糖皮质激素双向调节骨髓基质干细胞的分化机制

骨质疏松症是以骨量减少、骨显微结构退化为特征,以致骨脆性增高及骨折危险性增加的一种全身性疾病。糖皮质激素（ GC）因其具有非常好的抗炎和免疫调节作用而被临床上广泛使用,而过量的使用糖皮质激素会导致骨质疏松症,称为糖皮质激素性骨质疏松症,属于继发性骨质疏松症。骨髓基质干细胞（ BMSCs）具有多向分化潜能,能够分化为成骨细胞和脂肪细胞,两者相互竞争,此消彼长。糖皮质激素在正常范围内可促进BMSCs向成骨或成脂方向分化,而在这一过程中,多种调控因子可与其联合作用,促进或抑制BMSCs的成骨分化。本文就糖皮质激素对BMSCs的双向调节机制作一综述,阐述各因素与糖皮质激素的联合协同作用,从成骨最大化的角度探讨激素性骨质疏松的防治方法。     

Users of low-dose glucocorticoids have increased bone loss rates: A longitudinal study

Although high doses of glucocorticoids are believed to cause bone loss, the effects of low glucocorticoid doses are still controversial. Our study examined the effects of low-dose glucocorticoids on the rate of bone loss at three appendicular bone sites. The study population was a cohort of elderly Japanese-Americans, 1094 women and 1378 men. The women were all postmenopausal. At the baseline examination the mean age of the women was 64 years (range 45–81), and the mean age of the men was 68 years (range 61–82). Glucocorticoid users (19 women and 21 men) had used oral systemic or inhaled glucocorticoids on a regular schedule for more than 1 month (mean use was 2.1 years for the women and 1.9 years for the men). The most common dose was equivalent to 5 mg/day of prednisone; fewer than 15% of users had taken doses equivalent to 10 mg/day or more. Changes in bone mass at the calcaneus, distal radius, and proximal radius were documented using bone densitometry at 1 to 2-year intervals over an 8-year period. The initial bone mass of the glucocorticoid users and controls was similar at the baseline examination. The subsequent loss rates among females during glucocorticoid use, however, were approximately double that of the controls. Among males, bone loss rates during glucocorticoid use were 2–3 times that of controls for the calcaneus and radius sites. The differences between glucocorticoid users and controls persisted after adjusting for confounding variables such as age and use of thiazides and estrogens. We conclude that users of low-dose glucocorticoids have increased rates of bone loss at appendicular sites among both elderly women and men.     

Bone repletion in vitro: evidence for a locally regulated bone repair response to PTH treatment

Normally bone formation and resorption are balanced by coupling, but in some conditions such as dietary Ca deficiency, bone resorption exceeds formation, resulting in bone loss (termed bone depletion in previous animal studies). When conditions causing depletion return to normal, a compensatory decrease in resorption and increase in formation occurs, leading to replacement of the lost bone. To test if this recovery process, termed bone repletion, might be locally regulatedupling we determined whether cellular and metabolic changes associated with repletion in vivo would as in co occur in vitro in neonatal mouse calvaria.

To increase resorption and decrease formation, serumfree cultures were treated with parathyroid hormone (10 nM bovine PTH_1–84). Although formation was inhibited ([³H]proline incorporation into [³H]hydroxyproline), the number of bone cells increased during PTH treatment. To simulate repletion, PTH was removed after 3–9 days. Within 6 days of removal of PTH, resorption (osteoclast numbers and ⁴⁵Ca release) decreased to control levels and bone formation increased to exceed untreated control levels. Autoradiographs of [³H]proline incorporation suggested an increase in the number of active bone forming cells (compared to untreated controls) after removal of PTH. These cellular and metabolic changes were similar to changes which occur during depletion and repletion in vivo. The results support the hypothesis that reversal of the resorptive processes initiated by PTH in organ cultures can occur in the absence of circulating factors. The apparent increase in the components of bone formation that were observed after PTH withdrawal may have resulted from generation of increased numbers of osteoblastic cells during PTH treatment.     

