第二代骨吸收生化标志物——血清抗酒石酸酸性磷酸酶5b的临床研究

目的　对血清TRACP5b作为骨吸收生化标志物进行临床研究。方法　利用BoneTRAP试剂盒 ,对 473例正常及骨质疏松患者血清做TRACP5b检测及分析。结果　男性和女性 ,随年龄的增加 ,特别是男性更年期及女性绝经期后 ,血清TRACP5b水平均不断升高 ,表明骨吸收增强 ;当患有骨质疏松后 ,血清中TRACP5b的水平高于同龄人 ,且男性及女性骨质疏松患者血清TRACP5b水平均高于正常人 ,统计学提示有显著性差异。结论　血清TRACP5b是一个较好的骨吸收监测指标     

血清耐酒石酸酸性磷酸酶5b等骨代谢指标在大鼠去卵巢中晚期的变化

目的观察大鼠去卵巢中晚期血清耐酒石酸酸性磷酸酶5b(TRACP5b)的变化以及探讨其意义。方法7月龄SD大鼠70只,实验开始时随机取10只处死作为基线,其余60只分为去卵巢(OVX)和假手术(Sham)两组。3周各组取10只处死,余下的15周处死。处死前称量体重,分离血清测定耐酒石酸酸性磷酸酶5b和碱性磷酸酶(ALP),取左胫骨分7个区测定各骨密度(BMD)。结果OVX组血清TRACP5b呈下降趋势,3周已低于Sham组,15周降到(1.76±0.74)U/L,显著性低于Sham组(3.01±1.06)U/L(P<0.05)。3周OVX组血清ALP为(8.02±1.54)ng/ml,明显高于Sham组(6.61±0.75)ng/ml(P<0.05),15周两组间的差异消失。3周OVX组体重出现增长,15周明显高于Sham组(P<0.05)。3周OVX组胫骨1区BMD明显低于Sham组(P<0.05),15周OVX组胫骨1和2区的BMD均明显低于Sham组(P<0.05)。OVX组胫骨2、3、4和6区15周BMD较3周有明显增长(P<0.05)。结论TRACP5b的变化提示大鼠去卵巢中晚期破骨细胞数目和活性下降,骨吸收处于低水平。     

高度糖基化终产物对破骨细胞酸性磷酸酶和抗酒石酸酸性磷酸酶活性的影响

目的：观察高度糖基化终产物（AGE）对破骨细胞酸性磷酸酶（ACP）和抗酒石酸酸性磷酸酶（TRACP）活性的影响。方法：用人血清白蛋白和葡萄糖恒温孵育制备人AGE蛋白，应用酶消化法从人髂骨松质骨分离培养破骨细胞。将AGE蛋白与培养的破骨细胞共同孵育，通过酶动力学法测定培养液ACP和TRACP活性，结果：低浓度AGE蛋白（50－200μg/ml）对破骨细胞ACP和TRACP活性无显著，而高浓度（500－1000μg/ml）则使ACP和TRACP活性显著增高，且ACP活性的增高主要来自TRACP活性的增高。结论：上述结果提示AGE蛋白可能促进破骨细胞的破骨功能。     

Dexamethasone and 1,25(OH)2 vitamin D3 modulate the synthesis of insulin-like growth factor-I in osteoblast-like cells

Summary In the present study, we have shown that insulin-like growth factor-I (IGF-I) was released by primary cultures of rat osteoblast-like (ROB) cells into the conditioned medium (CM). Dexamethasone (DEX) caused a dose-dependent inhibition of the IGF-I. At 10⁻⁸ M, DEX reduced IGF-I level to 70% of the control value (P<0.05); at 10⁻⁷ M DEX, the IGF-I level was further reduced to 60% of the control (P<0.01). The active vitamin D metabolite 1,25-dihydroxycholecalceferol [1,25(OH)₂D₂] slightly increased the IGF-I level, but the increase was not statistically significant. However, in combined treatments of 10⁻⁷ M DEX and 10⁻⁸ M of 1,25(OH)₂D₃, the inhibition of DEX was partially antagonized by the presence of 1,25(OH)₂D₃. Studies with metabolically radiolabeled IGF-I by immunoprecipitation indicated the changes of IGF-I in the CM reflected synthesis of the protein by the cells. The alteration of IGF-I level may mediate some of the actions of these steroid hormones on bone cells.     

