Gene and Protein Expression During Differentiation and Matrix Mineralization in a Chondrocyte Cell Culture System

Endochondral bone formation occurs through a series of developmentally regulated cellular stages, from initial formation of cartilage tissue to calcified cartilage, resorption, and replacement by bone tissue. Nasal cartilage cells isolated by enzymatic digestion from rat fetuses were seeded at a final density of 10⁵ cell/cm² and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum in the presence of ascorbic acid and β-glycerophosphate. First, cells lost their phenotype but in this condition they rapidly reexpressed the chondrocyte phenotype and were able to form calcified cartilaginous nodules with the morphological appearance of cartilage mineralization that occurs in vivo during endochondral ossification. In this mineralizing chondrocyte culture system, we investigated, between day 3 and day 15, the pattern expression of types II and X collagen, proteoglycan core protein, characteristic markers of chondrocyte differentiation, as well as alkaline phosphatase and osteocalcin associated with the mineralization process. Analysis of labeled collagen and immunoblotting revealed type I collagen synthesis associated with the loss of chondrocyte phenotype at the beginning of the culture. However, our culture conditions promoted extracellular matrix mineralization and cell differentiation towards the hypertrophic phenotype. This differentiation process was characterized by the induction of type X collagen mRNA, alkaline phosphatase, and diminished expression of type II collagen and core protein of large proteoglycan after an increase in their mRNA levels before the mineralizing process. These results revealed distinct switches of the specific molecular markers and indicated a similar temporal expression to that observed in vivo recapitulating all stages of the differentiation program in vitro. Received: 12 December 1996 / Accepted: 26 June 1997     

Human Osteoarthritic Cartilage Matrix Vesicles Generate Both Calcium Pyrophosphate Dihydrate and ApatiteIn Vitro

Calcium crystals in osteoarthritic (OA) joints promote enzymatic degradation of articular tissues. Matrix vesicles provide a nidus for calcium crystal formation in chick epiphyseal and mature porcine articular cartilage. In order to examine a potential role for matrix vesicles from OA cartilage in generating pathologic crystals, we sought to determine whether vesicles derived from human OA cartilage (OAMV) could mineralize; and we characterized the resultant mineral species. OAMV were isolated and examined for alkaline phosphatase (AP) and nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity. OAMV ATP-dependent and independent mineralization were measured in a radiometric biomineralization assay, and newly formed OAMV crystals were examined using Fourier transform infrared spectroscopy (FTIR) and compensated polarized light microscopy. The mean specific activity of OAMV AP was approximately 6 times higher and NTPPPH activity 11 times lower than that of previously characterized, mature, porcine, articular cartilage vesicles. OAMV progressively precipitated ⁴⁵Ca over time both in the presence and absence of ATP. The FTIR spectra of mineral formed in ATP-dependent assays most closely resembled the standard spectrum for calcium pyrophosphate dihydrate (CPPD). The FTIR spectra of OAMV mineral formed in the absence of ATP closely resembled apatite. These data support the hypothesis that OAMV may form mineral phases of two key crystals found in degenerating cartilage and provide further evidence for the role of matrix vesicles in pathologic articular cartilage biomineralization.     

Different Behavior of Human Osteoblast-Like Cells Isolated from Normal and Heterotopic Bone In Vitro

In this study, a characterization of human bone-forming cells responsible for heterotopic ossification was carried out in vitro. The biological and biochemical cell characteristics of the heterotopic osteoblast-like (HOB) cells were compared with those of orthotopic osteoblast-like (OB) cells from normal bone and stromal bone marrow cells believed to contain a subpopulation of osteogenic precursor cells. We found that HOB's from the spongiosa of heterotopic ossification required less time until the beginning of migration and the achievement of confluence in vitro compared with OBs from femoral shaft spongiosa. The fraction of mitotically active cells assessed by a clonogenic assay was higher as well in HOB cells. The in vitro studies of mitogenesis and the efficiency of colony formation of osteogenic cells indicate that with increasing differentiation and relative age they become more dependent on growth factors in the medium, otherwise the morphology of osteoblast-like cells changes and they pass irreversibly into the postmitotic stage of the cell cycle. The activity of the alkaline phosphatase is distinctly higher in the HOB than in the OB cells, HOB cells exhibit a lower level of osteocalcin expression compared with OB cells. No significant difference was found between OB and HOB cells in the amount of procollagen of type I sequestered by the cells. After 30 days, HOB and OB cells formed a mineralized matrix on exposure to 2 mM β-glycerophosphate. Since HOBs were isolated from heterotopic bone that had developed within 3–6 months after hip surgery, the differences in cellular behavior compared with OBs may be attributed to the relatively young age of HOB cells. Received: 29 March 1996 / Accepted: 21 May 1997     