严重烧伤后糖皮质激素受体变化在应激性胃粘膜损害中的作用

目的　探讨糖皮质激素受体变化在烧伤后应激性胃粘膜损害中的作用。　方法　观察大鼠严重烧伤后不同时相胃粘膜组织糖皮质激素受体的动态变化及胃粘膜损伤情况。　结果　伤后6h皮质醇含量明显升高 ,12h达高峰 (P <0 .0 5～ 0 .0 1) ,而胃粘膜细胞胞浆内糖皮质激素受体水平显著下降 (P <0 .0 5～ 0 .0 1) ,胃粘膜损伤指数于伤后 6、12、2 4、4 8h显著增加 (P <0 .0 5～ 0 .0 1)。　结论　严重烧伤后糖皮质激素受体减少是应激性胃粘膜损害的重要因素     

Demonstration of the presence of independent pre-osteoblastic and pre-adipocytic cell populations in bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSC) are defined as plastic-adherent, clonal cells that are common progenitors for osteoblasts and adipocytes. An inverse relationship between bone and fat has been observed in several clinical conditions and has been suggested to be caused by re-directing MSC differentiation into one particular lineage. However, this inverse relationship between bone and fat is not consistent and under certain in vivo conditions, bone and fat can change independently suggesting separate precursor cell populations. In order to test for this hypothesis, we extensively characterized two plastic-adherent clonal MSC lines (mMSC1 and mMSC2) derived from murine bone marrow. The two cell lines grew readily in culture and have undergone more than 100 population doublings with no apparent differences in their growth rates. Both cell lines were positive for the murine MSC marker Sca-1 and mMSC1 was also positive for CD13. Both cell lines were exposed to in vitro culture induction of osteogenesis and adipogenesis. mMSC1 and not mMSC2 were only able to differentiate to adipocytes evidenced by the expression of adipocyte markers (aP2, adiponectin, adipsin, PPARgamma2 and C/EBPa) and the presence of mature adipocytes visualized by Oil Red O staining. On the other hand, mMSC2 and not mMSC1 differentiated to osteoblast lineage as demonstrated by up-regulation of osteoblastic makers (CBFA1/RUNX2, Osterix, alkaline phosphatase, bone sialoprotein and osteopontin) and formation of alizarin red stained mineralized matrix in vitro. Consistent with the in vitro results, mMSC2 and not mMSC1, were able to form bone in vivo after subcutaneous implantation in immune-deficient (NOD/SCID) mice. Our data suggest that contrary to the current belief, bone marrow contains clonal subpopulations of cells that are committed to either osteoblast or adipocyte lineage. These cell populations may undergo independent changes during aging and in bone diseases and thus represent important targets for therapy.     

辛伐他汀部分阻止尾悬吊大鼠股骨近端骨量的丢失

目的：观察辛伐他汀体内给药对尾悬吊大鼠骨量和骨髓基质干细胞增殖﹑分化的影响。方法18只9周龄雄性SD大鼠被随机分成3组,每组6只：第一组（G1）,正常对照组,每天蒸馏水灌胃;第二组（G2）,尾悬吊组,每天蒸馏水灌胃;第三组（G3）,尾悬吊大鼠每天20 mg/kg辛伐他汀灌胃。实验持续3周,所有大鼠在最后一次灌胃的第二天被处死,取大鼠右侧股骨用双能X线骨密度仪测量骨密度。取大鼠左侧股骨和胫骨骨髓细胞向成骨细胞定向培养,并作如下检测：碱性磷酸酶活性和von Kossa染色分别在细胞培养第16天和25天检测。在细胞培养第21天,采用Real-time RT-PCR检测BMP-2、RANKL mRNA的表达。结果尾悬吊组大鼠骨量低于对照组。全长骨密度（ tBMD）及远端骨密度（ dBMD） G1组显著高于G2、G3组,近端骨密度（ pBMD） G1组显著高于G2组,但与G3组没有区别, G3组高于G2组但没有显著差别。细胞外基质矿化能力（ von Kossa染色）、ALP比活性以及RANKL、BMP-2 mRNA水平各组间均没有显著差别。结论尾悬吊3周可致大鼠骨骨质疏松,辛伐他汀体内给药可部分阻止股骨近端骨量丢失,但不能显著促进骨髓基质干细胞向成骨细胞分化。     

Casodex (a nonsteroidal antiandrogen) reduces cancellous,endosteal, and periosteal bone formation in estrogen-replete female rats

Testosterone has been implicated in the preservation of the skeleton in women and female rats. In this study we investigated the role of androgens in estrogen-replete female rats by giving Casodex (pure nonsteroidal anti-androgen) daily and analyzing the effects on the skeleton using static and dynamic histomorphometric parameters after 3 weeks. There was a significant reduction in the bone formation rate in both the cancellous bone of the tibial metaphysis and in the periosteal and corticoendosteal diaphyseal bone (90%, 30%, and 100% reduction, respectively, compared with control animals, which was unaccompanied by a change in the indices of bone resorption. Casodex had no effect on cancellous bone volume and cortical bone area but this can be accounted for by the short duration of the experiment. The serum levels of dehydroepiandrosteronesulfate and androstenedione were significantly reduced in the Casodex-treated rats compared with the control animals and there was no difference in the plasma levels of estrone, estradiol, or testosterone between the two groups. This study demonstrates that androgens play a physiological role in regulating osteoblast activity in female rats.     

