Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2

We have examined the effects of BMP-2 on the expression of bone matrix proteins in both human bone marrow stromal cells (HBMSC) and human osteoblasts (HOB) and their proliferation and mineralization. Both HBMSC and HOB express BMP-2/-4 type I and type II receptors. Treatment of these two cell types with BMP-2 for 4 weeks in the presence of β-glycerophosphate and ascorbic acid results in mineralization of their matrix. BMP-2 increases the mRNA level and activities of alkaline phosphatase and elevates the mRNA levels and protein synthesis of osteopontin, bone sialoprotein, osteocalcin, and α1(I) collagen in both cell types. Whereas the mRNA level of decorin is increased, the mRNA concentration of biglycan is not altered by BMP-2. No effect on osteonectin is observed. The effect of BMP-2 on bone matrix protein expression is dose dependent from 25 to 100 ng/ml and is evident after 1–7 days treatment. In the presence of BMP-2, proliferation of HBMSC and HOB is decreased under either serum-free condition or in the presence of serum. Thus, BMP-2 has profound effects on the proliferation, expression of most of the bone matrix proteins and the mineralization of both relatively immature human bone marrow stromal preosteoblasts and mature human osteoblasts. J. Cell. Biochem. 67:386–398, 1997. © 1997 Wiley-Liss, Inc.     

Sulfated hyaluronic acid and dexamethasone possess a synergistic potential in the differentiation of osteoblasts from human bone marrow stromal cells

The development of novel bioactive biomaterials is urgently needed to meet the needs of an aging population. Both sulfated hyaluronic acid and dexamethasone are candidates for the functionalization of bone grafts, as they have been shown to enhance the differentiation of osteoblasts from bone marrow stromal cells in vitro and in vivo. However, the underlying mechanisms are not fully understood. Furthermore, studies combining different approaches to assess synergistic potentials are rare. In this study, we aim to gain insights into the mode of action of both sulfated hyaluronic acid and dexamethasone by a comprehensive analysis of the cellular fraction, released matrix vesicles, and the extracellular matrix, combining classical biochemical assays with mass spectrometry–based proteomics, supported by novel bioinformatical computations. We found elevated differentiation levels for both treatments, which were further enhanced by a combination of sulfated hyaluronic acid and dexamethasone. Single treatments revealed specific effects on osteogenic differentiation. Dexamethasone activates signalling pathways involved in the differentiation of osteoblasts, for example, CXC-motif chemokine receptor type 4 and mitogen-activated protein kinases. The effects of sulfated hyaluronic acid were predominantly linked to an alteration in the composition of the extracellular matrix, affecting the synthesis, secretion, and/or activity of fibrillary (fibronectin and thrombospondin-2) and nonfibrillary (transglutaminase-2, periostin, and lysyloxidase) extracellular matrix components, including proteases and their inhibitors (matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-3). The effects were treatment specific, and less additive or contrary effects were found. Thus, we anticipate that the synergistic action of the treatment-specific effects is the key driver in elevated osteogenesis.     

A dual role of cholesterol in osteogenic differentiation of bone marrow stromal cells

Osteoblasts, the chief bone-forming cells, are differentiated from mesenchymal stromal/stem cells. Disruption of this differentiation process can cause osteoporosis, a bone disease characterized by low bone mass and deteriorated bone structure. Cholesterol has been implicated in pathogenesis of osteoporosis, and was recently identified as an endogenous activator of Hedgehog (Hh) signaling. However, its pathological and physiological roles in osteoblast differentiation are still poorly understood. Moreover, it is unclear whether these potential roles played by cholesterol are related to its capability to modulate Hh pathway. In this study, we investigated the role of exogenous versus endogenous cholesterol in osteogenesis and Hh pathway activation using ST2 cells, a bone marrow stromal cell line. We found that exogenous cholesterol significantly inhibited alkaline phosphatase (ALP) activity and messenger RNA expression of osteoblast markers genes (Alpl, Sp7, and Ibsp) while modestly activating expression of Gli1 (a readout of Hh signaling) under both basal osteogenic culture condition and Wnt3a treatment. Similarly, exogenous cholesterol suppressed osteogenic response of ST2 cells to sonic Hh (Shh) or purmorphamine (Purmo) treatment, which, however, was accompanied by diminished induction of Gli1, indicating the involvement of a Hh-dependent mechanism. Interestingly, depletion of endogenous cholesterol also reduced Shh-induced ALP activity and Gli1 expression. Likewise, cholesterol depletion inhibited osteogenic response to Purmo, although it did not affect Gli1 induction. Taken together, our findings have demonstrated that cholesterol plays a dual role in osteoblast differentiation likely through both Hh-dependent and -independent mechanisms.     