乳酸对成骨样细胞成骨表型及碱性磷酸酶基因表达的影响研究

目的 明确聚乳酸降解终产物乳酸对细胞成骨表型和基因表达的影响机理,为聚乳酸支架的精确设计提供参考意义。方法 配制乳酸浓度为8、16、22、27 mmol/L的培养基,将不含乳酸培养基作为对照组,检测培养基的pH和渗透压。利用培养基培养大鼠成骨样细胞Ros17/2.8,观察细胞形态,测定细胞I型胶原、碱性磷酸酶（alkaline phosphatase,ALP）活性和骨钙素含量,盐酸四环素染色钙结节并定量分析。采用实时定量PCR法检测细胞ALP mRNA表达。结果 与对照组相比,随着乳酸浓度增加,培养基pH下降,渗透压升高,成骨样细胞形态改变,数量减少。低浓度乳酸组（8、16 mmol/L）的I型胶原量都显著高于对照组,其余组与对照组相比无显著差异。含乳酸组的细胞ALP活性都低于对照组,并且乳酸浓度越高,细胞ALP活性越低。含乳酸组的骨钙素量与对照组之间没有显著差异。随着乳酸浓度增加,含乳酸组细胞所形成的钙结节阳性面积减少,都低于对照组。低浓度乳酸组（8、16 mmol/L）的细胞ALP mRNA表达量都低于对照组。结论 聚乳酸降解终产物乳酸可抑制细胞ALP mRNA的表达,降低ALP活性。随着乳酸浓度增加,乳酸抑制作用增强,最终降低细胞的成骨矿化能力。此外,乳酸不会抑制细胞分泌I型胶原和骨钙素。     

Characterization of gap junctions between osteoblast-like cells in culture

Summary The structure of gap junctions in osteoblast-like cells (OBs) and the connexins (cx) that build up these structures were characterized by ultrastructural, immunocytochemical, and molecular techniques. Ultrastructural studies revealed numerous gap junctions which were mostly located on processes of neighboring cells. Immunofluorescence labeling using two different antibodies (specific to mouse live cx26 and cx32 and to a peptide-specific rat heart gap junction protein cx43) gave evidence that in OBs, gap junctions consist mainly of cx43. The presence of cx43 in cultured OB was also confirmed by Western blot analysis. Dye-coupling with Lucifer yellow led to a staining of up to 30 neighboring cells. Parallel intracellular recordings showed that membrane potential amplitude changes (4–5 mV) are typically related to those in the coupled cells. Thus, there is morphological and functional evidence for intercellular communication between OB in culture. OBs in culture express the same connexins as observed in vivo and may serve as a model to investigate electrophysiological events in response to different stimulation signals.     

Potential role of tartrate‐resistant acid phosphatase 5b (TRACP 5b) as a surrogate marker of late loosening in patients with total hip arthroplasty: A cohort study

In a cohort study, the role of the active tartrate‐resistant acid phosphatase (TRACP 5b), a marker of bone‐resorbing osteoclasts, for the assessment of loosening after total hip arthroplasty (THA), was analyzed, as well as its correlation with osteolysis and multinucleated cell appearance in the retrievals. Eighty THA patients, who went consecutively to the orthopedic department, were asked to participate, and 54 accepted and were enrolled in the study. Finally, 46 subjects were analyzed, clinical‐radiographic evaluation was considered the gold standard, serum TRACP 5b was blindly measured, and a cut‐off was obtained, by performing a ROC Curve. Based on the gold standard, patients were split by 19 stable and 27 loosened subjects, and results were matched. TRACP 5b was significantly higher in loosened patients than in stable ones (p < 0.001). A good specificity (89.5%), positive predictive value (90.0%), and likelihood ratio (6.33) were calculated, that provided strong evidence of loosening with TRACP 5b levels higher than the cut‐off. Moreover, TRACP 5b and osteolysis (Fisher's exact test, p = 0.03) were found significantly correlated. TRACP 5b has been proven a reliable marker, specifically related to resorbing‐multinucleated cells, to ascertain late loosening in THA, and could support standard procedures, if confirmed by performing prospective studies. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:887–892, 2010     

