Insulin-like growth factor-I induces early osteoblast gene expression in human mesenchymal stem cells

Human adult mesenchymal stem cells (hMSCs) differentiate into an osteogenic lineage if the appropriate differentiative cues, such as dexamethasone or bone morphogenetic protein 2 (BMP-2), are present. This study was undertaken to determine the role of insulin-like growth factor I (IGFI) in the regulation of early osteoblast differentiation in hMSC. Previous studies have shown that IGF-I, regulates bone formation and remodeling by participating in the differentiation of mature cells of osteoblast lineage. We hypothesized that IGF-I exerted its effects early, but the effects were too subtle to be detected. Therefore, engineered hMSCs to produce IGF-I via adenoviral transfection and used quantitative real-time PCR (qPCR) to assess marker gene expression. Here we show that IGF-I up-regulates Type I collagen, Runx2, and alkaline phosphatase (Alp) gene expression in hMSCs, genes indicative of early osteogenic differentiation. We also observed mineral deposition in the absence of dexamethasone (Dex) in hMSC cultures treated with recombinant human BMP-2 after transduction with Ad-IGF-I. In conclusion Igf-I transduction up-regulated markers of osteoblastic differentiation and in conjunction with recombinant BMP-2-induced matrix mineralization independently of Dex (see Salasznyk et al., Stem Cells Dev 14(6):608-620, 2005, this issue).     

Chemotactic migration of human mesenchymal stem cells and MC3T3-E1 osteoblast-like cells induced by COS-7 cell line expressing rhBMP-7

During bone development, remodeling, and repair, bone morphogenetic proteins (BMPs) induce the differentiation of mesenchymal progenitor cells (MPCs) that enter into the osteoblastic lineage, and enhance the recruitment of MPCs and osteogenic cells. The process of migration is believed to be regulated, in part, by growth factors stored within the bone matrix, which are released by bone resorption. In this study, primary human mesenchymal stem cells (hMSCs) and MC3T3-E1 osteoblasts were examined for chemotaxis in response to recombinant human BMP-7 (rhBMP-7) produced in COS-7 cells (co-culture system). In order to produce BMP-7 transfected cells (BTCs), which serve as suppliers of rhBMP-7 under in vitro culture conditions, the encoding DNA was transferred into the pTARGET expression vector and introduced into COS-7 cells by conventional genetic engineering techniques. In cell culture studies, the rhBMP-7 produced in BTCs stimulated the specific activity of ALP, the production of cAMP in response to PTH, and the synthesis of osteocalcin. Migration assays were conducted with a computer-aided time-lapse video-microscopy system, to allow the rapid and precise analysis of cell migration and for the dynamic measurement of cell position and morphology. The migration distance and speed of the MC3T3-E1 cells, or hMSCs, co-cultured with BTCs, using a band-type seeding method, were significantly increased (p < 0.001), compared to those of the MC3T3-E1 cells (or hMSCs) only. In conclusion, these studies revealed that rhBMP-7 plays a role in the migration of bone-forming cells, and that the co-culture model (co-culture of bone-forming cells with BMP-7-producing cells) using a computer-aided, time-lapse video-microscopy system, is useful for the chemotactic migration assay of other chemotactic growth factors.     

Uptake of nickel from 316L stainless steel into contacting osteoblastic cells and metal ion interference with BMP-2-induced alkaline phosphatase

Bone cells contacting nickel (Ni)-containing implant materials may be affected by Ni species via disturbed signaling pathways involved in bone cell development. Here we analyze effects of the Ni-containing steel 316L and major metal constituents thereof on bone morphogenetic protein-2 (BMP-2)-induced alkaline phosphatase (ALP) of MC3T3-E1 cells. While cells grew normally on 316L, cellular Ni content increased 10-fold vs. control within 4 days. With respect to the major components of 316L, Ni2+ (3-50 microM) was most inhibitory to BMP-2-induced ALP, whereas even 50 microM Fe3+, Cr3+, Mo5+, or Mn2+ had no such effect. In line with this, BMP-2-induced ALP was significantly reduced in cells on 316L. This effect was not prevented by the metal ion chelator diethylenetriaminepentaacetic acid (DTPA). Instead, DTPA abolished the stimulatory effect of BMP-2 on ALP, pointing to chelatable metal ions involved. Zn2+, as one possible candidate, antagonized the Ni2+ inhibition of BMP-2-induced ALP in both MC3T3-E1 and human bone marrow stromal cells. Results show that cells contacting 316L steel are exposed to increased concentrations of Ni which suffice to impair BMP-2-induced ALP activity. Zn2+, as a competitor of this inhibition, may help to restore normal osteoblastic function and bone development under these conditions.     

