1α(OH)D 3) and both the biochemical and osseous responses measured. The most consistent changes seen were an increase in serum calcium concentration to normal, a decrease in immunoreactive parathyroid hormone toward normal, an increase in the extent of the calcification front and a decrease in the extent of fibrous dysplasia in the marrow cavity. Two important parameters which did not change significantly were the serum alkaline phosphatase activity and the osteoid volume. These data, in conjunction with that from previous studies, indicate that therapy with 1α,25(OH) 2D 3 or 1α(OH)D 3 does not heal the osteomalacia of renal osteodystrophy, but that it does suppress the secondary hyperparathyroidism, and ameliorate the osteitis fibrosa seen in patients with chronic renal disease. They raise the likelihood that additional factors, such as metabolites of vitamin D other than 1α,25(OH) 2D 3, play a role in regulating bone formation and/or mineralization.

Bone disease and hyperparathyroidism in chronic renal failure: the effect of 1alpha-hydroxyvitamin D3.

SUMMARYPlasma biochemistry, iliac crest bone biopsy and calcium and phosphate absorption were performed in ninety patients with chronic renal failure, forty‐one of whom were on maintenance haemodialysis. The studies were repeated in a number of these patients after at least 6 months treatment with 1α‐hydroxyvitamin D3 (lα‐OHD3).Before lα‐OHD3 treatment all patients had some abnormality in bone histology; there was an increase in the osteoid volume, the osteoid and resorbing surfaces and a decrease in the calcification fronts. There was no differences between those on and those not on haemodialysis, although the amount of osteoid increased with the length of dialysis. Plasma parathyroid hormone (PTH) was increased in all patients in the presence of hypo‐normo‐ or hypercalcaemia, but the plasma calcium was higher in the haemodialysed patients. Malabsorption of calcium and phosphorus was present but it was significantly worse in the non‐dialysed patients. Hyperphosphataemia was more marked in the non‐dialysed patients and plasma alkaline phosphatase was raised in a number of patients. Plasma PTH correlated positively with plasma alkaline phosphatase and plasma calcium but was unrelated to any of the bone histology parameters.Treatment with lα‐OHD3 corrected the malabsorption of calcium and phosphorus and cured the proximal myopathy. There was a rise in plasma calcium and phosphate, but the rise in phosphate could be prevented with aluminium hydroxide in most patients. Only in 56% of patients was a rise in plasma calcium associated with a suppression of PTH, which did not fall into the normal range. In twenty‐six patients repeat biopsies on lα‐OHD3 showed improvement in histology.It is concluded that lα‐OHD3 corrects the malabsorption and improves the bone histology and the myopathy of renal failure. Tertiary or autonomous hyperparathyroidism occurred in 44% of patients.

The metabolic heterogeneity of glycosaminoglycans of the different zones of the epiphyseal growth plate and the effect of ethane-1-hydroxy-1, 1-diphosphonate (EHDP) upon glycosaminoglycan synthesis in vivo.

The synthesis of total and unextractable glycosaminoglycans of rabbit epiphyseal growth plate was studied by determination of the incorporation in vivo of 35S-sulphate into various glycosaminoglycans separated by microfractionation procedures using CPC and ECTEOLA columns. In the narrow hypertrophic zone, hyaluronic acid and chondroitin sulphate of low molecular weight were found to be unextractable with 3M guanidinium chloride for 24 h, thus being firmly bound within the tissue. While the unextractable chondroitin sulphate showed a sharply reduced synthesis rate in the hypertrophic zone compared with that in the columnar and resting zones, a rapid increase in the synthesis of extractable chondroitin sulphate toward the calcification front is indicated. At microfractionation, solubility profiles indicated that the unextractable chondroitin sulphate was of predominantly lower molecular weight and/or charge density compared to that of the whole tissue, most evident in the hypertrophic zone. The distribution pattern of incorporated 35S showed a close correspondence to the distribution of the various chondroitin sulphate fractions both for extracted and unextracted tissue. In the hypertrophic zone, higher radioactivity was found for chondroitin sulphate of predominantly low molecular weight and/or charge density in extracted compared to unextracted tissue. Low doses of EHDP caused no change of distribution of the various glycosaminoglycans but generally inhibited their synthesis.

Scanning electron microscopy of dental calculus.

The morphologic structure of anorganic dental calculus was studied by means of the scanning electron microscope. From surface observations, calculus is apparently composed of two components with distinguishable patters of calcification. One component is formed by the precipitation of minute calcific crystals on microorganisms and intermicrobial substances (plaque matrix). Such calcified masses, often spherical in shape, have a sponge-like appearance with empty spaces representing the former sites of entombed and degenerated organisms. Thus, intracellular calcification is not evident at this stage of calculus development. The other component, although having at least one common calcification front with the former, does not appear to be directly associated with microbial calcification. It exhibits a configuration of generally larger crystal growths of varying shapes and sizes. These two calcification patterns are comparable, both in distribution and size, to what has been observed by means of the transmission electron microscope, and what Schroeder has designated as "types A & B centers of mineralization," respectively. The calcific precipitation in type A centers have been identified by X-ray diffraction as hydroxyapatite. It is, therefore, speculated that the crystal patters in type B centers might represent other known forms of calcium phosphates present in calculus, such as octacalcium phosphate, whitlockite and brushite.

