Thiazolidinediones cause compaction of nuclear heterochromatin in the pluripotent mesenchymal cell line C3H10T1/2 when inducing an adipogenic phenotype.

OBJECTIVE: To characterize the nuclear changes induced in vitro by thiazolidinediones (TZDs) in a murine pluripotent mesenchymal cell line. STUDY DESIGN: The C3H10T1/2 cell line, which can differentiate either in osteoblast or in adipocyte, was cultured in the presence of pioglitazone (5 microM) or rosiglitazone (0.5 microM) for 6, 8 and 9 days (D). Quantitative real-time polymerase chain reaction analysis evaluated the expression of key genes of the adipocytic or osteoblastic differentiation (PPARgamma[peroxisome proliferatoractivated receptor gamma], Runx2 [runt-related transcription factor 2] and alkaline phosphatase). Cells were stained with Oil Red O for lipids, and chromatin was counter-stained with hematoxylin. Cells were photographed at x 1,000 magnification and analyzed with texture analysis software. Nuclear area, mean gray level and run-length parameters were calculated. RESULTS: PPARgamma was significantly expressed from D6 (normalized ratio > 7) in TZD groups (ratio >27 at D9). No significant differences were found for either Runx2 or alkaline phosphatase expression versus control at D6 or D9. Cells cultured with TZDs began to differentiate into adipocytes with numerous lipid droplets which appeared at D6. Nuclear area decreased suddenly at D6 for both TZDs, and the mean gray level increased. Run-length parameters changed significantly due to chromatin compaction. CONCLUSION: TZDs provoked differentiation of C3H10T1/2 into adipocytes, leading to inactivation of genes that were highly compacted into heterochromatin.

Thiazolidinediones induce osteocyte apoptosis by a G protein-coupled receptor 40-dependent mechanism.

Thiazolidinediones (TZDs) represent an interesting treatment of type 2 diabetes mellitus. However, adverse effects such as heart problems and bone fractures have already been reported. Previously, we reported that pioglitazone and rosiglitazone induce osteocyte apoptosis and sclerostin up-regulation; however, the molecular mechanisms leading to such effects are unknown. In this study, we found that TZDs rapidly activated Erk1/2 and p38. These activations were mediated through Ras proteins and GPR40, a receptor expressed on the surface of osteocytes. Activation of this pathway led only to osteocyte apoptosis but not sclerostin up-regulation. On the other hand, TZDs were capable of activating peroxisome proliferator-activated receptor-γ, and activation of this signaling pathway led to sclerostin up-regulation but not osteocyte apoptosis. This study demonstrates two distinct signaling pathways activated in osteocytes in response to TZDs that could participate in the observed increase in fractures in TZD-treated patients.

Relationships between bone mass and micro-architecture at the mandible and iliac bone in edentulous subjects: a dual X-ray absorptiometry, computerised tomography and microcomputed tomography study.

OBJECTIVES: To compare bone volume, bone mineral density, cortical thickness and bone micro-architecture in a series of paired mandibular and iliac bone samples analysed by various imagery techniques to see whether relationships exist between the various techniques and between mandibular and iliac bone. MATERIALS AND METHODS: Bone samples from the mandible and ilium were harvested in 20 cadavers and analysed by dual energy X-ray absorptiometry (DXA), computerised tomography (CT) on a conventional hospital machine and microCT. RESULTS: Significant correlations were found between Hounsfield density obtained by CT, and bone mass determined by microCT but not with DXA values. Cortical thickness measurements were well correlated between CT and microCT. No relationships were found between mandibular and iliac bone, when considering mineral density, cortical thickness, bone volume or micro-architecture. CONCLUSION: In clinical practice, CT remains the most appropriate routine means for bone qualitative and quantitative evaluation at the mandible. In this ex vivo study, these results confirm that mandibular bone status does not reflect the axial skeletal one and assist in the placement of implants with dental prostheses in old or osteoporotic patients.

Cellular and molecular effects of thiazolidinediones on bone cells: a review.

Thiazolidinediones represent a class of molecules used in the treatment of type 2 diabetes mellitus. Despite interesting effects in lowering blood glucose and HbA1c levels durably, an augmentation of the fracture risk in women has emerged in the past years. This review is providing the readers with information about the cellular and molecular mechanisms involved in bone and bone cells in response to these drugs.

In vitro assessment of osteoblast and macrophage mobility in presence of ß-TCP particles by videomicroscopy.