The effect of TNFα secreted from macrophages activated by titanium particles on osteogenic activity regulated by WNT/BMP signaling in osteoprogenitor cells

Wear particles are the major cause of osteolysis associated with failure of implant following total joint replacement. During this pathologic process, activated macrophages mediate inflammatory responses to increase osteoclastogenesis, leading to enhanced bone resorption. In osteolysis caused by wear particles, osteoprogenitors present along with macrophages at the implant interface may play significant roles in bone regeneration and implant osteointegration. Although the direct effects of wear particles on osteoblasts have been addressed recently, the role of activated macrophages in regulation of osteogenic activity of osteoblasts has scarcely been studied. In the present study, we examined the molecular communication between macrophages and osteoprogenitor cells that may explain the effect of wear particles on impaired bone forming activity in inflammatory bone diseases. It has been demonstrated that conditioned medium of macrophages challenged with titanium particles (Ti CM) suppresses early and late differentiation markers of osteoprogenitors, including alkaline phosphatase (ALP) activity, collagen synthesis, matrix mineralization and expression of osteocalcin and Runx2. Moreover, bone forming signals such as WNT and BMP signaling pathways were inhibited by Ti CM. Interestingly, TNFα was identified as a predominant factor in Ti CM to suppress osteogenic activity as well as WNT and BMP signaling activity. Furthermore, Ti CM or TNFα induces the expression of sclerostin (SOST) which is able to inhibit WNT and BMP signaling pathways. It was determined that over-expression of SOST suppressed ALP activity, whereas the inhibition of SOST by siRNA partially restored the effect of Ti CM on ALP activity. This study highlights the role of activated macrophages in regulation of impaired osteogenic activity seen in inflammatory conditions and provides a potential mechanism for autocrine regulation of WNT and BMP signaling mediated by TNFα via induction of SOST in osteprogenitor cells.     

RGD-grafted thermoreversible polymers to facilitate attachment of BMP-2 responsive C2C12 cells

The purpose of this study was to design thermoreversible biomaterials for enhanced adhesion of bone morphogenetic protein-2 (BMP-2)-responsive cells. Peptides containing the arginine-glycine-aspartic acid (RGD) sequence were conjugated to N-isopropylacrylamide (NiPAM) polymers via amine-reactive N-acryloxysuccinimide (NASI) groups. In monolayer cultures, the adhesion of BMP-2-responsive C2C12 cells to RGD-grafted NiPAM/NASI surfaces was significantly higher than adhesion on ungrafted NiPAM/NASI surfaces. Although the morphology of cells adhered to RGD-grafted NiPAM/NASI surfaces was comparable to cells adhered on tissue culture polystyrene (TCPS), long-term cell growth was limited on the NiPAM/NASI surfaces, even for RGD-grafted surfaces. Treatment of C2C12 cells with recombinant BMP-2 induced dose-dependent osteoblastic differentiation as assessed by alkaline phosphatase (ALP) activity. In the absence of BMP-2, cells cultured on NiPAM/NASI polymers (either grafted with RGD peptide or not) expressed significantly higher levels of ALP activity than the cells cultured on TCPS, indicating that the polymer surfaces induced some osteoblastic activity in C2C12 cells without the need for BMP-2. We conclude that NiPAM-based thermoreversible biomaterials, despite their limited ability to support cell growth, allowed an enhanced expression of the chosen osteogenic marker (ALP) by C2C12 cells in vitro.     