Strontium ions promote in vitro human bone marrow stromal cell proliferation and differentiation in calcium-lacking media

In order to investigate the influence of calcium and strontium ion concentration on human bone marrow stromal cells and their differentiation to osteoblasts, different cell culture media have been used. Even though this study does not contain a bone substitute material, the reason for this study was the decrease of cation concentration by many biomaterials, due to induced apatite precipitation. As a consequence, the reduced calcium ion concentration is known to affect osteoblastic development. Therefore, the main focus was put on the question, whether an increased strontium concentration (in the range of mM) might be suitable to compensate the lack of calcium ions. The effect of solely strontium ions—with only calcium in the media resulting from fetal calf serum—was investigated. Commercially available calcium-free medium (modified α-MEM) was tested in comparison with media with varied calcium ion concentrations (0.9, 1.8, and 3.6 mM), or strontium ion concentration (0.4, 0.9, 1.8, and 3.6 mM). In case of calcium, higher concentrations cause increased proliferation, while differentiation was shifted to earlier points of time. Differentiation was increased by solely strontium ions only at 0.4–0.9 mM, while proliferation was highest for 0.9–1.8 mM. From these results, it can be concluded that strontium is able to compensate a lack of calcium to a certain degree. Thus, in contrast to calcium ion release, a strontium ion release from bone substitute materials might be applicable for stimulation of bone regeneration without influencing the media saturation.     

人骨髓基质细胞向成骨细胞分化过程中差异表达基因的筛选

为了探讨人骨髓基质细胞（bone marrow stromal cells,BMSCs）向成骨细胞分化过程中差异表达的基因,本实验采用体外培养人BMSCs,诱导向成骨细胞分化。分别选取培养12和21d的细胞作为驱动方（driver）和实验方（tester）,进行抑制消减杂交,构建cDNA消减文库,将挑选出的阳性克隆与GenBank人基因库中己公布的核酸序列进行同源性比较分析。结果表明,从培养21d的BMSCs中,筛查出5个差异基因,与人基因库中己知基因的同源性分别达到90％以上。有兴趣的是,核心蛋白聚糖和Bax inhibitorl在培养2ld的BMSCs中差异表达。RT-PCR检测显示,核心蛋白聚糖基因在培养21d的细胞中高表达,而在12d的细胞中未检测到表达;Bax inhibitorl基因在培养21d细胞中的表达明显高于12d的细胞。     

Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells

Dexamethasone is capable of directing osteoblastic differentiation of bone marrow stromal cells (BMSCs) in vitro, but its effects are not lineage-specific, and sustained exposure has been shown to down-regulate collagen synthesis and induce maturation of an adipocyte subpopulation within BMSC cultures. Such side effects might be reduced if dexamethasone is applied in a regimented manner, but the discrete steps in osteoblastic maturation that are stimulated by dexamethasone are not known. To examine this, dexamethasone was added to medium to initiate differentiation of rat BMSCs cultures and then removed after a varying number of days. Cell layers were analyzed for cell number, rate of collagen synthesis, expression of osteocalcin (OC), bone sialoprotein (BSP) and lipoprotein lipase (LpL), and matrix mineralization. Withdrawal of dexamethasone at 3 and 10 days was found to enhance cell number relative to continuous exposure, but did not affect to decrease collagen synthesis slightly. Late markers of osteoblastic differentiation, BSP expression and matrix mineralization, were also sensitive to dexamethasone and increased systematically with exposure while LpL systematically decreased. These results indicate that dexamethasone acts at both early and late stages to direct proliferative osteoprogenitor cells toward terminal maturation.     

Adenovirus-mediated BMP2 expression in human bone marrow stromal cells

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.     