ß-TCP is widely used to repair bone defects due to its good biocompatibility, macroporosity (favoring bone ingrowth) and bioresorbability. However, cell interactions with the biomaterial at the first times of implantation remain largely unknown. We have observed cell behaviors in direct contact with ß-TCP particles using long-term culture under videomicroscopy. Osteoblastlike cells (SaOs-2) and macrophages (J774.2 and mouse peritoneal macrophages) were cultured in the presence of ß-TCP particles. For each experiment, images from 20 independent fields were acquired and stored every 15 min during 8 days. At the end of the culture, they were combined to generate time lapse videos; coverslips were fixed and observed by scanning electron microscopy (SEM). SaOs-2 proliferation was determined by counting cells on six different and independent fields at days 1, 3, and 6. Videos showed the capacity of cells to displace the particles. Dynamic follow-up showed active proliferation of SaOs-2 occurring in the direction of the particles. J774.2 and peritoneal macrophages did not proliferate but came in direct contact with the particles and actively eroded them. SEM showed that cells were stretched and fixed onto the surface and seemed to climb from the coverslip to the particles. The long-term culture under videomicroscopy allowed a better understanding of the colonization process of ß-TCP particles by osteoblastlike cells and macrophages. Data obtained from long-term videomicroscopy are in agreement with in vivo observations confirming the interest of ß-TCP to promote osteogenesis.

Sinus lift augmentation and beta-TCP: a microCT and histologic analysis on human bone biopsies.

Sinus lift elevation is an interesting method to restore bone mass at the maxilla in edentulated patients. We have investigated the histological effects of beta tricalcium phosphate (beta-TCP) combined with autograft bone for sinus lift elevation. A series of 14 patients who were candidate for dental implantation were grafted with beta-TCP granules and morcellized autograft bone harvested at the chin. beta-TCP was characterized by scanning electron microscopy and optical profilometry. Before implant placement, a small bone biopsy (2mm in diameter) was done. The amount of residual material and newly formed bone were determined by microcomputed tomography. Histological analysis was done on undecalcified sections stained by Goldner's trichrome and osteoclast identification (TRAcP). beta-TCP served as a template for bone apposition by osteoblasts onto the granules' surface. The material was simultaneously resorbed by TRAcP positive osteoclasts and macrophages. Fragments of the material remained buried in bone trabeculae as long as 12 months post-graft but the formed bone onto the granules surface had a lamellar texture. beta-TCP combined with autograft bone appears a suitable biomaterial for sinus lift augmentation before the placement of bone implants. The material favors the apposition of lamellar bone by osteoblasts and is simultaneous resorbed by two types of cells.

The cathepsin K inhibitor AAE581 induces morphological changes in osteoclasts of treated patients.

Inhibitors of Cathepsin K (Cat-K) are recognized as an interesting way to inhibit osteoclast (OC) activity. OCs from patients treated with the anticathepsin-K inhibitor AAE581 (balicatib) were found enlarged. They contained numerous vacuoles filled with tartrate resistant acid phosphatase (TRAcP), an intracellular enzyme that terminates the degradation of collagen internalized in OC transcytotic vesicles. In a phase 2 clinical study, 675 patients with postmenopausal osteoporosis received the Cat-K inhibitor AAE581 at 0, 5, 10, 25, or 50 mg/D during 1 year. Eleven patients had a transiliac bone biopsy, studied undecalcified. Histoenzymatic detection of TRAcP was used to identify and count OC number. The histomorphometrist was not aware of the randomization of patients at the time of analysis. OC were unstained in one patient because of a failure in the fixation protocol, but easily observable in the 10 remaining patients. Whatever the received dose, treated patients exhibited a characteristic aspect of the OC cytoplasm which appeared filled of deeply-stained brown vacuoles, making cells looking like bunches of grape. These round vacuoles, evidenced on TRAcP-stained sections, were due to the accumulation of intracytoplasmic TRAcP. This led to a moderate enlargement of the OC size when compared to a series of control osteoporotic patients. AAE581 did not induce OC apoptosis at any dosage but it modified OC morphology. Cat-K inhibition (inhibiting the extracellular collagen breakdown) is associated with a compensatory accumulation of intracellular TRAcP that could not be used to complete protein degradation. TRAcP is also known to be degraded by Cat-K.

Migration of wear debris of polyethylene depends on bone microarchitecture.