Dye and electric coupling between osteoblast-like cells in culture

Summary Primary cultures of osteoblast-like cells (OB) derived from calvarial fragments of newborn rats and juvenile guinea pigs formed numerous gap junctions between neighboring cellsin vitro. Intracellular injection of Lucifer yellow led to a staining of up to 30 adjacent cells. Parallel intracellular recordings showed that amplitudes of stimulated membrane potential changes (4–5 mV) were closely related between coupled cells. The coupling factor, which was derived from the ratio of these amplitudes, ranged between 0.1 and 0.8. The coupling factor (1) was not dependent on the membrane potential or the injected current strength; (2) strong acidosis (pH < 6.6) and hypercapnia (PCOZ > 80 mm Hg) did not affect electric or dye coupling; (3) elevation of intracellular cAMP level was ineffective; (4) rise of the extra- and intracellular Ca²⁺ concentration did not effect the electric coupling; (5) the anticonvulsant drugs carbamazepine and phenytoin impaired the coupling factor up to 59%. The findings show that cell-cell communication between OB via gap junctions proved stable under various conditions which, in other tissues, were found to reduce the coupling strength of gap junctions.     

High tartrate-resistant acid phosphatase (TRACP 5b) level in cystic fluid is a significant prognostic marker for postoperative recurrence in solitary bone cysts

Purpose:The pathogenesis of cystic fluid storage in solitary bone cysts remains unclear. We aimed to compare the results of the biochemical analysis of cystic fluid with clinical findings. We identified a significant marker of postoperative recurrence.Methods:Twenty-seven male and eight female patients were studied; the median age at diagnosis was 11 (5–23) years. The mean follow-up period was 60 months (range: 14–146 months). Clinical information including sex, age, affected site, radiological findings of phase (active or latent), surgical procedure, outcome, and biochemical analysis of serum and cystic fluid was obtained.Results:The 5-year healing rate was 64.0%. Biochemical analysis revealed that total protein and albumin values in the cystic fluid were significantly lower, compared to those in the serum. Levels of bone turnover markers, such as alkaline phosphatase, bone-specific alkaline phosphatase, and tartrate-resistant acid phosphatase 5b were remarkably elevated in the cystic fluid than in the serum. R values were 0.127, 0.076, and 0.095 for alkaline phosphatase, bone-specific alkaline phosphatase, and tartrate-resistant acid phosphatase 5b, respectively. Areas under the receiver operating characteristic curves, calculated to assess the association of alkaline phosphatase, bone-specific alkaline phosphatase, and tartrate-resistant acid phosphatase 5b levels in the cystic fluid with postoperative recurrence, were 0.57, 0.51, and 0.70, respectively.Conclusions:No clear correlation of bone turnover marker levels between the serum and cystic fluid was observed. The high tartrate-resistant acid phosphatase 5b level in the cystic fluid was associated with postoperative recurrence. The bone resorption caused by osteoclasts is considered to affect postoperative recurrence.Level of evidence:Level IV.     

Relationship between coronary and abdominal calcification score, serum osteoprotegerin (OPG), and serum tartrate-resistant acid phosphatase (TRACP) -5b in pre-dialysis CKD patients

Osteoprotegerin (OPG) inhibits interaction of the receptor-activator of nuclear factor-kappaB (RANK) ligand (RANKL) with its receptor RANK, which is expressed on osteoclasts. OPG appeared to accelerate vascular calcification in vitro by the inhibition of vascular osteoclast-like cells. On the contrary, early-onset arterial calcification was observed in OPG-deficient mice. We measured the coronary artery calcification score (CACS) and abdominal aortic calcification score (AAoCS) by multi-detector computed tomography in 30 pre-dialysis CKD patients (eGFR 20 mL/min on average). Biomarkers were measured, including serum OPG, soluble RANKL (sRANKL) and tartrate-resistant acid phosphatase (TRACP) -5b (the biomarker of osteoclasts independent of renal function). The median values of CACS and AAoCS were 54.4 and 1,088 Agatston units (AU), respectively. Serum OPG was increased and serum sRANKL was decreased. In a multivariate logistic regression analysis using CACS > or = 100 AU as the outcome variable, CACS was found to be positively correlated with serum corrected Ca x iP product and serum OPG, though it was not correlated with serum TRACP-5b. ROC curve analysis showed that the serum OPG cutoff value predicting CACS > or = 100 AU was 5.2 pmol/L (624 pg/mL). In a stepwise regression analysis, log (AAoCS + 1) was positively correlated with serum OPG alone, but it was not correlated with age, eGFR, serum albumin and bone alkaline phosphatase (BAP). No correlation was found between serum OPG and serum TRACP-5b. In conclusion, vascular calcification in pre-dialysis CKD patients was correlated with an increase in OPG, but was independent of serum TRACP-5b. The decrease in serum sRANKL may have been caused by the increase in OPG production.     

