Chondrocytes were isolated from fetal bovine limb cartilage and cultured in Eagle's minimum essential medium. The shape of cultured cells were not polygonal as growth-cartilage cells of rats or rabbits, but spindle-shaped like fibroblasts. Metachromasia was not found for three weeks in these cells by staining with toluidine blue, indicating that these cells did not synthesize abundant proteoglycans. However, when cultured with parathyroid hormon or hydrocortison, or both, these cells showed intense metachromasia.
Electron microscopic studies of the extracellular matrix of cells derived from fetal bovine limb cartilage cultured for three weeks under normal conditions, showed that needles of apatite mineral were deposited as clusters. Furthermore, matrix vesicles which contained needle-like crystals were seen in the extracellular matrix. The elemental composition of the crystalline matrix appeared to be composed primarily of calcium and phosphorus. Therefore, prolonged culture of cells derived from fetal bovine limb cartilage seems to be a good experimental system for studies on endochondral ossification.

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In our radiological longitudinal study, cortical thickness of rat tibia is measured on the lateral pictures soft-radiographed repeatedly under ether anesthesia in order to obtain time-related changes of D value (periosteal diameter) and d value (endosteal diameter).
While the measurement is made at the site of 2.0mm proximal to the tibiofibular bifurcation, it must be noted that there is a rise of the bifurcation due to periosteal bone apposition with aging during the course of experiment. The bone apposition to bifurcation would be 100μm at most on the assumption of a six-month period of experiment starting at the animal's age of 50 weeks.
When the coefficient of variation in reproducibility study and the rise of bifurcation are taken into consideration, an increase of more than 20μm in D value and of more than 30μm in d value are cosidered that there has been a distinct progress in periosteal bone formation and endosteal bone resorption, respectively. If there is a decrease of more than 20μm in d value, it is certain that there has been a progress in endosteal bone formation.

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The X-linked dominant mutation in the laboratory mouse (gene symbol Hyp) is regarded as a model of familial hypophosphatemic vitamin D resistant rickets in man.
We investigated the effect of 1α, 24(R)(OH)₂D₃, recently synthesized from fucosterol, on plasma calcium and phosphorus metabolism in Hyp mice.
Single administration of 1α, 24(R)(OH)₂D₃ 50ng (P. O.) significantly increased plasma calcium level only in the +/Y mice but not significantly in the Hyp/Y mice.
Successive 4 weeks administration of 1α, 24(R)(OH)₂D₃ significantly increased plasma calcium level in the +/Y mice but the elevation of this level in the Hyp/Y mice was not so high as that in the +/Y mice. Meanwhile, plasma phosphorus level was improved to the normal range in Hyp/Y mice with increased % TRP after administration for 4 weeks. In addition, histological study revealed reduction of osteoid tissue in Hyp/Y mice after administration of 1α, 24(R)(OH)₂D₃.
It is therefore expected that 1α, 24(R)(OH)₂D₃ will be effective for the treatment of familial hypophosphatemic vitamin D resistant rickets.

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As it is obscure whether the pattern of microradiodensitogram expresses precisely the distribution of bone mineral, the various parameters of microradiodensitogrammetric pattern are compared to those of well-established analyses of bone mass, especially of bone mineral distribution. Comparison of matched and unmatched parameters in both analyses was performed in 62 measurements of 52 cases between ages of 41 and 88 years.
Although the bones measured were different between microradiodensitogram (metacarpal bone) and bone mineral distribution (radial bone), correlation coefficients between matched and unmatched parameters were high except the bone width. For the further comparison, the results obtained by both methods were compared to the parameters of bone atrophy of axial skeleton, namely Singh's index and lumbar score.
From overall results, bone microradiodensitogram is non-invasively valid parameter to evaluate the minimal change of bone mass as like bone mineral distribution and the other wellestablished methods.

