Bone-like nodules formed by human bone marrow stromal cells: comparative study and characterization

Autologous bone marrow stromal cells have been proposed as an adjuvant in the treatment of bone nonunion. This cell therapy would require the establishment of culture conditions that permit the rapid expansion of these cells ex vivo while retaining their potential for further differentiation. Our aim was to achieve a full differentiation process using human bone marrow aspirates. We first analyzed the effects of mineralization medium (with ascorbic acid and phosphate) and dexamethasone (dex) during the primary culture of human bone marrow stromal (HBMS) cells on the proliferation/differentiation behavior of first-passage cells. The most appropriate schedule was then selected to further characterize this differentiation model. We showed that primary culture of HBMS cells in proliferation medium (DMEM supplemented with 10% fetal calf serum), with a 48-h treatment by mineralization medium and dex resulted in a better osteoblastic differentiation of first-passage cells than primary culture carried out in mineralization medium with or without dex. We showed that culture of HBMS cells under these conditions (primary culture in proliferation medium, followed by subculture in mineralization medium) led to the formation of specifically mineralized bone-like nodules similar to the ones observed with rat bone marrow stromal cells. Our nodules exhibited three distinct cell types, reproducing in vitro a tissue-like structure. This treatment demonstrated an optimal proliferation and expression of osteoblastic markers such as alkaline phosphatase, osteocalcin, and type I collagen. The primary culture allowed the multiplication of the number of adherent progenitor cells at the initial time of plating by a mean factor of 44,000, which was found to be negatively correlated with age. Thus, this differentiation model could provide a new tool to elaborate an autologous cell therapy designed to enhance osteogenesis.     

Inhibition of growth and differentiation of osteoprogenitors in mouse bone marrow stromal cell cultures by increased donor age and glucocorticoid treatment

Primary cultures of bone marrow stromal cells (BMSC) from long bones of young (4-5 months) and old (22-25 months) C57BL/6 male mice were used to study how donor age affects growth and differentiation of osteoblasts and their sensitivity to dexamethasone (DEX). We assessed changes in the number and area of alkaline phosphatase-positive bone-forming osteolastic colonies (CFU-ALP) and in the total number of colonies (CFU-F) that include ALP negative colonies. Cell proliferation and apoptosis, specific activity of ALP, were also measured for growth and differentiation. We found that the number of nucleated cells harvested from old mice was significantly higher (approximately 20% more) than that from young mice. However, the number of colonies formed by old cells was fewer and the total area less than those formed by young cells plated at the same density. Young and old cells responded similarly to DEX showing a dose-dependent decrease in colony number and area with more inhibition for area than number. DEX affected CFU-ALP more than CFU-F indicating a greater inhibition for osteoprogenitor cells than other cell types. Inhibition of cell attachment at early culture was the major cause for the DEX reduction of colony number and the major cause of area reduction was inhibition of cell proliferation. This was demonstrated by a severe dose-dependent lowering of bromodeoxyuridine (BrdU) incorporation to less than 40% of the control. Although the number of apoptotic cells in the DEX-treated cultures was higher, apoptosis was not a major factor since the number of apoptotic cells was less than 5% even with DEX treatment. Despite these negative effects on colony number and size, DEX-enhanced osteoblastic differentiation activity by stimulating ALP activity of the colonies up to 25-fold in the young and 5-fold in the old. Our data suggest that increased age lowered the number of osteoprogenitor cells and their growth in BMSC cultures. DEX decreased the attachment and proliferation of BMSC in culture. These changes reflect age-related and glucocorticoid-induced osteopenia. Mouse BMSC cultures therefore may serve as a useful in vitro model to study the mechanisms of type II osteoporosis.     