Bone Mineral Density and Bone Turnover in Patients with Knee Osteoarthritis Compared with Generalized Osteoarthritis

The aim of this study was to investigate bone mineral density (BMD) and bone turnover in patients with primary knee osteoarthritis (KOA) and to compare them with generalized OA (GOA) and nonGOA patients. A total of 88 postmenopausal primary KOA patients were studied. OA was graded by using knee radiographs. BMD of the lumber spine, femur, and radius, and biochemical markers of bone turnover, pyridinoline (Pyr), deoxypyridinoline (Dpyr), CTx, and osteocalcin were compared among each grade. BMD was also compared with 88 normal controls who were age and weight-matched. In 88 KOA patients, 56 were divided into 28 GOA and 28 non-GOA groups by grading hand radiographs. BMD and biochemical markers were compared between GOA and non-GOA. KOA patients had higher BMD at several skeletal sites compared with age- and weight-matched normals. A significant difference of BMD between each grade was observed between grades 0–1 and 3 (0.774 ± 0.143 versus 0.940 ± 0.185 g/cm², P < 0.001), grades 2 and 3 (0.781 ± 0.125 versus 0.940 ± 0.185 g/cm², P < 0.01) in the spine, and between grades 0–1 and 3 (0.505 ± 0.100 versus 0.564 ± 0.127 g/cm², P < 0.05) in the trochanter. A significant difference of biochemical bone markers was observed between grades 0–1 and 3 (P < 0.05) and between grades 2 and 3 (P < 0.05) in Pyr and grades 0–1 and 3 (P < 0.05) and between grades 1 and 4 (P < 0.05) in Dpyr, but not in osteocalcin and CTx. GOA patients had higher BMD of the spine (0.902 ± 0.175 versus 0.747 ± 0.138 g/cm², P < 0.01), trochanter (0.535 ± 0.107 versus 0.480 ± 0.107 g/cm², P < 0.05), and one-third of the radius (0.526 ± 0.068 versus 0.472 ± 0.089 g/cm², P < 0.05) and had significantly higher biochemical markers in Pyr and Dpyr than non-GOA patients. It is concluded that KOA patients had higher BMD at several skeletal sites. Biochemical bone markers were influenced by some degree of cartilage damage in OA patients. This tendency was stronger in GOA patients than in non-GOA patients. Received: 12 February 1999 / Accepted: 2 November 1999     

Characterization of Demineralized Bone Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: III. Gene and Protein Expression

Our previous studies of rat cranial defect repairs after the implantation of demineralized bone matrix (DBM) have demonstrated that healing occurs initially and principally by the direct induction and proliferation of osteoblasts derived principally from resident mesenchymal stem cells of the dura, and to a lesser extent by resident mesenchymal stem cells of the connective tissues beneath the skin flap. A small amount of cartilage is also synthesized after the direct process of ossification occurs. To further confirm the molecular phenotypes of the repair cells in rat cranial defects, the present study evaluated mRNA expression and synthesis of collagens I, II, and X and osteocalcin in the DBM-induced repair tissue by Northern blot analyses, autoradiography after in vivo ³H-proline labeling of collagen, and immunohistochemistry. The results demonstrated that osteocalcin mRNA appeared in small amounts by day 4 and continued to increase over the experimental period. Much lesser quantities of collagen types II and X mRNAs appeared by day 6 and day 8, respectively. Collagen type I mRNA was present at all times examined but its expression significantly increased by day 5. Autoradiographic and immunohistochemical studies showed that type II collagen was not detected whereas type I collagen was synthesized on days 3–5. The data provide definitive molecular evidence confirming that the initial and by far the major pathway of cranial defects repair induced by implantation of DBM is by the direct induction of resident mesenchymal stem cells to osteoblasts and the direct formation of bone, which is spatially and temporarily distinct from the later formation of cartilage. Received: 30 November 1999 / Accepted: 21 March 2000     