他汀类药物促进骨生成作用的基础及临床研究

他汀类药物对机体可产生多种效用,不仅能降低血清胆固醇,减少心血管疾病的发生,减弱肾损伤,而且大量基础及临床试验研究证实该药物可明显地刺激骨生成。然而,他汀类药物促进骨生成确切的作用机制仍是一个备受争论的课题,在促进成骨的同时,其药物副作用及与其它药物的协同与抑制作用更值得进一步深入探讨。本文总结分析了近年来他汀类药物基础及临床研究的文献,综述了他汀类药物的作用机理及安全性,以供骨修复领域作临床参考。     

辛伐他汀体外促进骨形成作用的初步研究

目的 研究辛伐他汀(Simvastatin)对成骨细胞分化、增殖及骨形成的作用。方法 用RPMI 1640培养液分别对水囊引产胎儿及新生大鼠颅顶骨进行组织培养,并将等量的胎儿或大鼠的颅顶骨骨组织分为实验组(辛伐他汀1μmol/L)和对照组,除动态观察比较不同培养液中成骨细胞的增殖状况外,将培养7d后的颅顶骨组织块制成半薄或超薄病理切片,于光镜下和电镜下进行形态学观察,并用测微尺测量新生骨组织厚度,记录成骨细胞数;同时,对留取的培养液测定其中的碱性磷酸酶(AKP)及骨钙素(BGP)的含量。结果 在培养过程中,可见实验组胎儿和新生大鼠颅骨成骨细胞生长活跃,数量多;对照组成骨细胞生长缓慢,数量少,可见大量成纤维细胞。培养7d后可见实验组胎儿及大鼠颅骨新生骨组织的厚度均明显高于对照组(P<0.01);其单位长度(0.3 mm)新生骨组织内成骨细胞数均明显高于对照组(P<0.01)。透射电镜下可见实验组成骨细胞处于活化状态,胞浆内有丰富的细胞器,少见破骨细胞,而对照组成骨细胞处于衰老状态,细胞器少见,可见大量破骨细胞。实验组培养液中AKP及BGP均明显高于对照组(P<0.01)。结论 辛伐他汀在体外实验中具有促进成骨细胞分化、增殖和促进新骨形成的作用;他汀类药物可作为预防、治疗骨质疏松症的有价值的候选药物。     

Impaired Cortical Bone Acquisition and Osteoblast Differentiation in Mice with Osteoblast-Targeted Disruption of Glucocorticoid Signaling

To determine the role of endogenous glucocorticoids in bone, we previously developed transgenic mice in which a 2.3 kb fragment of the Col1a1 promoter drives 11beta-hydroxysteroid dehydrogenase 2 expression in mature osteoblasts. This transgene should inactivate glucocorticoids upstream of all receptor signaling pathways. In the present study, we show that femoral cortical bone area and thickness were approximately 10-15% lower in transgenic mice than in wild-type littermates. Femur length was unchanged, indicating that bone elongation was not affected in this model. Expression of osteocalcin mRNA, pOBCol2.3-GFP (a green fluorescent protein marker of mature osteoblasts), and the formation of mineralized nodules were impaired in ex vivo transgenic primary calvarial cultures. The extent of crystal violet staining in bone marrow cultures, indicative of the number of adherent stromal cells, was also decreased. These data suggest that endogenous glucocorticoids are required for cortical bone acquisition and full osteoblast differentiation. It appears that blocking glucocorticoid signaling in vivo leads to a decrease in the commitment and/or expansion of progenitors entering the osteoblast lineage.     