骨髓基质细胞共培养体系促进人脐血CD133+细胞的体外扩增

目的探讨来自白血病患者骨髓细胞的基质细胞层建立的扩增体系体外扩增人脐带血造血干/祖细胞的可行性。方法应用白血病患者缓解期骨髓基质细胞,结合造血细胞生长因子体外扩增人脐带血CD133 细胞(实验组),对扩增细胞的免疫表型、集落形成能力等生物学特性进行动态观察,并与单独应用造血细胞生长因子的常规扩增(对照组)进行比较。结果基质细胞层上接种的细胞易于收集;实验组在扩增各时间点的有核细胞数(NC)明显高于对照组,而且有核细胞中CD34 细胞、CD133 细胞、CD34 CD38-细胞以及CD34 CD133 细胞的比例也明显高于对照组;实验组扩增各时间点有核细胞各种集落的种植率明显高于对照组,有核细胞、CD34 细胞、CD133 细胞、CD34 CD38-细胞以及细胞集落的扩增倍数明显高于对照组。结论含有基质细胞的扩增培养体系,不仅可以加速人脐带血造血干/祖细胞的体外扩增,而且可以延缓造血干/祖细胞的分化。     

酒精对骨髓基质细胞增殖及分化的影响

目的 观察酒精对骨髓基质细胞增殖及分化的作用．探讨骨质疏松的病理学机理。方法 以0.09mol/L酒精加入骨髓基质细胞培养物中，测定增殖的骨髓基质细胞数及培养液中骨钙素含量。通过苏丹Ⅲ脂肪细胞染色计数观察酒精作用时间对脂肪细胞分化的影响。结果 实验组骨髓基质细胞数及培养液中骨钙素含量明显低于对照组，脂肪细胞的数量随酒精作用时间延长而增多。结论 酒精抑制骨髓基质细胞增殖及向成骨方向分化，促进其向脂肪细胞分化，这可能与酒精中毒引起继发性骨质疏松时骨量减少、髓内脂肪组织增多有关。     

Oscillatory fluid flow affects human marrow stromal cell proliferation and differentiation.

Mechanical loading is an important regulator of bone formation and bone loss. Decreased osteoblast number and function are important cellular mechanisms by which mechanical disuse leads to decreased bone formation. Decreased osteoblast number may be a result of decreased osteoprogenitor proliferation, differentiation, or both. However, the effects of cellular level physical signals on osteoprogenitors are not well understood. In this study, we examined the effects of loading induced oscillatory fluid flow (OFF), a potent regulator of osteoblastic cell function, on marrow stromal cells (MSCs). MSCs subjected to OFF exhibited increased intracellular Ca2+ mobilization. In addition, MSCs exhibited increased proliferation and increased mRNA levels for osteopontin and osteocalcin genes. Collagen I and core binding factor 1 mRNA levels did not change. MSCs subjected to OFF also exhibited decreased alkaline phosphatase activity. These results suggest that MSCs are mechanosensitive and that Ca2+ may play a role in the signaling pathway.     

Properties and origin of osteoblasts

Osteoblastic and chondroblastic (i.e., osteogenic) cells belong to the stromal cell system, which is associated with bone marrow, and bone and is separate from the hematopoietic stem-cell system. Stromal stem cells are capable of producing reticular, fibroblastic, osteogenic, and adipose stromal lines. Marrow-derived osteogenic cells are a component of marrow stroma, which in vitro form fibroblastic-type colonies. These colonies are a heterogeneous population with varying enzymatic expressions and potencies that differentiate into fibroblastic, reticular, adipocytic, and osteogenic populations. It is postulated that these colonies are a component of the stem- and progenitor cell populations. Progenitors of osteogenic cells are widely distributed in the extraskeletal organs. On contact with an adequate inductor, they differentiate into chondro- and/or osteoblasts, thus producing ectopic (i.e., induced) cartilage and/or bone. Such osteoprogenitor cells were termed inducible osteoprogenitor cells, in contrast to the determined osteoprogenitor cells, which are present in the bone marrow stroma and produce bone spontaneously. To the class of determined osteoprogenitors also belong endosteal cells, periosteal cells, and osteoblastic established cell lines. There is no evidence of the presence of osteogenic cells in the blood and peritoneal fluid. The concept of mesenchymal cells as an osteoblastic precursor in adult organisms is open to question.     