Development and Application of a Synthetic Peptide-Based Osteocalcin Assay for the Measurement of Bone Formation in Mouse Serum

The mouse is frequently used as an animal model to study skeletal mechanisms relevant to humans. Biochemical markers of bone formation and resorption provide one of the key parameters for assessing skeletal metabolism. One biochemical marker that has proven to be useful in the studies of mouse skeletal metabolism is osteocalcin. Assay for osteocalcin is available in the mouse. The present study describes development of an osteocalcin radioimmunoassay (RIA) using a synthetic peptide. Intact osteocalcin purified from mouse bone extracts shows parallel displacement with synthetic peptide. Sensitivity of the RIA was 19 ng/ml. The average (n = 9) intra- and interassay coefficient of variation for two controls was less than 10%; the averaged recoveries were 106%. The osteocalcin concentration measured by peptide RIA shows a high correlation (r = 0.88, n = 117, P < 0.0001) with an intact osteocalcin assay. In addition, when the intact assay and peptide assays were applied to evaluate skeletal perturbation, similar results were obtained. Accordingly, osteocalcin levels measured by both intact and peptide-based RIA in 8-week C57BL/6J (n = 8) mice treated with PTH 1-34 were twofold higher compared with the vehicle-treated control group. Further studies of the application of the peptide-based RIA for osteocalcin revealed that osteocalcin levels in 4-week postovariectomized (OVX) C57BL/6N mice (n = 10) were 80% higher than the sham-operated (n = 10) mice receiving vehicle. OVX mice receiving weekly injections of estradiol (400 μg/kg body weight) were 38% lower compared with the OVX group treated with vehicle. In conclusion, the peptide-based RIA has analytical and a discriminative power similar to that of the intact osteocalcin assay but has the advantage that the resources for this assay are much easier to accrue. Received: 29 October 1999 / Accepted: 1 March 2000     

Effect of Polyethylene on Osteocalcin, Alkaline Phosphatase and Procollagen Secretion by Human Osteoblastic Cells

We have studied the direct effects of polyethylene particles on osteoblastic function in primary human bone cell cultures. The cells were obtained from trabecular bone fragments of patients undergoing knee reconstructive surgery. When the cells reached confluency, they were subcultured into two flasks, one untreated (control culture) and the other treated with polyethylene particles, and incubated until confluency. Osteoblastic function was evaluated by assaying osteocalcin, alkaline phosphatase, and C-terminal procollagen type I, with and without 1,25(OH)₂D stimulation, in the cell-conditioned medium. We found that addition of polyethylene to these osteoblastic cell cultures induced higher levels of secreted osteocalcin after 1,25(OH)₂D stimulation. Alkaline phosphatase levels increased whereas C-terminal procollagen type I levels decreased in the cell conditioned medium after polyethylene was added to the cultures. Treatment of the control cultures with 1,25(OH)₂D stimulated alkaline phosphatase levels and decreased C-terminal procollagen type I. However, these osteoblastic markers in 1,25(OH)₂D-treated cells did not change in cultures with polyethylene. This study demonstrates that polyethylene particles have a direct effect on osteoblastic markers in human bone cells in culture. Received: 2 May 1996 / Accepted: 17 June 1997     

Triiodothyronine, a Regulator of Osteoblastic Differentiation: Depression of Histone H4, Attenuation of c-fos/c-jun, and Induction of Osteocalcin Expression

Thyroid hormones influence growth and differentiation of bone cells. In vivo and in vitro data indicate their importance for development and maintenance of the skeleton. Triiodothyronine (T3) inhibits proliferation and accelerates differentiation of osteoblasts. We studied the regulatory effect of T3 on markers of proliferation as well as on specific markers of the osteoblastic phenotype in cultured MC3T3-E1 cells at different time points. In parallel to the inhibitory effect on proliferation, T3 down-regulated histone H4 mRNA expression. Early genes (c-fos/c-jun) are highly expressed in proliferating cells and are down-regulated when the cells switch to differentiation. When MC3T3-E1 cells are cultured under serum-free conditions, basal c-fos/c-jun expressions are nearly undetectable. Under these conditions, c-fos/c-jun mRNAs can be stimulated by EGF, the effect of which is attenuated to about 46% by T3. In addition, T3 stimulated the expression at the mRNA and protein level of osteocalcin, a marker of mature osteoblasts and alkaline phosphatase activity. All these effects were more pronounced when cells were cultured for more than 6 days. These data indicate that T3 acts as a differentiation factor in osteoblasts by influencing the expression of cell cycle–regulated, of cell growth–regulated, and of phenotypic genes. Received: 10 May 1996 / Accepted: 5 June 1997     