Bone morphogenetic proteins 5 and 6 stimulate osteoclast generation

Bone regeneration is required for fracture-healing, and different procedures have been used to promote osteogenesis. Recently, BMP-2 has been shown to induce bone formation in vivo and has been tested in clinical trials. A recent in vitro study evaluated the osteogenic activity of 14 BMPs on osteoblastic progenitor cells with an osteogenic hierarchical model in which BMP-2 and BMP-6 may play an important role in inducing osteoblast differentiation. Although the relative osteoinductive activity of each BMP is important, bone regeneration is a process consisting of bone formation and bone resorption. Therefore, it remains unclear which effects BMP-5 and -6 have on the generation of osteoclasts and by which mechanism osteoclastogenesis is stimulated. To compare osteoclastic potency of each BMP, primary murine bone marrow cells were treated with human recombinant BMP-2, BMP-5, or BMP-6 and 1,25-(OH)2 vitamin D3 and stained for the TRAP enzyme. Osteogenic activity of BMP-5 was determined by measuring induction of ALP-activity and proliferation after incubation with primary murine osteoblasts. For elucidating the molecular mechanism, primary bone marrow cells with various concentrations of OPG were added to the TRAP assay and mRNA levels of RANKL and OPG were measured after stimulation with BMP-5. The presented data show that BMP-5 and BMP-6, unlike BMP-2, enhanced the formation of murine TRAP+/MNCs in a biphasic curve. BMP-5 and -6 were less potent in stimulating osteoclastogenesis compared to BMP-2. Concerning the effects of BMP-5 on osteoblasts, there was a dose-dependent increase of ALP activity and proliferation up to a maximum dose of 300 ng/mL. At the mRNA level, BMP-5 increased the RANKL/OPG ratio. In conclusion, this study demonstrates that in contrast to BMP-2, BMP-5 and -6 influences the generation of osteoclasts in a biphasic mode. Both proteins might be very important regulators of bone homeostasis, and therefore, potent candidates for future treatment strategies of bone regeneration.     

Limited but heterogeneous osteogenic response of human bone marrow mesenchymal stem cells to bone morphogenetic protein-2 and serum

Although there are numerous reports describing the in vivo bone forming capability of recombinant human bone morphogenetic proteins-2 (rhBMP-2), studies have reported limited effects on human mesenchymal stem cells (hMSCs). However, the reasons for these discrepancies are not well understood. The aim of this study was to investigate the responsiveness of hMSCs to osteoinductive signals, focusing on rhBMP-2 and the effect of serum on that responsiveness. Human MSCs from six donors were analysed. When those cells were treated with osteoinduction medium including dexamethasone (Dex), alkaline phosphatase (ALP) activities increased in all cell lines. On the other hand, rhBMP-2-containing medium failed to increase ALP activity. When five different sera were used for cultivation and induction with rhBMP-2, ALP activities increased in two of them, but not in the others. The expression of BMP-2 antagonist noggin was induced in almost all combinations regardless of the responsiveness to rhBMP-2. On the other hand, the expression of follistatin showed significant variations depending on the serum and cell line. However, the expression did not correlate with the responsiveness to rhBMP-2. The results from this study showed limited but heterogeneous osteogenic response of hMSCs to rhBMP-2 and that the results are affected by the choice of serum. This fact should be concerned for the successful and effective clinical application of rhBMP-2.     

In vivo bone formation following transplantation of human adipose-derived stromal cells that are not differentiated osteogenically

A number of studies have shown in vivo bone regeneration by transplantation of osteogenic cells differentiated in vitro from adipose-derived stromal cells (ADSCs). However, the in vitro osteogenic differentiation process requires an additional culture period, and the dexamethasone that is generally used in the process may be cytotoxic. Here, we tested the hypothesis that ADSCs that are not differentiated osteogenically in vitro prior to transplantation would extensively regenerate bone in vivo when exogenous bone morphogenetic protein-2 (BMP-2) is delivered to the transplantation site. We fabricated a poly(dl-lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) composite scaffold with osteoactive HA that is highly exposed on the scaffold surface. This scaffold was able to release BMP-2 over a 4-week period in vitro. Human ADSCs cultured on BMP-2-loaded PLGA/HA scaffolds for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) mRNA, while cells on PLGA/HA scaffolds without BMP-2 expressed only ALP. To study in vivo bone formation, PLGA/HA scaffolds (group 1), BMP-2-loaded PLGA/HA scaffolds (group 2), undifferentiated ADSCs seeded on PLGA/HA scaffolds (group 3), and undifferentiated ADSCs seeded on BMP-2-loaded PLGA/HA scaffolds (group 4) were implanted into dorsal, subcutaneous spaces of athymic mice. Eight weeks after implantation, group 4 exhibited a 25-fold greater bone formation area and 5-fold higher calcium deposition than group 3. Bone regeneration by transplanted human ADSCs in group 4 was confirmed by expression of human-specific osteoblastic genes, ALP, collagen type I, OPN, OCN, and bone sialoprotein, while group 3 expressed much lower levels of collagen type I and OPN mRNA only. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of ADSCs without prior in vitro osteogenic differentiation, and that a PLGA/HA composite BMP-2 delivery system stimulates bone regeneration following transplantation of undifferentiated human ADSCs.     