Estrogen regulation of growth and alkaline phosphatase expression by cultured human bone marrow stromal cells.

Estrogen has been reported to regulate the growth and differentiation of cultured murine osteoprogenitor cells in bone marrow stroma. This study tested the ability of 17beta-estradiol (E2) to regulate growth and expression of alkaline phosphatase (ALP), an osteoblastic differentiation marker, in strains of normal human bone marrow stromal cells derived from different donors. In eight strains examined, E2 at 1 and 10 nM produced at most modest effectxs on growth and ALP activity. Growth inhibition, seen in 4 of the 8 strains, was more common than stimulation (2 of the 8 strains); the greatest observed E2 effect was an inhibition of ca. 50%. E2 altered ALP activity less dramatically than cell growth. Differences from control in total ALP per culture were seen in only two strains: one was a reduction, one an increase. Colony forming assays were used to determine if E2 changed the proportion of ALP-expressing cells in marrow stromal cell cultures. In contrast to growth experiments, ALP expression under colony forming conditions (200 cells per 35 mm-diameter well) was dependent on the type of serum supplementation used. Under permissive conditions using medium supplemented with 10% charcoal-treated fetal bovine serum, 10 nM E2 increased the number of ALP-positive colonies (cfu-ap) but not the total number of colonies formed (cfu-f). When cells cultured in the presence or absence of 10 nM E2 were replated at colony forming densities, significantly higher proportions of cfu-ap were found in 2 of 6 strains examined, while pretreatment with E2 affected the number of cfu-f in only 1 of the 6 strains. Similar results were obtained when colony formation was carried out in the presence of dexamethasone and ascorbate, although these agents themselves increased the formation of both cfu-f and cfu-ap. These results show that the direct effects of E2 on human marrow stromal cells are small and vary depending on the cell strain and on the experimental conditions; however, the E2 actions observed in this study were consistent with reports that E2 exerts direct actions on osteoblasts and osteoblast progenitor cells that favor rather than suppress their phenotypic expression.     

Imatinib promotes osteoblast differentiation by inhibiting PDGFR signaling and inhibits osteoclastogenesis by both direct and stromal cell-dependent mechanisms.

Several lines of evidence suggest that imatinib may affect skeletal tissue. We show that inhibition by imatinib of PDGFR signaling in osteoblasts activates osteoblast differentiation and inhibits osteoblast proliferation and that imatinib inhibits osteoclastogenesis by both stromal cell-dependent and direct effects on osteoclast precursors. INTRODUCTION: Imatinib mesylate, an orally active inhibitor of the c-abl, c-kit, and platelet-derived growth factor receptor (PDGFR) tyrosine kinases, is in clinical use for the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal cell tumors. Interruption of both c-kit and c-abl signaling in mice induces osteopenia, suggesting that imatinib might have adverse effects on the skeleton. However, biochemical markers of bone formation increase in patients with CML starting imatinib therapy, whereas bone resorption is unchanged, despite secondary hyperparathyroidism. We assessed the actions of imatinib on bone cells in vitro to study the cellular and molecular mechanism(s) underlying the skeletal effects we observed in imatinib-treated patients. MATERIALS AND METHODS: Osteoblast differentiation was assessed using a mineralization assay, proliferation by [(3)H]thymidine incorporation, and apoptosis by a TUNEL assay. Osteoclastogenesis was assessed using murine bone marrow cultures and RAW 264.7 cells. RT and multiplex PCR were performed on RNA prepared from human bone marrow samples, osteoblastic cells, and murine bone marrow cultures. Osteoprotegerin was measured by ELISA. RESULTS: The molecular targets of imatinib are expressed in bone cells. In vitro, imatinib increases osteoblast differentiation and prevents PDGF-induced inhibition of this process. Imatinib inhibits proliferation of osteoblast-like cells induced by serum and PDGF. In murine bone marrow cultures, imatinib inhibits osteoclastogenesis stimulated by 1,25-dihydroxyvitamin D(3) and partially inhibits osteoclastogenesis induced by RANKL and macrophage-colony stimulating factor. Imatinib partially inhibited osteoclastogenesis in RANKL-stimulated RAW-264.7 cells. Treatment with imatinib increases the expression of osteoprotegerin in bone marrow from patients with CML and osteoblastic cells. CONCLUSIONS: Taken together with recent in vivo data, these results suggest a role for the molecular targets of imatinib in bone cell function, that inhibition by imatinib of PDGFR signaling in osteoblasts activates bone formation, and that the antiresorptive actions of imatinib are mediated by both stromal cell-dependent and direct effects on osteoclast precursors.     