Clinical Validation of a New Immunoradiometric Assay for Intact Human Osteocalcin

The purpose of this study was to estimate clinical validity of a new available immunoradiometric assay for circulating intact human BGP (N-tact Osteo SP) by measuring this protein in a large number of normal subjects and patients with the most common metabolic bone diseases. One hundred normal subjects were studied in order to obtain our normal ranges (4.9 ± 1.7 ng/ml). The mean values found in 28 patients with primary hyperparathyroidism (17.5 ± 22.8 ng/ml, P < 0.001), 15 glucocorticoid-treated patients (1.9 ± 1.5, P < 0.001), 10 patients with hypoparathyroidism (1.5 ± 0.7, P < 0.001), 9 with hyperthyroidism (8.3 ± 3.8, P < 0.001), 8 with skeletal metastases (7.2 ± 2.3, P < 0.001), and 4 with humoral hypercalcemia of malignancy (2.42 ± 1.91, P < 0.005) were significantly different from mean values found in normal subjects. Mean decrease of serum osteocalcin T-score values was significantly greater when evaluated by N-tact Osteo SP assay in 15 steroid-treated patients (−1.4 ± 1.0) and 19 primary hyperparathyroid (PHPT) patients (3.6 ± 1.9), compared with the mean values obtained with the Elsa-Osteo assay (−0.67 ± 1.2, P < 0.002 and 4.3 ± 2.8, P < 0.04, respectively). We found significant correlations between the global skeletal uptake of 99mTc-methylendiphosphonate and serum BGP levels assayed by both N-tact Osteo SP (P < 0.01) and Elsa-Ost-Nat assay (P < 0.05). Our results indicate that this new immunoradiometric assay for the intact human osteocalcin has the potential for good discrimination between normal subjects and patients with both low and high bone turnover. Furthermore, our findings emphasize the fact that, in the absence of available standardized commercial assays, one should rely on only one assay because different results are obtained by different assays under different clinical conditions. Received: 22 January / Accepted: 22 September 1998     

Diurnal Variation in Total and Undercarboxylated Osteocalcin: Influence of Increased Dietary Phylloquinone

A diurnal variation exists in blood levels of the vitamin K-dependent bone protein osteocalcin. However, it is not known whether the carboxylated and undercarboxylated constituents of osteocalcin also vary. Therefore, osteocalcin and undercarboxylated osteocalcin were measured in specimens collected every 4 hours over a 24-hour period in nine healthy subjects (five males, four females) ages 20–33 years who were consuming a mixed diet containing 100 μg of phylloquinone. Osteocalcin and undercarboxylated osteocalcin were measured by radioimmunoassay (RIA) before and after treatment with barium sulfate. Although the percent undercarboxylated osteocalcin did not change, a diurnal variation was observed in total osteocalcin, carboxylated osteocalcin, and undercarboxylated osteocalcin, with peak concentrations at 4 a.m. and the lowest concentrations between 12 p.m. and 4 p.m. The difference between the total osteocalcin peak and trough concentrations averaged 28 ± 7 (SEM)%. There were no gender differences in these rhythms. The effect of dietary phylloquinone as a modulator of these rhythms was evaluated in a randomized study by increasing phylloquinone intake to 420 μg/day with fortified corn oil, split between the lunch and dinner meals. Total and carboxylated osteocalcin fluctuations and concentrations were not affected by the dietary treatment. The diurnal variation in undercarboxylated osteocalcin was abolished with supplementation and concentrations at 8 a.m. (14 hours following supplementation) (2.3 ± 0.2 ng/ml) were significantly lower than the unsupplemented levels (2.7 ± 0.2 ng/mL, P= 0.006). The percentage of undercarboxylated osteocalcin was similarly decreased after supplementation (19.7 ± 1.3%) in relation to the mixed diet cycle (24.2 ± 1.6%, P= 0.006) at 8 a.m. on the second day. Dietary supplementation induced a fluctuation in percentage undercarboxylated osteocalcin with a decline in levels starting at approximately 12 a.m. Therefore, additional dietary phylloquinone does not appear to modulate the total osteocalcin diurnal rhythm, but can influence its undercarboxylated component. Received: 14 June 1996 / Accepted: 22 August 1997     