IRAK-4对成骨细胞分化BMP-Smad通道的影响

目的　探讨白细胞介素-l受体相关激酶-4对成骨细胞分化过程BMP-Smad通道的影响。 方法　 C2C12细胞是肌卫星细胞,不同培养条件下有不同分化潜能。它可分化为成骨细胞,是研究体外成骨细胞分化的理想模型。C2C12细胞培养于培养板中,随机分为3组,每组加入不同培养物,模拟干细胞向成骨细胞分化过程中受到的不同刺激。检测ALP活性、Smad1 mRNA、P-Smad1蛋白表达,观察不同刺激对成骨细胞分化的影响。 结果　与正常对照组比较,BMP-2组ALP活性明显增加,与BMP-2组比,BMP2+IRAK-4siRNA转染组ALP活性增加,BMP2+IRAK-4siRNA转染组和BMP-2组比Smadl 　mRNA的表达只是轻微增加,P-Smad1蛋白表达明显增加。 结论　BMP-2可增强C2C12细胞成骨化,IRAK-4可抑制C2C12细胞被BMP-2诱导的成骨化,其机制可能是通过减弱BMP-Smad通道中Smad1磷酸化水平实现的。     

Ectopic osteogenesis of hBMP-2 gene-transduced human bone mesenchymal stem cells/BCB

We determined the feasibility of using scaffolds of adenoviral human BMP2 gene (AdBMP2)-modified human bone marrow mesenchymal stem cells (hBMSCs) and antigen-free bovine cancellous bone (BCB) to construct bone tissue. hMSCs were infected with AdBMP-2. Expression of BMP-2 and alkaline phosphatase confirmed successful secretion of active BMP-2. The osteogenic capability of a composite of AdBMP2-modified hMSCs with BCB was evaluated in athymic mice (group A). BCB (group B), hMSCs/BCB (group C), adenoviral β‐galactosidase genes (Adβgal)-transfected hMSCs/BCB (group D) were controls. Formation of bone tissue was assessed by histological methods 4 weeks and 8 weeks after implantation. Implanted cells were identified by human Y-chromosome-specific fluorescence in-situ hybridization (FISH). hMSCs differentiated into osteogenic cells, and bone formation was observed. Obvious bone formation was not noted at any time point in control groups. We hypothesize that the described method is a promising method for bone regeneration.     

Nell-1-induced bone regeneration in calvarial defects

Many craniofacial birth defects contain skeletal components requiring bone grafting. We previously identified the novel secreted osteogenic molecule NELL-1, first noted to be overexpressed during premature bone formation in calvarial sutures of craniosynostosis patients. Nell-1 overexpression significantly increases differentiation and mineralization selectively in osteoblasts, while newborn Nell-1 transgenic mice significantly increase premature bone formation in calvarial sutures. In the current study, cultured calvarial explants isolated from Nell-1 transgenic newborn mice (with mild sagittal synostosis) demonstrated continuous bone growth and overlapping sagittal sutures. Further investigation into gene expression cascades revealed that fibroblast growth factor-2 and transforming growth factor-beta1 stimulated Nell-1 expression, whereas bone morphogenetic protein (BMP)-2 had no direct effect. Additionally, Nell-1-induced osteogenesis in MC3T3-E1 osteoblasts through reduction in the expression of early up-regulated osteogenic regulators (OSX and ALP) but induction of later markers (OPN and OCN). Grafting Nell-1 protein-coated PLGA scaffolds into rat calvarial defects revealed the osteogenic potential of Nell-1 to induce bone regeneration equivalent to BMP-2, whereas immunohistochemistry indicated that Nell-1 reduced osterix-producing cells and increased bone sialoprotein, osteocalcin, and BMP-7 expression. Insights into Nell-1-regulated osteogenesis coupled with its ability to stimulate bone regeneration revealed a potential therapeutic role and an alternative to the currently accepted techniques for bone regeneration.     

