In bone histomorphometry tetracycline double-labeling is a very useful method to analyze the bone dynamics. In 1981, Hori, et al. has reported four “patterns” of double labels after double-labeling in beagles, with or without presence of post-double-labeled mineralized bone layer and/or osteoid seam.
In this study, eight cases (four males and four females) were examined. Their age ranged from 22 to 71 years. The labeling schedule was on the tetracycline for 3 days, off for 7 days and on again for 3 days. The interval period between the second labeling and the biopsy was 5 to 13 days. Bone biopsy was done in the iliac bone. The undecalcified specimen was stained with the Villanueva bone stain and embedded in polymethylmethacrylate. Then, with the Jung K-type microtome, it was cut at 5μm in thickness.
The fraction of pattern I, II, III and IV of doubly-labeled surface per total labeled surface was 37.1±15.5, 28.7±15.2, o and 1.6±2.6, respectively; while that of pattern I, II, III and IV of singly-labeled surface per total labeled surface was 8.1±7.5, 17.9±8.7, 0.3±0.7 and 6.3±5.5, respectively.
We have reported three “patterns” of double labels except for pattern III in four “patterns” and all four “patterns” of single labels in trabecular bone of eight human iliac bones. Furthermore, we have discussed its significance in bone histomorphometry. When the second interval period was longer than 7 days, the pattern I was of the largest, and when it was shorter than 6 days, the pattern II was of the largest in four “patterns”.

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Role of Zinc as an Activator of Bone Metabolism in Weanling Rats. Masayoshi YAMAGUCHI and Kouzi TAKAHASHI, Department of Environmental Biochemistry, Shizuoka College of Pharmacy, Shizuoka, Japan.
The effect of zinc on bone metabolism was investigated in weanling rats orally administered zinc sulfate (0.5, 1.0 and 5.0mg Zn/100g body weight) for 3d. Administration of zinc (0.5 and 1.0mg/100g) produced dose-dependent increases in the contents of zinc, DNA, collagen and calcium, and in the activity of alkaline phosphatase in the femoral diaphysis. Of the above biochemical indices, significant increases in collagen content and alkaline phosphatase activity were observed at 1d of zinc administration, while DNA and calcium contents were significantly increased by the metal administration for 3d. With the dose of 5.0mg Zn/100g, the effect of zinc on bone component reduced slightly. These results suggest that comparatively low dose of zinc may play a physiological role as an activator of bone metabolism in weanling rats.

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The role of cytoskeleton in the expression of the differentiated phenotype of chondrocytes was investigated. Cytochalasin B, a microfilament disrupting agent, changed the shape of cultured rabbit costal chondrocytes from polygonal to nearly spherical and stimulated glycosaminoglycan synthesis (GAG), which is a differentiated phenotype of chondorcytes. Colchicine, a microtubule disrupting agent, changed them from polygonal to flattened and inhibited GAG synthesis. Induction of ornithine decarboxylase, the rate-limiting enzyme of polyamine biosynthesis, by parathyroid hormone (PTH), which is a good marker of differentiated chondorcytes, was markedly potentiated in the spherical cells which had been pretreated with cytochalasin B. In addition, pretreatment of chondorcytes with cytochalasin B enhanced PTH-stimulated-GAG synthesis, whereas pretreatment with colchicine inhibited the increases of ODC activity and GAG synthesis stimulated by PTH. Both cytochalasin B and colchicine inhibited DNA synthesis. The inhibition of DNA synthesis were observed after the appearance of the changes in the morphology of the cells and GAG synthesis. These findings suggested that intact microtubules and disruption of microfilaments are involved in regulating the expression of the differentiated phenotype of chondrcytes in culutre.

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A tumor promoter, 12-0-tetradecanoylphorbol-13-acetate (TPA), inhibited expression of the differentiated phenotype of chondrocytes in rabbit costal chondrocytes in culture, as judged by morphological changes and a decrease in glycosaminoglycan (GAG) synthesis. These effects were similar to those of retinoids [Takigawa et al. Mol. Cell. Biochem. 42: 145-153, 1982]. However, the morphology and decreased GAG synthesis in chondrocytes which had been pretreated with TPA were restored not only by dibutyryl cyclic AMP (DBcAMP) but also by parathyroid hormone (PTH), unlike retinoid-treated cells. Pretreatment with TPA did not prevent PTH-stimulated cAMP accumulation nor PTH-stimulated ornithine decarboxylase induction, in contrast to the inhibitory effect of retinoids. TPA-treated cells proliferated at a higher rate and gained a higher saturation density than the controls. TPA also increased DNA synthesis. These growth stimulatory effects were inhibited by PTH as well as DBcAMP.
From these results, it is suggested that: 1) Unlike retinoid-treated cells, TPA-treated cells retain some of the differentiated phenotype of the original cells, such as responsiveness to PTH. 2) cAMP plays an important role in restoration of expression of the differentiated phenotype of chondrocytes inhibited by both types of modulators of chondrocyte differentiation.