Expression of Bone Sialoprotein in Human Lung Cancer

Lung cancer belongs to the group of malignant lesions that specifically select bone as secondary implantation site. The molecular bases for this property, defined as osteotropism, is still largely unknown. The recent demonstration that human breast cancer cells express and attach to bone sialoprotein (BSP), a sulfated phosphoprotein rich in bone and other mineralized tissues, could provide a clue to elucidating bone metastases formation. BSP contains the integrin binding peptide Arg-Gly-Asp (RGD), as well as non-RGD cell attachment domain. Using an immunoperoxidase technique and a specific polyclonal antibody directed against a BSP synthetic peptide, we examined the expression of BSP in 48 lung lesions including 25 squamous carcinoma, 21 adenocarcinoma, and 2 bronchioloalveolar cancers, as well as 38 human ovarian carcinoma that constitute a group of generally nonosteotropic cancers. BSP was not specifically detected in normal lung tissue with the exception of cartilage associated with bronchi. Most of the adenocarcinoma (74%) and all squamous carcinoma of the lung examined exhibited detectable levels of BSP. Staining was mainly cytoplasmic and membrane associated. The two bronchioloalveolar lung cancers examined did not show detectable amounts of BSP. When microcalcifications were observed in pulmonary malignant lesions, they were usually associated with cancer cells expressing BSP. Only 21% of the ovarian cancers examined contained malignant cells with 2+ or 3+ positivity for BSP. We further demonstrated that in 8 of 10 additional lung cancers, BSP was detected at the mRNA level. Our observation is the first demonstration that BSP is expressed in non-small cell lung carcinoma. Lung cancer cells are now the second type of osteotropic malignant cells described to express BSP. Added to the observation that BSP expression is not frequent in ovarian carcinoma, a low osteotropic cancer, our study supports our hypothesis that BSP could play a role in determining the affinity of cancer cells to bone. Received: 30 August 1996 / Accepted: 23 April 1997     

Effects of Magnesium Deficiency on Magnesium and Calcium Content in Bone and Cartilage in Developing Rats in Correlation to Chondrotoxicity

Quinolone-induced arthropathy has been described in juvenile rats between 3 and 6 weeks of age, but not in adult rats. The mechanism of this chondrotoxic effect is probably related to the Mg²⁺-chelating properties of the drugs, since identical cartilage lesions were observed in magnesium-deficient juvenile rats without quinolone treatment. However, the reasons for the phase-specificity of the effect are unknown. In the present study, we fed a magnesium-deficient diet to Wistar rats at different postnatal developmental stages. Cartilage lesions were only observed in magnesium-deficient rats between 3 and 5 weeks of age, but not in rats receiving the magnesium-deficient diet during weeks 5 to 8, weeks 8 to 11, or months 15 to 16. The formation of cartilage lesions was not related to the magnesium concentration in plasma, since magnesium concentrations in plasma were similarly reduced in rats with and without cartilage lesions. However, chondrotoxicity correlated with magnesium content in articular cartilage. In articular cartilage (articular and epiphyseal cartilage in immature rats) and bone, magnesium content was more reduced in rats receiving the magnesium-deficient diet between 3 and 5 weeks of age as compared with rats receiving the magnesium-deficient diet during weeks 8 to 11 postnatally. It was not possible to reduce the magnesium content in bone tissue of 15-month-old Wistar rats, which suggests a lower magnesium turnover in aged rats. Magnesium content in epiphyseal cartilage of 2-week-old rats (total femoral head) was 41.9 ± 16.9 mmol/kg dry weight. The magnesium content in joint hyaline cartilage was significantly lower in 4-week-old rats (19.5 ± 3.6 mmol/kg dry weight) and increased subsequently again to 48.5 ± 9.2 mmol/kg dry weight (mean ± SD; n= 8 to 16). Increase of the magnesium content in femoral bone between weeks 4 and 6 postnatally was less pronounced (139 ± 10 and 175 ± 15 mmol/kg dry weight, respectively). Taken together, these data show that in 4-week-old rats, magnesium concentration in joint hyaline cartilage is significantly lower than at other times during postnatal development. Only at this developmental stage can cartilage lesions be induced by feeding rats a magnesium-deficient diet. This period correlates well with the sensitive phase of immature rats toward the chondrotoxic action of quinolones. Received: 30 September 1996 / Accepted: 31 December 1996     