Polarization and secretion of cathepsin K precede tartrate-resistant acid phosphatase secretion to the ruffled border area during the activation of matrix-resorbing clasts

The activation sequence of clasts (the designation clast was used because ultrastructurally in this tissue, it is not always possible to differentiate between chondroclasts sitting on cartilage and osteoclasts sitting on bone matrix) was studied in vivo using the healing of low-phosphate, vitamin D-deficiency rickets as a model system. Thus, the bones of 7-week-old rachitic animals were analyzed with a combination of morphological, biochemical, and molecular biological methods at 48 and 72 h, respectively, after change to normal food. A quantitative ultrastructural analysis showed that the number of clast profiles exhibiting the characteristic polarized features of actively resorbing cells, i.e., ruffled borders and clear zones, had reached normal levels after 48 h. By combining the data with quantitative analyses by the immunogold technique, we demonstrated that cathepsin K secretion was coupled to ruffled border formation in clasts irrespective of whether the number of polarized clasts was low (in rickets) or high (in healing). In contrast, the levels of tartrate-resistant acid phosphatase (TRAP) both between ruffles and in the outside matrix adjoining the ruffled border were low in polarized clasts both in rickets and at the early (48 h) healing time-point, but were increased at the latest (72 h) healing time-point. Interestingly, expression of TRAP and the cathepsin K at the mRNA level, as well as protein expression and the activity of TRAP, were not different during the healing sequence. Although the two enzymes are confined to the same clast populations, their secretion during the resorption process is apparently differentially regulated: cathepsin K secretion is coupled to ruffled border formation in clasts, whereas TRAP is secreted at a later stage during the resorption sequence, suggesting a role for secreted TRAP as a modulator of resorptive activity.     

Increased cathepsin K and tartrate-resistant acid phosphatase expression in bone of streptozotocin-induced diabetic rats

The effect of insulin-dependent diabetes mellitus (IDDM) on bone metabolism was evaluated using the streptozotocin (STZ)-induced diabetic rat 1 week after the induction of diabetes. The urinary excretion of cross-linked N-telopeptides of type I collagen (NTx) and deoxypyridinoline (Dpd) in diabetic rats increased to 3.6-fold and 1.2-fold the control level, respectively. The amount of hydroxyproline and calcium in the distal femur of diabetic rats significantly decreased to 76% and 90% of the control, respectively. The levels of serum osteocalcin and alkaline phosphatase (ALP) activity in the distal femur of the diabetic rats were significantly reduced to about 40% and 70% of the control levels, respectively. The decrease in the expression osteocalcin was observed in distal femur of the diabetic rats, although the level of ALP mRNA was unchanged. The activity and the mRNA level of tartrate-resistant acid phosphatase (TRAP) increased to 1.5- and 2.3-fold the control level, respectively, in distal femur of the diabetic rats. The activity, protein, and mRNA levels of cathepsin K of diabetic rats also elevated to about 2-, 2.3-, and 2-fold the control levels, respectively. These results suggest that IDDM contributes to bone loss through changes in gene expression of TRAP and cathepsin K in osteoclasts as well as osteocalcin in osteoblasts resulting in increased bone resorptive activity and decreased bone formation.     

Serum tartrate-resistant acid phosphatase 5b in monitoring bisphosphonate treatment with clodronate: a comparison with urinary N-terminal telopeptide of type I collagen and serum type I procollagen amino-terminal propeptide

Osteoclastic tartrate-resistant acid phosphatase activity in serum (S-TRACP 5b) was measured in postmenopausal women ( n =59, mean age 56.1 years) with vertebral osteopenia before and during 2-year treatment with an 800-mg daily dose of clodronate, with a non-amino bisphosphonate. Changes in TRACP 5b were compared with those in urinary excretion of type I collagen amino-terminal telopeptide (U-NTX), corrected for creatinine excretion, a well-established marker of bone resorption, and to serum type I procollagen amino-terminal propeptide (S-PINP), a marker of bone formation. Marker changes 1 year after start of treatment were correlated with changes in bone mineral density (BMD). The least significant change (LSC) for each marker and BMD was calculated from values for subjects receiving placebo. Responders to treatment were those exhibiting a change larger than LSC. In response to clodronate treatment S-TRACP 5b (mean change up to -18%) decreased less than did U-NTX (up to -51%) or S-PINP (up to -46%). Marker changes correlated with changes in lumbar spine and trochanter BMD. The most efficient marker for finding responders to treatment was S-PINP, which changed more than the LSC (32%) in 72% of the subjects at the 1-year time point and in 79% at the 2-year time point. S-TRACP 5b change exceeded the LSC (27%) in 40% and 34% of the subjects at each time point, while U-NTX change exceeded the LSC (55%) in 55% and 40%, respectively. We conclude that, in terms of the proportion of subjects exhibiting any change exceeding the LSC, S-TRACP 5b did not appear to be superior to U-NTX and S-PINP in the follow-up of clodronate treatment. The reason may lie in the mechanism of action of clodronate, which rather than reducing the number of TRACP 5b-secreting osteoclasts, reduces the activity of bone proteolytic enzymes and thus the rate of bone organic matrix degradation. This is seen in decreased amounts of type I collagen breakdown products (U-NTX), and through coupling of bone resorption with bone formation, in a decrease in circulating levels of the marker that reflects new collagen formation (S-PINP).     