The mechanism of hip arthroplasties loosening is related to the migration of wear debris throughout the implant environment. In vivo, polyethylene particles (PE) were shown to infiltrate the bone implant interface and the medullary spaces of the cancellous bone. Our test hypothesis was that polyethylene particle migration is correlated to bone porosity. Bone samples with a high or low trabecular volume and microarchitecture were harvested in 20 calves and 20 human cadavers. They were extensively washed to remove marrow cells. Bone cylinders were filled with a light-curing monomer having the same viscosity as bone marrow. PE particles (7 and 33 microm) were deposited at the surface of the polymer. The bone cylinders were agitated during 7 days on an orbital shaker and the gel was left to polymerize at day light. X-ray microtomography was performed to characterize bone volume and microarchitecture. Cylinders were sectioned and observed under polarized light. The migration distance and rate were determined. Migration of PE particles strongly depended on trabecular bone volume and microarchitecture. We found a linear relationship (r = 0.61) between speed migration and bone volume and an exponential relationship between speed migration and bone architecture. The present in vitro model confirmed our hypothesis about the key role of bone microarchitecture in the migration of large PE wear particles. This is an explanation for the development of inflammatory reaction at distance from a prosthesis although our study did not include submicron particles.

Bone grafts cultured with bone marrow stromal cells for the repair of critical bone defects: an experimental study in mice.

Tissue engineering of autologous bone combined with osteoprogenitor cells is a suitable strategy for filling large bone defects. The aim of this study was to evaluate the osteogenicity of a xenogenic bone graft cultured with allogenic bone marrow stromal cells (BMSC) in a mouse critical size craniotomy. Bovine trabecular bone grafts were made free of bone marrow cells or debris and were delipidated. BMSC were harvested from C57BL/6-Tg(ACTbEGFP)1Osb/J mice (GFP+ cells) and were cultured 14 days on bone grafts in control or osteogenic medium. Engineered grafts were implanted in calvarial defect in C57BL/6 mice. Four groups were studied: graft with BMSC differentiated in osteoblasts (G-Ob), graft with BMSC (G-BMSC), graft without cells (G) and no graft. Calvariae were studied 2 and 8 weeks after implantation by radiographic and histomorphometric analyses. G group: the bone ingrowth was limited to the edges of the defect. The center of the graft was filled by a fibrovascular connective tissue. G-BMSC or G-Ob groups: bone formation occurred early in the center of the defect and did not increase between 2 and 8 weeks; the newly formed woven bone was partially replaced by lamellar bone. The preoperative osteoblastic differentiation of BMSC did not allow faster and better bone regeneration. After 2 weeks, GFP+ cells were observed around the grafted bone but no GFP+ osteocyte was present in the newly formed bone. No GFP+ cell was noted after 8 weeks. However, pre-implantation culture of the biomaterial with allogenic BMSC greatly enhanced the bone regeneration.

Viability of osteocytes in bone autografts harvested for dental implantology.

Bone autograft remains a very useful and popular way for filling bone defects. In maxillofacial surgery or implantology, it is used to increase the volume of the maxilla or mandible before placing dental implants. Because there is a noticeable delay between harvesting the graft and its insertion in the receiver site, we evaluated the morphologic changes at the light and transmission electron microscopy levels. Five patients having an autograft (bone harvested from the chin) were enrolled in the study. A small fragment of the graft was immediately fixed after harvesting and a second one was similarly processed at the end of the grafting period when bone has been stored at room temperature for a 20 min +/- 33 s period in saline. A net increase in the number of osteocyte lacunae filled with cellular debris was observed (+41.5%). However no cytologic alteration could be observed in the remaining osteocytes. The viability of these cells is known to contribute to the success of autograft in association with other less well-identified factors.

Iron inhibits hydroxyapatite crystal growth in vitro.

Hemochromatosis is a known cause of osteoporosis in which the pathophysiology of bone loss is largely unknown and the role of iron remains questionable. We have investigated the effects of iron on the growth of hydroxyapatite crystals in vitro on carboxymethylated poly(2-hydroxyethyl methacrylate) pellets. This noncellular and enzyme-independent model mimics the calcification of woven bone (composed of calcospherites made of hydroxyapatite crystals). Polymer pellets were incubated with body fluid containing iron at increasing concentrations (20, 40, 60 micromol/L). Hydroxyapatite growth was studied by chemical analysis, scanning electron microscopy, and Raman microscopy. When incubated in body fluid containing iron, significant differences were observed with control pellets. Iron was detected at a concentration of 5.41- to 7.16-fold that of controls. In pellets incubated with iron, there was a approximately 3- to 4-fold decrease of Ca and P and a approximately 1.3- to 1.4-fold increase in the Ca/P ratio. There was no significant difference among the iron groups of pellets, but a trend to a decrease of Ca with the increase of iron concentration was noted. Calcospherite diameters were significantly lower on pellets incubated with iron. Raman microspectroscopy showed a decrease in crystallinity (measured by the full width of the half height of the 960 Deltacm(-1) band) with a significant increase in carbonate substitution (measured by the intensity ratio of 1071 to 960 Deltacm(-1) band). Energy dispersive x-ray analysis identified iron in the calcospherites. In vitro, iron is capable to inhibit bone crystal growth with significant changes in crystallinity and carbonate substitution.