1,25 Dihydroxyvitamin-D3 Attenuates the Confluence-Dependent Differences in the Osteoblast Characteristic Proteins Alkaline Phosphatase, Procollagen I Peptide, and Osteocalcin

In the present study a cell culture model of primary human osteoblasts based on degrees of confluence was investigated by measuring basal and 1,25(OH)₂D₃stimulated levels of the osteoblast characteristic proteins alkaline phosphatase (AP), procollagen I-peptide (PICP), and osteocalcin (OC), as well as the corresponding gene expression. Primary osteoblast-like cell cultures from seven donors were treated in the second passage with 1,25(OH)₂D₃ (5 × 10⁻⁸ M for 48 hours) and investigated at four stages of confluence (stage I 50%, stage II 75%, stage III 100%, and stage IV 7 days postconfluence). In untreated cultures passing through the different stages of confluence, we saw a 1.8-fold increase of AP activity, a 2.3-fold increase of OC secretion, but a decrease of PICP levels to 0.36-fold. Gene expression showed only minor variation between the different confluence stages. 1,25(OH)₂D₃ did not significantly affect PICP production. Alkaline phosphatase protein was stimulated during proliferation until confluence, with no effect thereafter. Surprisingly, OC secretion and mRNA expression were stimulated in all four stages to the same absolute level independent of basal values. We conclude that our results correspond to other studies showing differentiation-stage dependent changes of basal levels of osteoblast-specific proteins. However, 1,25(OH)₂D₃ stimulation decreased the confluence-dependent difference for AP and abolished it for osteocalcin, thus leading to a more differentiated phenotype of the osteoblast. Therefore, 1,25(OH)₂D₃ stimulation might improve the reproducibility of results obtained at different confluence stages from cultures of clinical samples. Received: 25 November 1997 / Accepted: 2 September 1998     

Human Osteoclast Formation from Blood Monocytes, Peritoneal Macrophages, and Bone Marrow Cells

Mononuclear precursors of the human osteoclast have been identified in both bone marrow and the circulation in man, but osteoclast membership of the mononuclear phagocyte system (MPS) and its precise cellular ontogeny remain controversial. We isolated human hematopoietic marrow cells, blood monocytes, and peritoneal macrophages and incubated each of these cell populations with UMR106 osteoblast-like cells on glass coverslips and dentine slices in both the presence and absence of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃), macrophage-colony stimulating factor (M-CSF), and dexamethasone. Cells isolated from peripheral blood and peritoneal dialysis fluid were positive only for monocyte/macrophage markers (CD11a, CD11b, CD14, and HLA-DR) and negative for osteoclast markers [tartrate-resistant acid phosphatase (TRAP), vitronectin reception (VNR), and calcitonin (CT) receptors and did not form resorption pits on dentine slices after 24 hours in culture. Similarly marrow cells did not form resorption pits on dentine slices after 24 hours in culture. However, after 14 days in co-culture with UMR106 cells, in the presence of 1,25(OH)₂D₃ and M-CSF, numerous TRAP, CT receptor, and VNR-positive multinucleated cells capable of extensive lacunar resorption were formed in co-cultures of all these preparations. The presence of 1,25 (OH)₂D₃, M-CSF, and UMR106 were absolute requirements for osteoclast differentiation. It is concluded that precursor cells capable of osteoclast differentiation are present in the marrow compartment, the monocyte fraction of peripheral blood, and in the macrophage compartment of extraskeletal tissues and that these cells are capable of differentiating into mature functional osteoclasts. These findings argue in favor of osteoclast membership of the human MPS. Received: 3 January 1997 / Accepted: 14 November 1997     

Effects of Indomethacin Administration on Bone Turnover and Bone Mass in Adjuvant-Induced Arthritis in Rats