Differential growth factor control of bone formation through osteoprogenitor differentiation

The osteogenic factors bone morphogenetic protein (BMP-7), platelet-derived growth factor (PDGF)-BB, and fibroblast growth factor (FGF-2) regulate the recruitment of osteoprogenitor cells and their proliferation and differentiation into mature osteoblasts. However, their mechanisms of action on osteoprogenitor cell growth, differentiation, and bone mineralization remain unclear. Here, we tested the hypothesis that these osteogenic agents were capable of regulating osteoblast differentiation and bone formation in vitro. Normal human bone marrow stromal (HBMS) cells were treated with BMP-7 (40 ng ml(-1)), PDGF-BB (20 ng ml(-1)), FGF-2 (20 ng ml(-1)), or FGF-2 plus BMP-7 for 28 days in a serum-containing medium with 10 mM beta-glycerophosphate and 50 microg ml(-1) ascorbic acid. BMP-7 stimulated a morphological change to cuboidal-shaped cells, increased alkaline phosphatase (ALKP) activity, bone sialoprotein (BSP) gene expression, and alizarin red S positive nodule formation. Hydroxyapatite (HA) crystal deposition in the nodules was demonstrated by Fourier transform infrared (FTIR) spectroscopy only in BMP-7- and dexamethasone (DEX)-treated cells. DEX-treated cells appeared elongated and fibroblast-like compared to BMP-7-treated cells. FGF-2 did not stimulate ALKP, and cell morphology was dystrophic. PDGF-BB had little or no effect on ALKP activity and biomineralization. Alizarin Red S staining of cells and calcium assay indicated that BMP-7, DEX, and FGF-2 enhanced calcium mineral deposition, but FTIR spectroscopic analysis demonstrated no formation of HA similar to human bone in control, PDGF-BB-, and FGF-2-treated samples. Thus, FGF-2 stimulated amorphous octacalcium phosphate mineral deposition that failed to mature into HA. Interestingly, FGF-2 abrogated BMP-7-induced ALKP activity and HA formation. Results demonstrate that BMP-7 was competent as a sole factor in the differentiation of human bone marrow stromal cells to bone-forming osteoblasts confirmed by FTIR examination of mineralized matrix. Other growth factors, PDGF, and FGF-2 were incompetent as sole factors, and FGF-2 inhibited BMP-7-stimulated osteoblast differentiation.     