The fluorescent simultaneous azo dye technique for demonstration of tartrate-resistant acid phosphatase (TRAP) activity in osteoclast-like multinucleate cells

The purpose of our research was to show that the fluorescent simultaneous azo dye technique for tartrate-resistant acid phosphatase (TRAP) activity is useful for demonstration of osteoclasts under a fluorescent microscope and a confocal laser scanning microscope. Osteoclast-like multi-nucleate cells (MNC) were obtained from cultured mouse calvariae. The fluorescent simultaneous azo dye technique for TRAP activity was applied for cultured MNC. For the observation of specimens, both a conventional fluorescent microscope and a confocal laser scanning microscope for three-dimensional localization of TRAP-positive sites were used. Double staining with the azo dye technique was also adapted for mitochondria and tubrin staining. We found that the fluorescent simultaneous azo dye technique for TRAP activity could be used as a method of demonstrating MNC with a fluorescent microscope. We were also able to obtain the serial localization of three-dimensional sectioning images of TRAP-positive sites in MNC with a confocal laser scanning microscope. Moreover, with double staining of MNC as a modified version of the azo dye technique, we were able to observe both cell localization of TRAP-positive sites and other staining with epifluorescent illumination using the blue excitation method. Therefore, by using the fluorescent azo dye technique for TRAP activity, we can simultaneously observe not only the presence of osteoclasts, but also their characteristic construction and function.     

Detection of apoptotic gene expression in human osteoblast-like cells by cDNA microarrays

Global gene expression during the induction of ion pair-mediated apoptosis was evaluated by an apoptosis microarray system. Human bone marrow stromal cells were cultured in the presence of 10^–6M dexamethasone to promote osteogenesis. After 28 days, these cells expressed elevated alkaline phosphatase activity and maintained Cbfa1 expression even when challenged with an apoptogen. Apoptosis was initiated by treating cells with 3mM Ca²⁺ and 5mM Pi for 4h. ³²P-Labeled mRNA was hybridized to a human apoptosis microarray containing 205 cDNA fragments. We found that apoptosis influenced the expression of 15 genes mainly involved in cell cycle and cell signaling. These genes included IGFBPs and ERK1, known to play a role in cell survival; GST and GST mu, required for maintenance of thiol redox; TNFR1, a gene product that initiates cell death; and finally, BAD, a gene that encodes a proapoptotic protein. Real-time PCR analysis showed that the expression of ERK1, TNFR1, and GST was modulated by 1.89-, 2.66-, and 1.6 fold after 4h and by 1-, 1.91-, and 1.5 fold, respectively, after 8h treatment with the ion pair. In addition, we also measured the expression of Bcl-2 and Bax by quantitative RT-PCR. We noted that these two genes were increased 3.07 and 2.99 fold, respectively, after 8h treatment with the apoptogen. Results of this study suggest that the ion pair influenced ERK1 and TNFR1 signaling pathways and affected thiol metabolism, whereas Bcl-2 and Bax were expressed at late stages of the death process.     