Effects of FGF-2 release from a hydrogel polymer on bone mass and microarchitecture.

Bone substitutes are widely used for filling and restoring bone defects. Among them, methacrylic polymers are employed in load-bearing bones to seal hip prostheses. Incorporation of growth factors into a polymer device could be a way to enhance bone growth. In the present study, we evaluated the capacity of poly(2-hydroxyethyl methacrylate) - pHEMA - copolymerized with 2-vinyl pyrrolidone - VP - to release proteins. Fibroblast growth factor-2 (FGF-2) was incorporated into cylinders of p(HEMA-co-VP). FGF-2 release was studied by ELISA in vitro and cylinders were implanted in the femoral condyle of white New Zealand rabbits. After 2 months post-surgery, FGF-2 was able to enhance bone formation by increasing bone volume; this effect was evidenced by an increase in trabecular number and bone gain was mainly in the form of woven bone. At 3 months post-surgery, no difference could be evidenced between animals receiving vehicle or FGF-2. Animals receiving vehicle exhibited bone mass higher than at 2 months and woven bone was replaced by mature bone with a lamellar matrix. The hydrogel polymer allowed the release of FGF-2, which in return enhanced bone regeneration soon after surgery but the effect vanished rapidly.

Bone changes in myelofibrosis with myeloid metaplasia: a histomorphometric and microcomputed tomographic study.

Myelofibrosis with myeloid metaplasia (MMM) is a clonal disorder of the haematopoietic stem cell which can be associated with marrow fibrosis and/or osteosclerosis. Because bone progenitors and mature bone cells are influenced by the marrow microenvironment, cellular and tissular changes were assessed by histomorphometry in MMM. Thirteen patients, with a clinical proven MMM, had a bone biopsy of the iliac crest with double tetracycline labelling and osteoclast count. Histomorphometry was done at the 2D level (bone volume, osteoid parameters, bone histodynamic parameters and osteoclast count) and 3D level by microcomputed tomography. All patients had clusters of abnormal megakaryocytes in bone marrow. Newly apposed bone packets were observed in 12 patients and corresponded to an increased thickness of some bone units with new lamellae or focal areas of woven bone anchored on the pre-existing trabeculae. Osteoid parameters were unchanged, only bone formation rate appeared considerably increased in seven patients. There was a net tendency for decrease in osteoclast number and conversion of trabecular pillars into plates. An uncoupling of bone remodelling was evidenced with an increased life-span of osteoblasts associated with a normal/reduced osteoclast activity. A very complex network of factors is candidate to explain bone changes observed in MMM.

Absence of renal lesions in C57BL/KaLwRij mice with advanced myeloma due to 5T2MM cells.

Renal failure is one of the main complications in multiple myeloma (MM) and histopathological lesions are due to light chains accumulation in the kidney. The 5T2MM mouse model closely mimics osteolytic lesions observed in clinics. We studied the occurrence of pathological changes in the kidney of mice inoculated with 5T2MM myeloma cells. No renal lesions due to light chain deposition were observed after histological, immunological staining and dosage of creatinine in serum and urine. PTH levels decreased in 5T2MM mice, confirming the absence of secondary hyperparathyroidism. Osteolytic lesions appear to be the unique consequence of 5T2MM cells inoculation.

The influence of processes for the purification of human bone allografts on the matrix surface and cytocompatibility.