Osteogenic differentiation of multipotent mesenchymal stromal cells isolated from human bone marrow and subcutaneous adipose tissue

Cellular populations with phenotypes similar to multipotent mesenchymal stromal cells were isolated from two different sources, including human bone marrow (BM) and subcutaneous adipose tissue (SAT). Comparative analysis of the efficiency of differentiation in the direction of osteogenesis has revealed morphological changes confirmed by staining with Alizarin red and von Kossa in bone marrow cells at the 14th day and in adipose tissue cells at the 28th day of cultivation in the medium with inductors. Analysis of expression of the osteopontin, osteocalcin, and bone sialoprotein genes in RT-PCR reactions has detected essential differences in the potential of these cells to differentiate into bone tissue cells. Cells isolated from BM of both the control and experimental groups were positive for octeopontin (OP) on the 14th day. Unlike these cells, in cells isolated from SAT in medium without an inductor, no product of OP gene expression was identified. In the cells subjected to differentiation, OP appeared at day 14. In the BM cells, octeocalcin (OC) was found at the 14th day, while the bone sialoprotein (BS) was found at the 21st day of cultivation in induction medium. In cells isolated from SAT, OC, and BS were not detected, even at the 28th day after the beginning of induction.     

人骨髓基质细胞的长期培养及其对病毒的敏感性

为了长期培养骨髓基质细胞和研究其对病毒的敏感性,我们采用静置贴壁培养法,体外长期培养了胎儿、儿童和成人骨髓基质细胞,并将传至５代以上的肌样骨髓基质细胞采用微量细胞病变（ＣＰＥ）法,开展了对５种病毒的敏感性试验。结果表明,人骨髓基质肌样细胞对滤泡性口腔炎病毒,脊髓灰质炎病毒、Ⅰ型和Ⅱ型单纯疱疹病毒均敏感,能产生明显的ＣＰＥ,其效价（ＴＣＩＤ５０）可达１０＾－３～１０＾－４,其中胎儿骨髓基质肌样细胞对     

Inflammatory T cells rapidly induce differentiation of human bone marrow stromal cells into mature osteoblasts

Activated T cells secrete multiple osteoclastogenic cytokines which play a major role in the bone destruction associated with rheumatoid arthritis. While the role of T cells in osteoclastogenesis has received much attention recently, the effect of T cells on osteoblast formation and activity is poorly defined. In this study, we investigated the hypothesis that in chronic inflammation activated T cells contribute to enhanced bone turnover by promoting osteoblastic differentiation. We show that T cells produce soluble factors that induce alkaline phosphatase activity in bone marrow stromal cells and elevated expression of mRNA for Runx2 and osteocalcin. This data indicate that T cell derived factors have the capacity to stimulate the differentiation of bone marrow stromal cells into the osteoblast phenotype. RANKL mRNA was undetectable under any conditions in highly purified bone marrow stromal cells. In contrast, RANKL was constitutively expressed in primary osteoblasts and only moderately up-regulated by activated T cell conditioned medium. Interestingly, both bone marrow stromal cells and osteoblasts expressed mRNA for RANK, which was strongly up-regulated in both cell types by activated T cell conditioned medium. Although, mRNA for the RANKL decoy receptor, osteoprotegerin, was also up-regulated by activated T cell conditioned medium, it's inhibitory effects may be mitigated by a simultaneous rise in the osteoprotegerin competitor TNF-related apoptosis-inducing ligand. Based on our data we propose that during chronic inflammation, T cells regulate bone loss by a dual mechanism involving both direct stimulation of osteoclastogenesis, by production of osteoclastogenic cytokines, and indirectly by induction of osteoblast differentiation and up-regulation of bone turnover via coupling.     

In vitro interactions between bone marrow stromal cells and hippocampal slice cultures

Bone marrow stromal cells (BMSCs) are capable of differentiating into various cell types including brain cells. Several groups have also demonstrated trophic effects of MSC grafts in experimental ischemia models. However, the underlying molecular mechanisms of these effects are not fully understood. We developed an “in vitro graft model” which consisted in a coculture of GFP-expressing BMSCs and hippocampal organotypic slice cultures. Total marrow cells (MCs) or BMSCs after one (BMSC^1P) or five passages (BMSC^5P) were transplanted on hippocampal slices. During the 10 days of our experiments, MCs and BMSC^1P migrated toward the tissue, but their total number remained constant. Conversely, the number of BMSC^5P decreased over the 10 days of the experiment, and no migration could be detected. Using immunohistochemistry, we observed that the hippocampal slices induced the expression of neural antigens in very few grafted cells, but MCs and BMSC^1P improved the conservation of the hippocampal slice culture. Similar experiments using BMSC^5P did not produce any significant change. We conclude that the number of passages greatly influence BMSCs survival rate, migration and neuroprotective capacities.     