Absence of mouse pleiotrophin does not affect bone formation in vivo

Pleiotrophin (Ptn) is an extracellular matrix protein that regulates hippocampal synaptic plasticity and learning behavior in vivo. Since the overexpression of Ptn in transgenic mice leads to increased bone formation, we analyzed whether a deficiency in Ptn expression would have a negative effect on bone remodeling. Bones from Ptn-deficient mice and wild-type littermates were analyzed using radiography, μCT imaging and undecalcified histology. Biomechanical stability was determined in a three-point-bending assay. Cellular activities were assessed using dynamic histomorphometry and the determination of urinary collagen degradation products. Skeletons of Ptn-deficient mice have no gross abnormalities, displayed a normal size, and showed no differences in growth plate organization compared to wild-type littermates. There were no obvious differences in bone mass as determined by radiographic and μCT imaging. The absence of a bone remodeling phenotype in Ptn-deficient mice was further confirmed using static histomorphometry and biomechanical testing. Finally, the number, morphology, and function of osteoclasts, osteoblasts, and osteocytes were not altered in Ptn-deficient mice compared to wild-type littermates. The complete skeletal analysis of Ptn-deficient mice presented here demonstrates that the lack of Ptn in mice does not affect bone formation in vivo. Therefore, Ptn does not play a significant role in normal bone physiology.     

Skeletal effects of short-term prednisolone therapy in children with steroid-responsive nephrotic syndrome

Background. Osteoporosis or bone fracture can be induced in nephrotic children treated long-term with high doses of glucocorticoids. The purpose of this study was to determine whether short-term prednisolone therapy affects the skeleton in children with steroid-responsive nephrotic syndrome (NS). Methods. Bone mineral density (BMD) and biochemical parameters of mineral and skeletal homeostasis in nine children (four girls, five boys) aged between 2 and 7 years at the first episode of NS were measured. Prednisolone was started at 60 mg/m² for 4 weeks, then decreased every 2 weeks for 12 weeks. All patients were steroid-responsive and had no relapse. All patients were examined at 0, 4, and 12 weeks of prednisolone therapy, and 16 weeks after the cessation of prednisolone therapy. Results. The Z-scores (BMD) before prednisolone therapy (−0.79 ± 0.51) were slightly low. The Z-scores at 4 weeks (−1.37 ± 0.46) and at 12 weeks of therapy (−1.22 ± 0.36) were significantly lower than those at 16 weeks after the cessation of prednisolone therapy (−0.29 ± 0.29). There was also a significant decrease in the mean serum levels of alkaline phosphatase, osteocalcin, and urinary deoxypyridinoline during the short-term prednisolone therapy. However, BMD and biochemical parameters of mineral and skeletal homeostasis returned to normal values at 16 weeks after the cessation of prednisolone therapy. Conclusions. Skeletal effects of short-term prednisolone therapy for 16 weeks were transient in children with steroid-responsive NS without relapse. Received: August 28, 2000 / Accepted: November 18, 2000     

Relationship of bone morphogenetic protein expression during osteoblast differentiation to wild type p53

We have previously shown p53 to have a specific role in osteoblast differentiation by its ability to regulate expression of certain bone specific proteins. In this study, we show mineralized matrix formation in vivo to be directly related to the presence of wild type p53 in osteoblastic osteosarcoma cells. In order to further understand the importance of p53 in differentiation, we investigated the relationship between p53 and Bone Morphogenetic Proteins (BMPs) (BMP 1, 2, 3A, 3B (GDF-10), 4, 5, 6, 7, 8A and 8B) during osteoblast differentiation. The expression of several BMPs were tested using RNase Protection Assay in differentiating ROS17/2.8 osteoblastic osteosarcoma cells. The expression of BMPs 1, 2, 3a, 3b and 7 showed time dependent modulation during in vitro differentiation. In order to determine if p53 has a role in this process, we used a murine osteosarcoma cell line stably expressing a temperature sensitive p53. Cells were exposed to ascorbic acid and glycerophosphates to hasten in vitro osteoblast differentiation and maintained either at 32 or 37 degrees C for expression of the wild type or mutant p53 phenotype. The expression of BMP-2, BMP-4 and BMP-7 were modulated in a p53 dependent fashion. We were able to confirm the p53 dependency of BMP-2 independently by RT-PCR. While BMP-2 expression was evident in the presence of both wild type and mutant p53, regulated expression was seen only in cells expressing wild type p53. Transient over expression of wild type p53 did not result in the same BMP-2 response as stable expression showing that the presence of p53 may be important for an orderly development of osteoblast differentiation rather than a direct effect on gene expression. The functional relationship between p53 and these bone specific markers is discussed.     