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A method is presented for isolating and cultivating odontoblasts from the root area of rat incisors. A homogeneous layer of odontoblasts attached to predentine with dentine was digested with 0.25% trypsin-0.05% collagenase (1:1) mixed solution. The isolated cells were subcultured at every 20 days in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum. Relative proliferation rates of each subcultured cells were over 10 fold. Chromosome numbers (2n=42) in the subcultures after 6 and 12 subculture generations were over 80%. Doubling time of the subcultures were, on an average, approximately 48 hours. Primary cultures, which survived over 50 days in monolayer, were fibroblast-like cell, mesenchymal cell forms except that it revealed epithelioid cell form, shift from epithelioid cell to fibroblast-like cell form in a time of growth phase. Subcultured cells which secreted the extracellular matrix, at the periphery of the matrix, showed odontoblast-like characteristic polarity of the cells. Odontoblast cultures biochemically retaining original alkaline phosphatase (ALPase) activity before cultivation histochemically showed ALPase activity on the periphery of the cells, the cell surface, the extracellular matrix and the cells surrounding the matrix. Thus these results suggest that odontoblast subcultures retain the functional properties of odontoblasts. Possibilities saying that odontobalst subcultures may recreate primordial pulp cells and differentiate again into odontoblasts are discussed.

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Experiments on identification of cultivated odontoblasts isolated from rat incisors were made. Subcultured cells which secreted the extracellular matrix, at the periphery of the matrix, showed odontoblast-like characteristic polarity of the cells and intense presence of alkaline phosphatase (ALPase) activity on the extracellular matrix and on subcultured cells surrounding the matrix. These matrices increased in number and size with an increase of number of subculture generations and became identifiable with the nake eye. The matrix showed intense reaction to cytochemical stainings for hydroxyproline and calcium. Hydroxyproline in medium secreted from subcultured cells increased with incubation time, associated with an increase of proliferation rates of subcultured cells. Energy and wavelength dispersive X-ray microanalysis of the matrix revealed the intense presence of calcium, phosphorus and sulfur, related to the presence of glycoproteins, phosphoproteins and acid mucopolysaccarides. However, subcultured cells grown in the presence of L-azetidine-2-carboxylic acid were not able to secrete the extracellular matrix containing calcium and hydroxyproline and showing the intense presence of ALPase activity. Thus these results suggest that the extracellular matrix are dentine-like matrix consisting of collagenous proteins including calcium, phosphorus and sulfur and showing the intense presence of ALPase activity.

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Recent progress in calcium metabolism has revealed the therapeutic usefulness of active vitamin D₃ analogues for senile osteoporosis. However, it has not been well understood the mechanism of accelerated bone mineralization by active vitamin D₃ in human senile osteoporosis.
In this report, we examined the effects of 1α-hydroxycholecalciferol (1α-OH D₃) on calcium and phosphate metabolism in the elderly subjects. The treatment of 2μg/day of 1α-OH D₃ for a week in 6 subjects significantly increased the intestinal calcium absorption rate, serum levels of phosphate and renal reabsorption of phosphate (% TRP). The administration of 0.5μg/day of 1α-OH D₃ for 2 weeks in 6 subjects did not change these parameters. Furthermore, no significant changes in the intestinal phosphate absorption, serum level of calcium, iPTH, vitamin D metabolites, blood urea nitrogen, creatinine and Al-Pase activity were observed by the treatment of both doses of 1α-OH D₃. The increase in serum phosphate levels after the treatment of 2μg/day of 1α-OH D₃ might be due to the increased renal phosphate reabsorption.
These data suggest that increased intestinal calcium absorption and accelerated renal reabsorption of phosphate explain the mechanism of accelerated bone mineralization of active vitamin D₃ analogues.