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A 46-year-old woman was admitted to Juntendo University Hospital on suspicion of Sjögren syndrome complicated with distal renal tubular acidosis and a slight hypocalcemia. Serum levels of 25-hydroxyvitamin D and 1α, 25-dihydroxyvitamin D were within normal limit (11.9ng/ml and 43.8pg/ml, respectively), but that of 24, 25-dihydroxyvitamin D was less than the detection limit (0.025ng/ml) of the assay. Neither treatment of metabolic acidosis nor daily administration of 1α-hydroxyvitamin D₃ (0.5μg/day) for 3 days affected the serum level of 24, 25-dihydroxyvitamin D. However daily administration of 100μg/day of vitamin D₂ for 7 days increased the serum level of 24, 25-dihydroxyvitamin D to 0.7ng/ml. Serum levels of parathyroid hormone were within normal range. Histological examinations of biopsy specimen from iliac crest revealed that there is a moderate increase in osteoid seams. This case poses a question about the tubular localization of 25-hydroxyvitamin D-24hydroxylase and the biologic action of 24, 25-dihydroxyvitamin D in humans.

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The effects of Cytochalasin B and 1, 25-dihydroxycholecalciferol (1, 25-(OH)₂D₃) on alkaline phosphatase and sucrase activities were studied using the organ culture system of 20-day-old chick embryonic duodena. A significant inhibitory effect of cytochalasin B on duodenal alkaline phosphatase activity was observed, although this inhibitory effect was cancelled by high concentration of 1, 25-(OH)₂D₃ (625nM). Duodenal sucrase activity was influenced by neither cytochalasin B (up to 10μg/ml) nor 1, 25-(OH)₂D₃ (62.5-625nM). On the other hand, alkaline phosphatase activity in the culture media was rather high compared with the activity in tissue in the presence of cytochalasin B. From these results, it was suggested that cytochalasin B could compete the site of 1, 25-(OH)₂D₃ action in the duodenal microvilli.

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We have analyzed the trabecular bone of the ilium from six postmenopausal women by using a quantitative histomorphometry, to distinguish osteoporosis from physiological osteopenia in aging. Three of these 6 subjects, with mild Osteopenia and relatively slow rate of bone loss, probably representing physiological osteopenia in aging, showed a decrease of mean wall thickness (MWT). Relative thickness of interstitial bone between two packets in the trabecular bone (%IBT) tended to increase with advancing age. Osteoblastic lifespan, estimated by the bone formation period (sigma f), was shortened in proportion to a decrease of MWT. These findings suggest that the shortening of osteoblastic lifespan and the resulting decrease of bone formation are responsible for the bone loss in physiological Osteopenia in aging.
In contrast to these 3 subjects, the remaining 3 with considerably small bone mass and rapid bone loss, were thought to have osteoporosis as a pathological process. They exhibited thin trabeculae (small MTT) with a concomitant decrease of mean thickness of interstitial bone (MIBT). One of them, with multiple compression fractures of the vertebrae, had typical lowturnover osteoporosis characterized by the absence of tetracycline apposition. Since there was no difference in the MWT between “osteoporotics” and “physiological osteopenics”, the thin trabeculae in the bone of osteoporosis was apparently due to a decrease in the amount of interstitial bone representing the remnant of the pre-existing bone resorbed by osteoclasts. This evidence suggests that osteoporosis is characterized by the enhanced resorption of pre-existing bone (interstitial bone). Furthermore, the findings in low-turnover osteoporosis which showed smallest MIBT among the subjects despite scanty resorption surface, might indicate that osteoporosis, regardless of the current activity of bone remodeling, should have undergone a period of high-turnover phase, sometime in the past.
In conclusion, the mechanisms of bone loss appear to be different between postmenopausal osteoporosis and physioloigcal osteopenia in aging. Postmenopausal osteoporosis may therefore be regarded as a discrete clinical entity rather than an extreme condition of physiological bone loss with aging.

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A high molecular weight factor(s) was extracted from fetal bovine cartilage with 1M guanidine hydrochloride. This factor inhibited neither the proliferation of sarcoma 180 cells in culture nor the growth of their ascites form. However, it strongly inhibited the growth of solid sarcoma 180 in vivo.

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The normal variation and bilateral symmetry of bone remodeling at different sites in iliac trabecular bone were studied in 2 male and 2 female adult beagle dogs to determine a suitable site for biopsy. Bone remodeling parameters were measured in transverse sections at 5mm intervals using an image analyzer. Significant differences were found in sections at different sites in the same animal in the values for the trabecular bone specific volume, mean trabecular or seam thickness, fractional formation surface, mineral apposition rate, tetracycline labelling rate, corrected mineral appositional rate, bone formation rate and period and/or trabecular bone specific surface, but these differences were not significant in adjacent sites between 10mm and 15mm from the crest. The state of bone remodeling at a site 15mm from the crest was found to be representative of that of all sections of the ilium. Less than about 10% mean variation was observed in almost all parameters of bone remodeling in symmetrical sections, but no significant differences were found in bone remodeling in sections from different sites of the left and right side except in the mean seam thickness. Symmetrical variation in the mean seam thickness of a site 10mm from the crest seemed to be different from those of three other sites. It is concluded, therefore, that symmetrical transverse sections should be taken sequentially at sites of both ilia of beagle dogs about 15mm from the crest to evaluate alteration(s) in trabecular bone remodeling associated with metabolic bone diseases or with therapeutic treatments.

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