We examined the bone turnover and bone mass in adjuvant-induced arthritis in rats and assessed the effects of indomethacin in this model. One hundred ten SD rats, 6 weeks of age, were assigned to 11 groups and injected with adjuvant or solvent in the right foot. Adjuvant-injected rats were orally administered indomethacin at doses of 0 (vehicle), 0.1 (low), 0.5 (medium), and 1.5 (high) mg/kg body weight from the start (day 0). Animals were sacrificed on days 0, 14 (acute phase), and 28 (chronic phase). In the arthritic-control group, serum osteocalcin level and bone mineral content of the fourth lumbar body (L4) and the femur were significantly reduced on day 14. Serum alkaline-phosphatase was increased on day 28. Trabecular bone volume of L4 was decreased on day 14, and the value was further decreased on day 28. Bone formation rate (BFR/BS) was significantly reduced on day 14, and then osteoclast number (Oc.N/BS) increased on day 28. Indomethacin treatment dose-dependently prevented increases in paw volume and osteoclast number. In the high dose group, these indices were maintained at the same level with those in the normal group. However, indomethacin treatments were not able to maintain the parameters of bone formation such as serum osteocalcin and BFR/BS values, and the trabecular bone mass decrease was only partially prevented. These data clearly indicated both reduced bone formation and increased bone resorption as the causes of bone loss in adjuvant-induced arthritis in rats. Increased bone resorption seemed to be due to the increased activity of prostaglandins, but bone formation defect would be related to other factors in this animal model. Received: 13 January 1996 / Accepted: 3 May 1996     

Plasma Leptin Values in Relation to Bone Mass and Density and to Dynamic Biochemical Markers of Bone Resorption and Formation in Postmenopausal Women

After the menopause it has been noted that heavier women conserve bone better than those with lower body weight. The protective effect of obesity on bone mass has been ascribed to a high body fat content. The present study of 54 postmenopausal women was undertaken to determine whether circulating plasma levels of leptin, the newly described hormone produced in adipocytes, were correlated with age-adjusted total body bone mineral content (BMC) or bone mineral density (BMD), or with dynamic biochemical markers of bone resorption or of bone formation. Leptin values were strongly correlated with all measures of adiposity (P < 0.001). Age-adjusted values for BMC and BMD, respectively, were also positively correlated (P < 0.001) with body weight (r = 0.643, r = 0.502), total fat mass (r = 0.557, r = 0.510) and with plasma leptin concentrations (r = 0.480, r = 0.551), confirming a positive relationship between fat mass and bone mass. By contrast, no significant correlations were observed between plasma leptin and dynamic markers of bone resorption (urinary deoxypyridinoline/creatinine r =−0.105, hydroxyproline/creatinine r =−0.193) or formation (plasma osteocalcin r = 0.103). Because there was no evidence for an association between ciculating plasma levels of leptin and biochemical markers of either osteoclastic or osteoblastic activity we conclude it is unlikely that circulating leptin plays any significant direct role in controlling bone cell activity. Our results do not support the hypothesis that leptin mediates the bone-sparing effects of obesity. Received: 23 September 1997 / Accepted: 11 May 1998     

Age-Related Changes in Serum Undercarboxylated Osteocalcin and its Relationships with Bone Density, Bone Quality, and Hip Fracture

The effect of the degree of carboxylation of osteocalcin (OC) on the properties of bone is unclear. The aim of this study was to relate serum concentrations of total OC (tOC) and undercarboxylated OC (ucOC), measured with a two-site immunoassay, to bone mineral density (BMD) at the femoral neck and ultrasonic transmitted velocity (UTV) at the os calcis in 257 women aged 60–99 years, 22 of whom had sustained a hip fracture. There was an increase in tOC (r = 0.19, P= 0.003) and ucOC (r = 0.20, P= 0.002) with age. No significant difference in tOC or ucOC between subjects with and without hip fracture was found. Serum tOC was negatively correlated with femoral neck BMD (r =−0.23, P= 0.0001) and os calcis UTV (r =−0.29, P= 0.0001) and partial correlations indicated that these relationships were independent of age. Serum ucOC also correlated negatively with os calcis UTV (r =−0.21, P= 0.001) and less strongly with femoral neck BMD (r =−0.13, P= 0.052). After adjusting for age, only the relationship between ucOC and os calcis UTV remained significant (r =−0.16, P= 0.017). It is concluded that in women over 60 years, the increase in tOC reflects an age-related rise in bone remodeling, whereas the increase in ucOC reflects an age-related fall in vitamin K status. The stronger relationship of ucOC with UTV than BMD suggests that the rise in ucOC may perhaps relate more to changes in bone quality than mineral content. Higher serum ucOC concentrations in subjects with a history of hip fracture could not be confirmed. Received: 8 January 1997 / Accepted: 29 September 1997     