Different industrial processes exist to purify allogenic bone, providing safe and cleaned blocks for bone allografting. However, they often make use of chemical reagents that can be aggressive for the bone matrix. Bone samples were processed with several soaking techniques used in industry: NaHCO3, H2O2, NaOH and H2O2+NaOH combined; the consequences on the bone matrix and cytocompoatibility were evaluated on femoral heads from osteoarthritic patients. Alterations of matrix were searched by histochemistry, atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cytocompatibility was evaluated by coculturing human osteoblast-like cells (SaOS-2) on bone slices. Collagen fibers were dramatically altered at the surface of bone treated with H2O2, NaOH (and their association), but not with NaHCO3. A marked reduction in the number of hydroxyapatite crystals was observed on the trabecular surfaces by TEM and morphological changes were evidenced in SEM and AFM. Argyrophilic proteins of the bone matrix were removed by H2O2 and NaOH (and their association), but not by NaHCO3. As a consequence, attachment, spreading, proliferation and alkaline phosphatase activity of SaOS-2 were reduced by H2O2 and NaOH treatments. Strong oxidizing reagents altered matrix integrity by modifying collagenous and non-collagenous proteins. Whether these changes have clinical consequences on the bone bonding and osseointegration in human necessitate further investigations.

Medullar fat influences texture analysis of trabecular microarchitecture on X-ray radiographs.

Alteration of trabecular architecture is a predictor of fracture risk in osteoporosis. Until now, microarchitecture analysis is difficult to evaluate in routine clinical practice for osteoporosis. Texture analysis on X-ray images has been advocated to be a suitable method to assess microarchitecture in bone diseases. The X-ray acquisition conditions have been often taken into consideration; however, the influence of anatomical conditions on texture parameters has received little interest. Because fat is a well-known problem with computed tomography and densitometry, we have designed a cadaver study to compare the influence of marrow lipids on numerous texture parameters. Twenty-one human distal radii were obtained, radiographed, and analyzed using a software that measures: heterogeneity, skeletonized parameters, run-lengths and fractal dimensions. Texture parameters were measured before, and after an extensive delipidation period lasting 3 weeks. Quality of the radiographs was improved after defatting. Delipidation had a very significant effect on measurements: afterwards defatting, the images were less blurred, and a better delineation of trabeculae and marrow cavities was obtained. This provoked an increase of parameters based on the grey level distribution but had no influence on parameters describing the reticulated honeycomb microarchitecture of the trabeculae (i.e., fractal dimension). Some parameters appeared anisotropy-sensitive, due to the different constitution and size of the trabeculae. The fat content of bone marrow induces noise that can modify some texture parameters. One should take into account the fat content of the marrow when using texture analysis to compare patients with osteoporosis due to various etiologies.

Biocompatibility studies of the Anaconda stent-graft and observations of nitinol corrosion resistance.

PURPOSE: To validate the deployment, in vivo performance, biostability, and healing capacity of the Anaconda self-expanding endoprosthesis in a canine aortic aneurysm model. METHODS: Aneurysms were surgically created in 12 dogs by sewing a woven polyester patch onto the anterior side of the thoracic or abdominal aorta. Anaconda prostheses were implanted transfemorally for prescheduled periods (1 or 3 months). Aneurysm exclusion and stent-graft patency were monitored angiographically. Healing was assessed with histological analysis and scanning electron microscopy (SEM). Textile analysis determined the physical and chemical stability of the woven polyester material, while the biostability of the nitinol wires was evaluated with SEM and spectroscopy. RESULTS: All prostheses were intact at explantation. After 1 month, endothelial-like cells were migrating in a discontinuous manner both proximally and distally over the internal collagenous pannus at the device-host boundary. After 3 months, endothelialization had reached the midsections of the devices, with a thicker collagenous internal capsule. Patches of endothelial-like cells were sharing the luminal surface with thrombotic deposits. However, the wall of the device at the level of the aneurysm was generally poorly healed, with multiple thrombi scattered irregularly over the luminal surface. The polyester fabric was intact except for some filaments that were ruptured adjacent to the sutures and some abrasion caused by the nitinol wires. No evidence of corrosion was found on the nitinol stents. CONCLUSIONS: This Anaconda stent-graft has demonstrated its ability to exclude arterial aneurysms. The device used in this study was an experimental prototype, and the manufacturer has incorporated new immobilization features into the model for clinical use. The constituent materials appear to be suitable in terms of biocompatibility, biofunctionality, and short-term durability.

Mandibular bone loss in an animal model of male osteoporosis (orchidectomized rat): a radiographic and densitometric study.