Effects of vitamin D3, 17β-estradiol, vasoactive intestinal peptide, and glutamate on electric coupling between rat osteoblast-like cells in vitro

Osteoblast-like cells express receptors for various hormones and neurotransmitters that induce widespread actions in the bone to which intercellular communication and its modulation may contribute. Therefore, we examined the effects of the osteotropic hormones vitamin D₃ (vitD₃) and 17β-estradiol (17β-E₂) as well as the neurotransmitter vasoactive intestinal peptide (VIP) and the excitatory amino acid glutamate (Glu) on gap junctions between rat osteoblast-like (ROB) cells in vitro. Electric coupling was measured by simultaneous intracellular recordings from neighboring cells. The coupling factor (c_f) was calculated from membrane potential changes induced by alternate current injections into both cells. In ROB cells c_f was increased by 5 × 10⁻⁸ mol/L vitD₃ to 130 ± 13% (mean ± SD; n = 6) of the initial value within 5–20 min. This effect was not reversible after washing with control saline for 10–15 min. In six cell pairs, c_f was not affected by vitD₃ (94 ± 5%). In three cell pairs superfusion of 10⁻⁸ mol/L E₂ reduced c_f to 80 ± 6% within 10 min, whereas, in two cell pairs, this hormone improved c_f to 140% within 20 min. Exposure of VIP (3 · 10⁻⁸ mol/L) did not alter c_f in the majority of cells (99 ± 3%; n = 11). In five cell pairs, c_f was improved within 5–15 min to 133 ± 12%, whereas, in one cell pair, c_f was reduced to 22% by VIP. In contrast, brief application of Glu (5 · 10⁻³ mol/L) decreased c_f to 75 ± 5% (n = 5), whereas, in nine other cell pairs, c_f was not affected (96 ± 5%). The findings indicate that cell-cell coupling of gap junctions between bone cells can be altered by actions of hormones and transmitters in a cell-pair-specific way, which may depend on their functional state.     

Characterization of a tartrate-resistant acid phosphatase (ATPase) from rat bone: hydrodynamic properties and N-terminal amino acid sequence

Certain physicochemical properties of rat bone tartrate-resistant acid ATPase (TrATPase), including the size and shape of the enzyme, potential subunit composition, and detergent binding, have been elucidated. SDS-polyacrylamide gel electrophoresis in combination with immunoblot analysis showed that the bone TrATPase has a molecular weight of 33,000 D and is composed of disulfide-linked polypeptides of 20,000 and 16,000 D. The enzyme contains 1.7 mol Fe per mol enzyme. Hydrodynamic studies allowed calculation of the Stokes radius (24 A), the sedimentation coefficient (3.19S), the partial specific volume (0.748 ml/g), the frictional ratio (0.995), and the axial ratio (1.0). The amount of detergent bound to the protein was determined to 4 mol of Triton X-100 per mol enzyme. The molecular weight of bone TrATPase derived from these parameters was 31,900 D. N-terminal amino acid sequence analysis of the Mr 20,000 subunit indicated a high degree of similarity with TRAP enzymes from spleen, uterus, placenta, hairy cell leukemia, and osteoclastoma. It is concluded that rat bone TrATPase belongs to the type 5 (tartrate-resistant and purple) acid phosphatase family. The similarities in the N-terminal amino acid sequences, iron content, and physicochemical properties of TRAP enzymes indicate a close structural relationship between type 5 acid phosphatases expressed in different tissues. The findings that TrATPase has a spherical shape and binds low amounts of detergent suggest that the enzyme is a soluble protein, compatible with the view that TrATPase is secreted by the osteoclast.     

Bone blood flow and In vitro proliferation of bone marrow and trabecular bone osteoblast-like cells in ovariectomized rats

Summary Ovariectomy in the rat induces a rapid osteopenia associated with an elevated bone turnover. One hundred and twenty-day-old rats were ovariectomized (OVX) or sham-operated (n=6–8 per group and per time period studied). ⁴⁵Ca accretion rate and bone blood flow (microspheres trapping technique) in the femurs were determined at 28, 42, 84, and 119 days after ovariectomy. Both parameters were markedly increased by 84 days and subsided thereafter. At the 42nd day, when bone turnover was maximal, bone marrow and trabecular bone cultures were obtained from shamoperated and ovariectomized animals (n=10/group). Proliferation rate of bone marrow cells and trabecular osteoblast-like cells estimated by fibroblast colony-forming units (FCFU) efficiency and cell counting was markedly increased in primary and secondary cultures in ovariectomy. These data fitted well with the enhanced number of osteoblasts observed in situ in the long bone metaphyses of estrogen-depleted animals. As estrogens were shown in the literature to inhibit proliferation of the red cell line and of other hemopoietic lines, it is possible that estrogens, through a general mechanism, inhibit hemopoietic and stromal lines and also the proliferation of bone marrow-derived trabecular bone cells.