Bone morphogenetic protein-2 and transforming growth factor-β2 interact to modulate human bone marrow stromal cell proliferation and differentiation

Osteoprogenitor cells in the human bone marrow stroma can be induced to differentiate into osteoblasts under stimulation with hormonal and local factors. We previously showed that human bone marrow stromal (HBMS) cells respond to dexamethasone and vitamin D by expressing several osteoblastic markers. In this study, we investigated the effects and interactions of local factors (BMP-2 and TGF-β₂) on HBMS cell proliferation and differentiation in short-term and long-term cultures. We found that rhTGF-β₂ increased DNA content and stimulated type I collagen synthesis, but inhibited ALP activity and mRNA levels, osteocalcin production, and mineralization of the matrix formed by HBMS cells. In contrast, rhBMP-2 increased ALP activity and mRNA levels, osteocalcin levels and calcium deposition in the extracellular matrix without affecting type I collagen synthesis and mRNA levels, showing that rhBMP-2 and rhTGF-β₂ regulate differentially HBMS cells. Co-treatment with rhBMP-2 and rhTGF-β₂ led to intermediate effects on HBMS cell proliferation and differentiation markers. rhTGF-β₂ attenuated the stimulatory effect of rhBMP-2 on osteocalcin levels, and ALP activity and mRNA levels, whereas rhBMP-2 reduced the rhTGF-β₂-enhanced DNA synthesis and type I collagen synthesis. We also investigated the effects of sequential treatments with rhBMP-2 and rhTGF-β₂ on HBMS cell differentiation in long-term culture. A transient (9 days) treatment with rhBMP-2 abolished the rhTGF-β₂ response of HBMS cells on ALP activity. In contrast, a transient (10 days) treatment with rhTGF-β₂ did not influence the subsequent rhBMP-2 action on HBMS cell differentiation. The data show that TGF-β₂ acts by increasing HBMS cell proliferation and type I collagen synthesis whereas BMP-2 acts by promoting HBMS cell differentiation. These observations suggest that TGF-β₂ and BMP-2 may act in a sequential manner at different stages to promote human bone marrow stromal cell differentiation towards the osteoblast phenotype. J. Cell. Biochem. 68:411–426, 1998. © 1998 Wiley-Liss, Inc.     

The effect of different implant biomaterials on the behavior of canine bone marrow stromal cells during their differentiation into osteoblasts

We investigated the effects of different implant biomaterials on cultured canine bone marrow stromal cells (BMSC) undergoing differentiation into osteoblasts (dBMSC). BMSC were isolated from canine humerus by marrow aspiration, cultured and differentiated on calcium phosphate scaffold (CPS), hydroxyapatite, hydroxyapatite in gel form and titanium mesh. We used the MTT method to determine the effects of osteogenic media on proliferation. The characteristics of dBMSC were assessed using alizarin red (AR), immunocytochemistry and osteoblastic markers including alkaline phosphatase/von Kossa (ALP/VK), osteocalcin (OC) and osteonectin (ON), and ELISA. The morphology of dBMSC on the biomaterials was investigated using inverted phase contrast microscopy and scanning electron microscopy. We detected expression of ALP/VK, AR, OC and ON by day 7 of culture; expression increased from day 14 until day 21. CPS supported the best adhesion, cell spreading, proliferation and differentiation of BMSCs. The effects of the biomaterials depended on their surface properties. Expression of osteoblastic markers showed that canine dBMSCs became functional osteoblasts. Tissue engineered stem cells can be useful clinically for autologous implants for treating bone wounds.     

Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells

The bone morphogenetic proteins were originally identified based on their ability to induce ectopic bone formation in vivo and have since been identified as members of the transforming growth factor-β gene superfamily. It has been well established that the bone morphogenetic cytokines enhance osteogenic activity in bone marrow stromal cells in vitro. Recent reports have described how bone morphogenetic proteins inhibited myogenic differentiation of bone marrow stromal cells in vitro. In vivo, bone marrow stromal cells differentiate along the related adipogenic pathway with advancing age. The current work reports the inhibitory effects of the bone morphorphogenetic proteins on adipogenesis in a multipotent murine bone marrow stromal cell line, BMS2. When exposed to bone morphogenetic protein-2, the pre-adipocyte BMS2 cells exhibited the expected induction of the osteogenic-related enzyme, alkaline phosphatase. Following induction of the BMS2 cells with adipogenic agonists, adipocyte differentiation was assessed by morphologic, enzymatic, and mRNA markers. Flow cytometric analysis combined with staining by the lipophilic fluorescent dye, Nile red, was used to quantitate the extent of lipid accumulation within the BMS2 cells. By this morphologic criteria, the bone morphogenetic proteins inhibited adipogenesis at concentrations of 50 to 500 ng/ml. This correlated with decreased levels of adipocyte specific enzymes and mRNAs. The BMS2 pre-adipocytes constitutively expressed mRNA encoding bone morphogenetic protein-4 and this was inhibited by adipogenic agonists. Together, these findings demonstrate that bone morphogenetic proteins act as adipogenic antagonists. This supports the hypothesis that adipogenesis and osteogenesis in the bone marrow microenvironment are reciprocally regulated.     

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).     

Silencing of RB1 and RB2/P130 during adipogenesis of bone marrow stromal cells results in dysregulated differentiation

Bone marrow adipose tissue (BMAT) is different from fat found elsewhere in the body, and only recently have some of its functions been investigated. BMAT may regulate bone marrow stem cell niche and plays a role in energy storage and thermogenesis. BMAT may be involved also in obesity and osteoporosis onset. Given the paramount functions of BMAT, we decided to better clarify the human bone marrow adipogenesis by analyzing the role of the retinoblastoma gene family, which are key players in cell cycle regulation.

Our data provide evidence that the inactivation of RB1 or RB2/P130 in uncommitted bone marrow stromal cells (BMSC) facilitates the first steps of adipogenesis. In cultures with silenced RB1 or RB2/P130, we observed an increase of clones with adipogenic potential and a higher percentage of cells accumulating lipid droplets.

Nevertheless, the absence of RB1 or RB2/P130 impaired the terminal adipocyte differentiation and gave rise to dysregulated adipose cells, with alteration in lipid uptake and release. For the first time, we evidenced that RB2/P130 plays a role in bone marrow adipogenesis.

Our data suggest that while the inactivation of retinoblastoma proteins may delay the onset of last cell division and allow more BMSC to be committed to adipocyte, it did not allow a permanent cell cycle exit, which is a prerequisite for adipocyte terminal maturation.     

Matrix remodeling associated 7 promotes differentiation of bone marrow mesenchymal stem cells toward osteoblasts

The matrix remodeling associated 7 (MXRA7) gene had been ill-studied and its biology remained to be discovered. Inspired by our previous findings and public datasets concerning MXRA7, we hypothesized that the MXRA7 gene might be involved in bone marrow mesenchymal stem cells (BMSCs) functions related to bone formation, which was checked by utilizing in vivo or in vitro methodologies. Micro-computed tomography of MXRA7-deficient mice demonstrated retarded osteogenesis, which was reflected by shorter femurs, lower bone mass in both trabecular and cortical bones compared with wild-type (WT) mice. Histology confirmed the osteopenia-like feature including thinner growth plates in MXRA7-deficient femurs. Immunofluorescence revealed less osteoblasts in MXRA7-deficient femurs. Polymerase chain reaction or western blot analysis showed that when WT BMSCs were induced to differentiate toward osteoblasts or adipocytes in culture, MXRA7 messenger RNA or protein levels were significantly increased alongside osteoblasts induction, but decreased upon adipocytes induction. Cultured MXRA7-deficient BMSCs showed decreased osteogenesis upon osteogenic differentiation induction as reflected by decreased calcium deposition or lower expression of genes responsible for osteogenesis. When recombinant MXRA7 proteins were supplemented in a culture of MXRA7-deficient BMSCs, osteogenesis or gene expression was fully restored. Upon osteoblast induction, the level of active β-catenin or phospho-extracellular signal-regulated kinase in MXRA7-deficient BMSCs was decreased compared with that in WT BMSCs, and these impairments could be rescued by recombinant MXRA7 proteins. In adipogenesis induction settings, the potency of MXRA7-deficient BMSCs to differentiate into adipocytes was increased over the WT ones. In conclusion, this study demonstrated that MXRA7 influences bone formation via regulating the balance between osteogenesis and adipogenesis in BMSCs.