In humans, hypogonadism is associated with osteoporosis and can be studied by densitometry (DXA) on the vertebrae or long bones. There is some controversy about the relationships between bone loss in these sites and in the mandible. Osteoporosis has been suggested as a risk factor for dental problems. In the rat, orchidectomy (ORX) is associated with an increased bone resorption resulting in bone loss. We have studied the time effects of ORX on the alveolar bone in the rat. Forty-eight male Wistar rats were divided into four groups and studied over 2, 4, 8 and 16 weeks. In each group, six rats were ORX and six sham-operated (SHAM) animals were used as control. The mandible of each rat was dissected. Numeric radiographs, centered on the molar region, were obtained. Bone loss was observed qualitatively at 16 weeks in ORX animals. Quantitative modifications were confirmed by texture analysis of numeric radiographs using the run-length technique. The bone mineral content (BMC) and density (BMD) of the hemimandible and in a region centered on the molars were measured by DXA. The coefficient of variation (CV) for BMC was poor on the whole bone and no differences could be observed even at 16 weeks. For BMC of the molar region, the CV was improved and significant bone loss occurred in the ORX group at 16 weeks ( P<0.016). This study confirms that in the male rat, the reduction of sex hormones induced by ORX is associated with a decrease in bone mass in the mandible.

Proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures: effects of dexamethasone and calcitriol.

During bone loss, osteoblast population can be replaced by adipose tissue. This apparent reciprocal relationship between decreased bone density and increased fat formation can be explained by an imbalance in the production of bone-forming and fat-forming cells in the marrow cavity. Thus, osteoblast and adipocyte pathways seem more closely and inversely related. In the present study, we investigated the effects of dexamethasone (dex) and calcitriol [1,25(OH)(2)D(3)] on proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures. Stromal cells were grown in primoculture in presence of dex and subcultivated in presence of dex and/or 1,25(OH)(2)D(3). Total cell proliferation, osteoblast and adipocyte-cells number, and -mRNA specific markers were used to study the effects of hormonal treatment on stromal cells. Total cell proliferation was stimulated by dex and inhibited by 1,25(OH)(2)D(3). Dex increased osteoblast and adipocyte cell population whereas calcitriol decreased bone-forming cell number and increased fat cell population. The presence of both hormones led to a strong decrease in osteoblastic cells and to a strong increase in adipocytic cell number. Dex induced mRNA osteoblastic markers expression like bone sialoprotein (BSP) and osteocalcin (OC) and an adipocyte marker expression, the fatty acid binding protein aP2. Calcitriol decreased the dex-induced BSP expression but stimulated slightly OC and aP2 mRNA. The effects of both hormones was to increase strongly OC and aP2 mRNA. These results support that, in rat bone marrow, adipocyte proliferation and differentiation are stimulated by glucocorticoids and calcitriol which act synergically, whereas osteoblastic cell proliferation and differentiation are increased by dex and inhibited by 1,25(OH)(2)D(3).

Phenotypic effects of continuous or discontinuous treatment with dexamethasone and/or calcitriol on osteoblasts differentiated from rat bone marrow stromal cells.

Osteoblasts are target cells for glucocorticoids and calcitriol, and their phenotype is greatly modified by these hormones. We investigated the effect of continuous or discontinuous hormonal exposure to osteoblasts derived from rat bone marrow stromal cells in long-term subcultures. Stromal cells were grown in primoculture in presence of dexamethasone (dex), but in following subcultures, dex and/or calcitriol were added just after seeding or after a 7-day hormone-free period. Cell proliferation, alkaline phosphatase (ALP) histochemical staining, and enzymatic bioactivity measurement, osteocalcin (OC), ALP and bone sialoprotein (BSP) mRNA expression were used to study the differential effect on osteoblastic phenotype of various conditions of treatment by dex and calcitriol. In primoculture, the osteoblastic differentiation was confirmed by the formation of calcified nodules and by strong expression of ALP, OC, and BSP mRNAs. In subcultures, proliferation of stromal cells was stimulated by dex and inhibited by calcitriol and by both hormones. Cell proliferation was not modified by hormonal lack during 7 days. Continuous hormonal treatment by dex strongly enhanced OC and BSP mRNAs, but apparently did not modified ALP mRNAs expression. Continuous treatment by calcitriol decreased ALP and the dex-induced BSP expression and stimulated the OC mRNAs level, strongly when associated with dex. The population of ALP+ cells and ALP bioactivity were strongly increased by dex, whereas calcitriol or both hormones decreased them. When the subcultures were undergone without hormonal treatment during 7 days, all osteogenic mRNAs strongly decreased even after hormonal recovery. Dex, calcitriol, and both hormones inhibited ALP mRNAs. OC messengers were only weakly detectable with both hormones. ALP+ cell population and ALP bioactivity were decreased after 14 days of hormonal treatment recovery. These results support that continuous presence of glucocorticoids appears as a major key for the permanent expression of the osteoblastic phenotype that is inhibited by calcitriol, in the rat bone marrow.