Low Bone Density with Normal Bone Turnover in Ovariectomized and Streptozotocin-Induced Diabetic Rats

Diabetes and estrogen deficit are known causes of osteopenia, diabetes being associated with a low bone turnover and estrogen deficit with a high bone turnover. In the present work, we studied the effect of combined ovariectomy and diabetes on bone mineral content (BMC) and bone mineral density (BMD) and several bone markers in the rat. Four groups of rats were studied: control (C), ovariectomized (O), diabetic (D), and ovariectomized and diabetic (DO). Twelve weeks after starting the experiments, BMC and BMD of the first six lumbar vertebrae were measured; a bone formation marker (BGP) and a bone resorption marker (free collagen cross-links, PYD) were also analyzed. Diabetic rats showed diminished gain in bone mass, BMC (D: 0.417 ± 0.028 g, DO: 0.422 ± 0.020 g) and BMDs (D: 0.171 ± 0.006 g/cm², DO: 0.174 ± 0.006 g/cm²) both being significantly (P < 0.001) lower than those of control (C: BMC 0.727 ± 0.024 g and BMD 0.258 ± 0.004 g/cm²) and ovariectomized (O: BMC 0.640 ± 0.044 g and BMD 0.240 ± 0.009 g/cm²) groups. Moreover, the BMC and BMD of the C group were significantly (P < 0.05) higher than that of the O group. BGP and PYD levels were significantly (P < 0.01) higher in the O group (BGP: 138.2 ± 16.8 ng/ml, PYD: 270.2 ± 17.8 nM/mM) than those found in the control rats (BGP: 44.7 ± 4.8 ng/ml, PYD: 165.6 ± 12.5 nM/mM); the D group showed significantly (P < 0.01) lower values (BGP: 27.4 ± 14.6 ng/ml, PYD: 55.0 ± 7.4 nM/mM) than those of the control group. The DO group showed similar levels (BGP: 43.4 ± 5.1 ng/ml, PYD: 146.7 ± 14.6 nM/mM) to those found in the C group. Although bone marker levels in the O and D groups were in accordance with those expected in these situations, in the DO group the corresponding levels are apparently ``normal.' Also, the decrease of gain in bone mass observed after combining estrogen deficit and diabetes (DO group) did not seem to be more marked than that caused by diabetes alone. Received: 7 January 1997 / Accepted: 7 August 1997     

Matrix Vesicles Mediate Mineralization of Human Thyroid Cartilage

Mineralization and ossification of human thyroid cartilage first starts after the end of adolescence when the previously cartilaginous human skeleton has become ossified and the epiphyseal discs are in the process of closing. However, the mechanisms involved in mineralization and ossification of human thyroid cartilage are not well understood. Ultrastructural analysis of human thyroid cartilage revealed that mineralization started close to cartilage canals in a matrix containing gigantic collagen fibers (asbestoid fibers). Matrix vesicles were detected in mineralized areas and were often associated with needle-like crystals. For the first time we were able to isolate matrix vesicles from human thyroid cartilage by mild enzymatic digestions and ultracentrifugation. These particles were oval and varied in size; some were heavily calcified. They were enriched in alkaline phosphatase, calcium, and inorganic phosphate, suggesting that the particles contain Ca²⁺-P_i complexes. Immunoblot analysis of these vesicles revealed the presence of annexins II, V, and VI, membrane-associated, channel-forming proteins, which allow influx of Ca²⁺ into the vesicles and intralumenal crystal growth. In addition, the vesicles were associated with types II and X collagen, suggesting that this association not only anchors the vesicles to the extracellular matrix, but, as shown previously, also stimulates Ca²⁺ influx into these particles. In conclusion, matrix vesicles isolated from human thyroid cartilage contain all the components, enabling them to initiate and mediate the mineralization process in human thyroid cartilage. Received: 21 July 1999 / Accepted: 2 November 1999