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Two cases of hypoparathyroidism with ankylosing spinal hyperostosis (ASH) are presented.
Case 1 was a 52-year-old man complaining of numbness in the upper extremities and difficulty in walking due to spasticity. Skeletal survey revealed ASH including a continued type of ossification of the posterior longitudinal ligament (OPLL) in the cervical spine, which appeared large enough to cause spastic tetraplegia. Subsequently, the patient experienced an attack of tetany which led to the diagnosis of pseudohypoparathyroidism. Tetany and hypocalcemia have been well controlled by 1, 25(OH)₂D₃ and the numbness and spasticity of extremities have improved to such an extent that surgical intervention to the cervical spine is not necessary at present.
Case 2 was a 60-year-old housewife who had undergone subtotal thyroidectomy for hyperthyroidism 30 years ago. Hypoparathyroidism occurred postoperatively presenting with tetany, and later sciatica. Skeletal survey and other radiological examinations revealed ASH without OPLL, and lumbar spinal canal stenosis. Laminectomy for lumbar spinal canal stenosis was performed and ergocalciferol and calcium lactate were recommended to control hypocalcemia.
Following the laminectomy, however, she was treated only with calcium lactate at another hospital for seven years, the hypocalcemic state having been left uncorrected. The latest skeletal survey revealed advancement of ASH including a new development of OPLL in the cervical spine.

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The experimental renal failure was made by the method of 5/6 nephlectomy in adult male rats. 1α, 25(OH)₂vit.D₃ and 1α(OH)vit.D₃ at the dose of 0.05, 0.1, 0.2μg/kg were administered to these rats for 7 weeks. And the effects were evaluated by blood chemistry, urinanalysis and bone histomorphometry.
At the beginning of drug administration, i.e. 6 months after the nephrectomy, renal failure with bone change was evident. After administration of these agents, serum Ca showed increase and PTH revealed slight decrease but Al-P was still elevated. Bone histomorphometry showed significant decrease of resorption surface, number of osteoclast and volume of fibrous tissue. On the contrary tetracycline labeling rate and mineral appositional rate showed decrease, so osteoid was still abundant in some cases. No significant difference was observed between two drugs or among the doses.

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To clarify the role of vitamin D (D) in the regulation of the endocrine pancreas, we have studied insulin secretion in D-deficient, D₃-long term replete and 1, 25(OH)₂D₃-short term replete rats, using the isolated perfused rat pancreas.
In the D-deficient rats, the insulin release by 16.7mM glucose exhibited a marked reduction compared to that in the D₃-long term and the 1, 25(OH)₂D₃-short term replete rats, and this was especially more noticeable in the first phase of insulin release depending on the intracellular ionic calcium (Ca) pool. It is said that the potentiating effect of cyclic AMP (cAMP) on insulin release is due to intracellular translocation of Ca⁺⁺ from the organell-bound pool and we found that the insulin release in the D-deficient rats in response to 1mM dibutyl-cAMP and 5.5mM glucose decreased compared to that in the 1, 25(OH)₂D₃-short term replete rats. These results suggest that the decreased insulin release in the D-deficient rats is due to a reduction of intracellular Ca⁺⁺ storage in the B cell, which we believe is a result of long term low plasma Ca levels in the D-deficient rats. On the other hand, somatostatin secretion from the D cell was not significantly different in the D-deficient and D₃-long term replete rats.

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The effects of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) on endosteal bone resorption induced by oophorectomy in adult rats were investigated by a radiological follow-up method for cortical measurement of tibia as well as by microradiological and histochemical method on nondecalcified cross-section of long bone. The animals were given 10mg of HEBP parenterally once every other day for a period of 16 weeks and were compared with the oophorectomized control group.
Histochemical observation showed that HEBP induced the formation of osteoid projecting from endosteal surface into the marrow cavity. The time-course changes of endosteal diameter in the longitudinal study showed that HEBP suppressed endosteal resorption and even increased the cortical thickness towards the marrow cavity. These findings suggest that HEBP may be an agent capable of inhibiting the progress of osteoporosis, further activating endosteal new bone formation resulting in bone mass increase.

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