地塞米松对骨髓基质干细胞体外增殖的影响

目的 观察地塞米松对小鼠骨髓基质干细胞体外增殖的作用。方法 取雄性6周龄ICR小鼠股骨骨髓基质细胞进行体外培养，在细胞培养不同时间点加入10^-8mol／L地塞米松（实验组）或保持基础培养条件（对照组），用CellTiter方法分别检测两组的细胞增殖情况，并予比较。结果 在细胞培养不同阶段开始应用地塞米松的实验组中，细胞增殖能力较对照组降低，随着培养时间延长实验组细胞数量明显少于对照组。实验组中细胞形态较对照组更趋成熟。结论 地塞米松在促进骨髓干细胞定向分化早期抑制骨髓基质干细胞体外增殖。     

Role of epidermal growth factor receptor in osteoblastic differentiation of rat bone marrow stromal cells

In an attempt to understand the role of epidermal growth factor (EGF) and its receptor (EGF-R) in osteoblastic cell differentiation, the changes in [¹²⁵I]-EGF binding capacity, synthesis of EGF-R protein, and expression of EGF-R mRNA were investigated during osteoblastic differentiation of cultured bone marrow stromal cells which were collected from the femora of young adult rats. In addition, the ability of EGF to suppress osteoblastic differentiation was also studied. Dexamethasone at a concentration of 0.1 mM increased the expression of osteoblastic markers by bone marrow stromal cells cultured in alpha-modified minimum essential medium (-MEM) con taining 1% fetal bovine serum (FBS), 50 mg/ml ascorbic acid, and 10 mM -glycerophosphate, as revealed by elevated alkaline phosphatase activity, an increase in osteopontin mRNA expression, and bone nodule formation. This osteoblastic differentiation was accompanied by a decreased expression of EGF-R mRNA, decreased synthesis of EGF-R protein, and a decreased number of EGF-binding sites without any change in affinity. When these cells were incubated with dexamethasone and EGF in combination throughout the culture, they exhibited significantly lower levels of all osteoblastic markers than did dexamethasonetreated cells, indicating suppression of osteoblastic differentiation by EGF. In contrast, EGF treatment of the cells induced expression of EGF-R mRNA. Thus, a decrease in EGF binding associated with osteoblastic differentiation could lead to decreased responsiveness of bone marrow cells to EGF, whereas the EGF-induced increase in expression of EGF-R could facilitate the inhibition of cell differentiation by EGF. These findings suggested that upregulation of EGF-R on bone marrow stromal cells antagonizes their differentiation, and thus possibly functions as a negative regulator of osteoblastic differentiation.     

Effects of aluminum on rat bone cell populations

Summary Aluminum (Al) loading is associated with reduced bone formation and osteomalacia in human and certain animal models. However, uncertainty exists as to the cellular effect(s) of Al as both inhibition and stimulation of osteoblast proliferation have been reported. Furthermore, the extent to which Al affects osteoprogenitor cell populations is unknown. To determine the cellular effects of Al in the rat, an animal model in which Al bone disease has been produced, we compared thein vitro effect of 10–50 Al on the proliferation and hydroxyproline collagen formation of marrow osteoprogenitor stromal cell populations and perinatal rat calvarial osteoblasts. In subconfluent cultures, Al suppressed proliferation of both marrow fibroblast-like stromal cells and calvarial osteoblasts. In confluent cultures, however, Al selectively stimulated periosteal fibroblast and osteoblast DNA synthesis and collagen (hydroxyproline) production, both in the presence or absence of 1,25-dihydroxyvitamin D. Osteocalcin was not detected in osteoblast-conditioned media or extracellular matrix. These observations suggest that the bone formation defect associated with Al toxicity in growing rats may be a function of impaired patterns of osteoprogenitor/osteoblast proliferation. Furthermore, the Al-stimulated increase in collagen formation is consistent with the development of osteomalacia in Al-toxic humans and animals. The mechanism by which Al stimulated DNA synthesis and collagen production in more mature cultures awaits further study.     