Differential effects of glucocorticoids on human osteoblastic cell metabolism in vitro

Clinical observations suggest that the onset and severity of glucocorticoid (GC) induced osteoporosis is dependent on the duration of the GC treatment and the applied GC compound. To test whether these in vivo observations are reflected by different in vitro effects of various synthetic GCs on human bone cell metabolism we isolated human osteoblast-like cells (HOC) from bone biopsies of healthy (no clinical symptoms of arthritis or arthrosis) adults who underwent selective orthopedic surgery. HOC were identified as bone cells by 1,25-vitamin D₃-stimulated increase of specific alkaline phosphatase (ALP) activity, secretion of osteocalcin and type-I procollagen peptide, and the ability to form mineral in vitro. We investigated the effects of dexamethasone (dexa), methylprednisolone (mpred), prednisolone (pred), and deflazacort (defla) on DNA-synthesis, ALP, and osteocalcin (OC)- and type-I procollagen peptide secretion of HOC in vitro. In summary, (1) GC exposure stimulates DNA synthesis after 6–12-hour treatment periods; (2) dex and mpred strongly inhibit DNA (48-hour treatment) and collagen synthesis but stimulate ALP, whereas pred and defla exhibit smaller effects on DNA synthesis, ALP, and collagen production; and (3) all tested glucocorticoids inhibit OC secretion by HOC in vitro. Thus, the effect of GC on DNA synthesis of HOC varies with the duration of GC exposure, and dex and mpred more potently affect HOC metabolism in vitro than pred and defla.This work was presented in part at the 23rd European Symposium on Calcified Tissues in Heidelberg 1993.     

Glucocorticoid-induced inhibition of osteoblastic bone formation in ewes: A biochemical and histomorphometric study

The mechanisms underlying glucocorticoid-induced osteoporosis in humans are a defect in bone formation associated with increased bone resorption. The latter may be due to elevated parathyroid hormone (PTH) levels induced by the impairment of intestinal calcium absorption caused by corticosteroids. In this study we analysed the effects of corticosteroids in old ewes, a potential model for the study of human bone turnover. Two groups of seven 9-year-old female sheep were selected. The first group was injected intramuscularly with a daily dose of 30 mg methylprednisone (MP) during the first 2 months and 15 mg during the last month. After 2 and 3 months of treatment, blood samples were taken. At the end of the experiment the animals were slaughtered and the iliac crest kept for bone histomorphometry. Serum osteocalcin (sOC) rapidly and markedly decreased in the MP-treated group compared with controls (–77%;p<0.01). In contrast, at the end of the experiment serum calcium and PTH levels were similar in both groups. Histomorphometric analysis showed a significant reduction in the wall width of trabecular packets. Dynamic parameters reflecting bone formation at the tissue and cell levels were significantly lower in the MP-treated group than in controls, with a highly significant decrease in the mineralization rate (MAR: –63%,p<0.05) and double-labeled perimeter (dLPm/B.Pm: –92%p<0.05). The bone formation rate (BFR/B.Pm) also decreased by 84% and the adjusted apposition rate (Aj.AR) by 80%. The increase in the total formation period was mainly due to an increase in the inactive period. Significant correlations were found between sOC and MAR, dLPm/B.Pm and BFR/B.Pm (withr respectively 0.67, 0.76 and 0.51). In conclusion, the effects of corticosteroid on ewe bone remodeling are essentially characterized by a major bone formation defect without evidence of secondary hyperparathyroidism, although this cannot be totally excluded by our results. Ewes treated with glucocorticoids could represent a good model for evaluating the effects of drugs candidates for all bone conditions characterized by reduced bone formation resulting from osteoblastic depression.     

RNA interference for noggin enhances the biological activity of bone morphogenetic proteins in vivo and in vitro

Noggin is a major extracellular antagonist to bone morphogenetic proteins (BMPs) which binds to BMPs and blocks binding of them to BMP-specific receptors and negatively regulates BMP-induced osteoblastic differentiation. In this study, we investigated the effect of noggin silencing by transfection of small interfering RNA (siRNA) on BMP-induced osteoblastic differentiation in vitro and ectopic bone formation in vivo induced by recombinant human BMP-2 (rhBMP-2). Noggin mRNA expression was up-regulated in response to rhBMP-2 in C2C12 cells, a myoblastic cell line, in dose- and time-dependent fashion as determined by real-time RT-PCR assay. Silencing of noggin expression by transfection of noggin siRNA suppressed BMP-stimulated noggin expression, resulting in acceleration of BMP-induced osteoblastic differentiation. For in vivo noggin silencing, siRNA was injected locally into back muscles and transfected into local cells by electroporation, where rhBMP-2-retaining (5 μg) collagen disks had been surgically placed. The implants were harvested at 2 weeks after surgery from experimental and control group mice and analyzed by radiological and histological methods. As a result, bone mineral content of ossicles ectopically induced by rhBMP-2 was significantly increased by silencing of noggin. Our findings suggest that silencing of noggin enhances the osteoblastic differentiation of BMP-responding cells in vitro and new bone formation induced by rhBMP-2 in vivo by eliminating negative regulation of the effects of BMP. RNA interference might be useful for intensifying the effects of BMP in promoting new bone (callus) formation in repair of damaged bone.     