The Effects of Phosphate and Vitamin D Therapy on Osteopenic, Hypophosphatemic Osteomalacia of Childhood

Two severely osteopenic adolescent males with clinical manifestations of adult onset hypophosphatemic osteomalacia were resistant respectively to physiological and pharmacological doses of vitamin D. Clinical improvement ensued following initiation of pharmacological doses of vitamin D and phosphate. Non-decalcified bone biopsies obtained before and during therapy exhibited correction of an isolated mineralization in the second. The response to treatment was reflected in striking changes in tetracycline-labelled calcification fronts in both patients.

The ultrastructure and calcification of the scales of Tilapia mossambica (Peters).

The scales of Tilapia are surrounded by an envelope of scleroblasts responsible for the production of layers of collagen that constitute the bulk of the scale. The scleroblasts adjoining the lateral face of the oldest scale region gradually atrophy. New Collagen layers are deposited against the inner face of the scale, the adjoining scleroblasts showing evidence of high metabolic activity. Calcification occurs by inotropic deposition of crystals alongside the fibres. There is no sharp demarcation between calcified and non-calcified scale regions, a calcification front gradually moving towards newly formed collagen layers. It is felt that fish scales should be considered as calcified derivatives of dermal collagen layers.

Calcifediol in Chronic Renal Insufficiency

Quantitative histology of thin, nondecalcified sections was performed on sequential bone biopsy specimens from five patients undergoing long-term hemodialysis and treated with calcifediol (25-hydroxycholecalciferol) for periods of three to nine months. With increase of intestinal absorption of calcium and decline of circulating immunoreactive parathyroid hormone and alkaline phosphatase, the bones of each patient exhibited striking histological improvement. The group as a whole showed statistically significant decreases in osteoclast number and in the percentages of osteoid surface covered by active osteoblasts. Marrow fibrosis was either eliminated or strikingly decreased in each patient. Osteoid volume significantly declined in four of five patients. In patients with osteitis fibrosa as the predominant histological lesion, calcifediol therapy resulted in decreased calcification front activity. Increased activity was the result when osteomalacia predominated.

Comparison of mineralization of the tibial epiphyseal plate in immature rats following treatment with cortisone, propylthiouracil or after fasting.

Comparison of mineralization in the hypertrophic zone of the tibial epiphyseal plate in immature rats was carried out after treatment with cortisone, propylthiouracil, or after fasting. Under normal conditions, in the extracellular matrix at the calcification front, calcium and phosphate increased, sulfated mucopolysaccharides decreased, and matrix vesicles, which serve as the locus for the formation of hydroxyapatite crystals, increased. In propylthiouracil-treated rats, hydroxyapatite crystals were prominent, related to an increase in calcium deposition, a decrease of mitochondrial granules (thought to contain calcium and phosphate), an increase in the number of matrix vesicles, and to a marked decrease in the amount of sulfated mucopolysaccharide. In cortisone-treated rats, hydroxyapatite crystals were present but they were not as numerous as in the propylthiouracil-treated rats. Correspondingly, calcium deposition was slightly reduced, mitochondrial granules were more numerous than in the previous groups of rats, matrix vesicles were less numerous, and sulfated mucopolysaccharide were more prominent than in the propylthiouracil-treated rats. In fasted rats, hydroxyapatite crystals were markedly reduced or absent, and related to a decrease in calcium deposition, an increase in the number of mitochondrial granules (suggesting a delay in transport to the extracellular matrix). Matrix vesicles were markedly reduced in number, and sulfated mucopolysaccharide much more prominent than in either the cortisone or the propylthiouracil-treated rats.

The contrasting effects on bone histology of vitamin D and of calcium carbonate in the osteomalacia of chronic renal failure.

1. The histological appearances of cancellous bone from the ilium have been quantified in thirteen patients with osteomalacia due to chronic renal failure. 2. There was an excess of osteoid tissue and a reduction in the extent of the calcification front in that osteoid lamella lying next to calcified bone. 3. Administration of calcium compounds orally produced an increase of punctate calcification within the osteoid, but there was no change in the extent of the calcification front. 4. Administration of vitamin D produced a marked rise in the extent of the calcification front but no increase of punctate calcification within the osteoid. 5. This rise in the extent of the calcification front was not dependent on a rise in plasma calcium × plasma phosphorus product. 6. It is concluded that vitamin D has a direct action on the bone in patients with the osteomalacia of chronic renal failure.

Seasonal variation of histological osteomalacia in femoral-neck fractures.

<h2>Abstract</h2> In a series of 134 iliac-crest biopsies from unselected cases of fracture of the proximal femur, the proportion with evidence of osteomalacia varied with the season. The highest frequency of abnormal calcification fronts (43%) was observed in February to April and the lowest (15%) in August to October. The highest frequency of abnormal osteoid-covered surfaces (47%) was observed in April to June and the lowest (13%) in October to December. The overall frequency of osteomalacia in femoral-neck-fracture cases in Leeds seems to be about 37%. It is concluded that variation in hours of sunshine is responsible for a seasonal variation in osteomalacia in these cases and, possibly, in the elderly population as a whole.