Importance of 1,25-dihydroxyvitamin D3 and the nonadherent cells of marrow for osteoblast differentiation from rat marrow stromal cells

Although steroid hormones regulate mature osteoblast function, much less is known about their actions on osteoprogenitor cells. The possibility of steroid hormone regulation of early stages in osteoblast differentiation was investigated by measuring the growth and induction of the osteoblast marker enzyme alkaline phosphatase (AP) in rat bone marrow stromal cell cultures. Experiments were performed in charcoalstripped serum; conditions which markedly impaired stromal cell growth. However, growth could be stimulated by nonadherent marrow cell-derived conditioned medium. 1,25(OH)₂D₃, but not dexamethasone, 17β-estradiol, or retinoic acid, increased both stromal cell proliferation and AP activity. The increased proliferation with 1,25(OH)₂D₃ was nonadherent cell-dependent. BMP-2 also increased AP levels and acted in synergy with 1,25(OH)₂D₃. These results suggest that (i) nonadherent marrow cells may support stromal cell development, and (ii) 1,25(OH)₂D₃ as well as glucocorticoids may regulate osteogenesis from the bone marrow but a similar role for estrogen is not supported.     

IGF-I does not affect the proliferation or early osteogenic differentiation of human marrow stromal cells

The ability of insulin-like growth factor-I (IGF-I) to regulate the proliferation and differentiation of primitive osteogenic precursors (CFU-F) has been investigated in cultures of bone marrow stromal cells (BMSC) derived from a large cohort of adult human donors. Treatment with IGF-I (0.1-20 ng/mL, days 0-28) had no consistent effect on the number or size of colonies that formed or the proportion of colonies that expressed the developmental marker alkaline phosphatase (AP). At the end of primary culture, similar numbers of cells were harvested from the control and IGF-I-treated groups and there was no detectable difference in the expression of AP (activity or percentage of positive cells) or the developmental marker STRO-1. This was found to be the case whether IGF-I was added alone or in combination with 10 nM dexamethasone (Dx), a known inducer of osteogenic differentiation in this cell culture system. In contrast, cells derived from the same cohort of donors responded to treatment with fibroblast growth factor-2 (FGF-2) with an increase in the number and size of the colonies that formed, in proliferation and in the number of cells recovered in STRO-1(+)/AP(+) (osteoprogenitor) fraction. Further analysis revealed that the majority of BMSC expressed the alpha and beta subunits of the type 1 receptor for IGF-I (IGF-IR), in the expected 1:1 ratio. Treatment with Dx did not affect the expression of these receptor subunits (percentage of positive cells or number of sites per cell) but did increase the proportion of cells present in the IGF-I(+)/AP(+) fraction. The results of this investigation suggest that the beneficial effects of IGF-I on the skeleton are not mediated primarily via an effect on osteoprogenitor fraction and are thus consistent with the hypothesis that the effects of IGF-I are differentiation dependent and restricted largely to the more mature cells of the osteoblast lineage.     

Fibrillin‐1 Regulates Skeletal Stem Cell Differentiation by Modulating TGFβ Activity Within the Marrow Niche

A full understanding of the microenvironmental factors that control the activities of skeletal stem cells (also known as mesenchymal stem cells [MSCs]) in the adult bone marrow holds great promise for developing new therapeutic strategies to mitigate age‐related diseases of bone and cartilage degeneration. Bone loss is an understudied manifestation of Marfan syndrome, a multisystem disease associated with mutations in the extracellular matrix protein and TGFβ modulator fibrillin‐1. Here we demonstrate that progressive loss of cancellous bone in mice with limbs deficient for fibrillin‐1 (Fbn1^Prx1–/– mice) is accounted for by premature depletion of MSCs and osteoprogenitor cells combined with constitutively enhanced bone resorption. Longitudinal analyses of Fbn1^Prx1–/– mice showed incremental bone loss and trabecular microarchitecture degeneration accompanied by a progressive decrease in the number and clonogenic potential of MSCs. Significant paucity of marrow fat cells in the long bones of Fbn1^Prx1–/– mice, together with reduced adipogenic potential of marrow stromal cell cultures, indicated an additional defect in MSC differentiation. This postulate was corroborated by showing that an Fbn1‐silenced osteoprogenitor cell line cultured in the presence of insulin yielded fewer than normal adipocytes and exhibited relatively lower PPARγ levels. Consonant with fibrillin‐1 modulation of TGFβ bioavailability, cultures of marrow stromal cells from Fbn1^Prx1–/– limb bones showed improper overactivation of latent TGFβ. In line with this finding, systemic TGFβ neutralization improved bone mass and trabecular microarchitecture along with normalizing the number of MSCs, osteoprogenitor cells, and marrow adipocytes. Collectively, our findings show that fibrillin‐1 regulates MSC activity by modulating TGFβ bioavailability within the microenvironment of marrow niches. © 2015 American Society for Bone and Mineral Research.     