PGE2及cAMP在烧伤合并内毒素血症骨髓粒单系造血变化中作用的实验研究

目的　探讨前列腺素E2 (PGE2 )、环磷酸腺苷 (cAMP)在烧伤合并内毒素血症骨髓粒单系造血变化中的作用。　方法　 178只昆明种小鼠随机分为烧伤合并内毒素注射组、单纯烧伤组、单纯内毒素注射组和等渗盐水对照组。应用放射免疫分析和免疫组化法 ,动态检测各组小鼠致伤后 1周内 ,骨髓细胞环氧酶 2 (COX 2 )表达及PGE2 、cAMP含量 ,并对粒单系造血变化进行了动态观察。　结果　烧伤合并内毒素血症早期骨髓粒单系略呈增生反应 ,继而呈现造血抑制。骨髓细胞COX 2表达、骨髓细胞上清PGE2 和细胞内cAMP在 12h～ 5d显著升高 ,且cAMP与PGE2 呈显著正相关 (r=0 .978,P <0 .0 1) ,而与粒 巨噬细胞集落形成单位 (CFU GM)呈显著负相关 (r =- 0 971,P <0 .0 1)。　结论　PGE2 在烧伤合并内毒素血症骨髓粒单系抑制中起重要作用 ,抑制效应是通过其与特异性受体结合 ,激活腺苷酸环化酶 ,使骨髓细胞内cAMP升高     

Inflammation-mediated osteopenia (IMO) during acute inflammation in rats is due to a transient inhibition of bone formation

Summary Local inflammation was induced in rats through the subcutaneous injection of magnesium silicate. Trabecular bone volume of the tibia decreased progressively during a 3 week observation period following the inflammatory stimulus. The trabecular bone surface covered with osteoblasts was strikingly reduced during the first week but had normalized by the end of the third week. Calcification rate in the cortical bone of the tibia was reduced with a parallel reduction in endosteal osteoid seam width. Both calcification rate and tetracycline double-labeled surface of vertebral trabecular bone were reduced during the first 2 weeks. Neither total bone resorption surface nor active bone resorption surface were increased. There was a decrease in osteoclast numbers/mm² bone tissue associated with decreasing bone volume. Our data demonstrate a transient inhibition of bone formation during acute inflammation in the rat and indicate that changes in osteoblast function are part of the acute phase response following local inflammation.     

The role of glucocorticoids and prostaglandin E2 in the recruitment of bone marrow mesenchymal cells to the osteoblastic lineage: Positive and negative effects

The role of glucocorticoids in bone formation presents a problem because although pharmacological doses in vivo give rise to osteoporosis, physiological concentrations are required for osteoblast (OB) differentiation in vitro. To try and rationalize this dichotomy, we investigated the effect of dexamethasone on the recruitment of OB precursors present in bone marrow. Using the CFU-f assay, we can measure (1) total colony formation; (2) the osteoblastic differentiation of the colonies defined as their ability to express alkaline phosphatase, synthesize collagen, and to calcify; and (3) colony expansion as either average colony surface area or average colony number. In control cultures and in the presence of 10⁻¹⁰–10⁻⁹ M dexamethasone, colony formation and total cell number was maximal, but the addition of PGE₂ had no effect on colony number and very few colonies expressed the OB phenotype. In the presence of 10⁻⁸–10⁻⁷ M dexamethasone, colony numbers and total cell numbers were reduced but were increased by the addition of PGE₂, the average colony cell number and surface area were relatively unchanged and a proportion of the colonies expressed APase, calcified and synthesized collagen. In cultures containing 10⁻⁶–10⁻⁵ M dexamethasone, colony numbers were further reduced but were stimulated by the addition of PGE₂ and some colonies differentiated; however, colony expansion was dramatically reduced by up to 80%. These results suggest that physiological levels of glucocorticoids are necessary for OB differentiation and allow the control of OB recruitment by PGE₂. High levels of glucocorticoids drastically reduce proliferation of the OB precursors leading to glucocorticoid-induced osteoporosis. Received: 8 May 1995 / Accepted: 23 February 1996