Polymethylmethacrylate particles inhibit osteoblastic differentiation of MC3T3‐E1 osteoprogenitor cells

Orthopedic wear debris has been implicated as a significant inhibitory factor of osteoblast differentiation. Polymethylmethacrylate (PMMA) particles have been previously shown to inhibit the differentiation of osteoprogenitors in heterogeneous murine marrow stromal cell cultures, but the effect of PMMA particles on pure osteoprogenitor populations remains unknown. In this study, we challenged murine MC3T3‐E1 osteoprogenitor cells with PMMA particles during their initial differentiation in osteogenic medium. MC3T3‐E1 cultures challenged with PMMA particles showed a gradual dose‐dependent decrease in mineralization, cell number, and alkaline phosphatase activity at low particle doses (0.038–0.150% v/v) and complete reduction of these outcome parameters at high particle doses (≥0.300% v/v). MC3T3‐E1 cultures challenged with a high particle dose (0.300% v/v) showed no rise in these outcome parameters over time, whereas cultures challenged with a low particle dose (0.075% v/v) showed a normal or reduced rate of increase compared to controls. Osteocalcin production was not significantly affected by particles at all doses tested. MC3T3‐E1 cells grown in conditioned medium from particle‐treated MC3T3‐E1 cultures showed a significant reduction in mineralization only. These results indicate that direct exposure of MC3T3‐E1 osteoprogenitors to PMMA particles results in suppression of osteogenic proliferation and differentiation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:932–936, 2008     

Aluminum Accelerates Osteoblastic Differentiation But is Cytotoxic in Long-Term Rat Calvaria Cell Cultures

We have examined the effects of aluminum (Al) on osteoprogenitor proliferation and differentiation, cell survival, and bone formation in long-term rat calvaria (RC) cell cultures. RC cells were grown in α minimal essential medium containing 10% fetal bovine serum, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphate with or without Al added to final concentrations of 1 μM—1 mM. Al caused a dose-dependent increase in the number of bone nodules present at early times (day 11) but had no significant effect on nodule numbers at later times (day 17). Time course experiments showed that Al increased nodule number beginning from day 7. Alkaline phosphatase activity, assessed at four stages during the differentiation sequence of RC cell cultures (from 4 to 13 days) was stimulated by Al at all times. However, Al decreased colony formation, inhibited cell growth in late log phase, and decreased saturation density of the treated cultures. Al concentrations of 30 μM and above resulted in degeneration of the cell layer and an increasing fibrillar appearance of the matrix present in between or adjacent to nodules when cultures were maintained for more than 15 days. The presence of Al significantly decreased the viability of cells obtained from 13–17 days cultures, as determined by plating efficiency and trypan blue exclusion. We frequently observed cellular toxicity (in 8 of 10 experiments) in cultures containing 300 μM Al, and by days 17–19, cells, nodules, and matrix were disintegrating in these cultures. We conclude that Al accelerates the rate of osteoprogenitor cell differentiation and the formation of bone nodules while concomitantly inhibiting nodule mineralization. However, concentrations that accelerate differentiation appear to be cytotoxic in long-term cultures. Received: 29 April 1997 / Accepted: 9 December 1998