Differential effects of growth differentiation factor-5 on porcine dental papilla- and follicle-derived cells

In this study, the effect of growth differentiation factor-5 (GDF-5) on the growth and differentiation of porcine dental papilla- and follicle-derived cells was investigated. Furthermore, the effect was compared with that of BMP-2. Recombinant mouse GDF-5 (rmGDF-5) enhanced alkaline phosphatase (ALP) activity in dental papilla-derived cells in a dose-dependent manner, while ALP activity in dental follicle-derived cells was reduced. In rmGDF-5 stimulated dental papilla-derived cells, the expressions of odontoblast-marker genes were up-regulated. Conversely, recombinant human BMP-2 (rhBMP-2) enhanced ALP activity dose-dependently in both dental papilla- and follicle-derived cells. When combined, GDF-5 did not further enhance BMP-2-induced ALP activities. Rather, GDF-5 reduced BMP-2-induced ALP activities in both dental papilla- and follicle-derived cells. This suggests that affinity of GDF-5 to the shared receptors may be higher than that of BMP-2 in both cell types. These observations indicate that GDF-5 regulates differentiation of both dental papilla and follicle during odontogenesis, co-operatively with other growth factors such as BMP-2.     

Brg1调控重组人骨形态发生蛋白2诱导成骨细胞分化

背景：Brg1是依赖ATP的染色质改变复合物的核心催化亚基,该亚基在基因的转录调控、复制、重组,骨骼肌的分化、抑制肿瘤的发生等活动中起着重要的作用。 目的：探索Brg1基因在骨形态发生蛋白2诱导成骨细胞分化过程中的调控机制。 方法：采用胶原酶消化法进行小鼠颅骨成骨细胞的原代培养;分别用0,50,200 μg/L的重组人骨形态发生蛋白2诱导原代培养的成骨细胞的分化,摸索骨形态发生蛋白2的最佳作用剂量;实时荧光定量PCR和Western blot进行骨形态发生蛋白2对Brg1的作用时间的动力学分析;实时荧光定量PCR和钙钴染色法检测敲除Brg1对骨形态发生蛋白2诱导的成骨分化的影响;构建Dlx5腺病毒重组表达载体,实时荧光定量PCR和钙钴染色法检测Brg1在骨形态发生蛋白2诱导的成骨分化过程中对Dlx5的调控作用。 结果与结论：用自行合成的重组人骨形态发生蛋白2可诱导原代培养小鼠成骨细胞分化,200 μg/L剂量有着较好的诱导分化效果;重组人骨形态发生蛋白2可诱导Brg1基因转录水平和翻译水平表达水平上调;敲除Brg1可抑制重组人骨形态发生蛋白2诱导的成骨分化;Brg1能够调控Dlx5的表达水平。说明Brg1通过调控Dlx5的表达水平调控重组人骨形态发生蛋白2诱导的小鼠成骨细胞的分化。     

Osteogenic differentiation of human mesenchymal stem cells in collagen matrices: effect of uniaxial cyclic tensile strain on bone morphogenetic protein (BMP-2) mRNA expression

Human mesenchymal stem cells (hMSCs) differentiate down an osteogenic pathway with appropriate mechanical and/or chemical stimuli. This study describes the successful culture of hMSCs in 3D collagen matrices under mechanical strain. Bone marrow-derived hMSCs were seeded in linear 3D type I collagen matrices and subjected to 0%, 10%, or 12% uniaxial cyclic tensile strain at 1 Hz for 4 h/day for 7 or 14 days. Cell viability studies indicated that hMSCs remained viable throughout the culture period irrespective of the applied strain level. Real-time RT-PCR studies indicated a significant increase in BMP-2 mRNA expression levels in hMSCs strained at 10% compared to the same day unstrained controls after both 7 and 14 days. An increase in BMP-2 was also observed in hMSCs subjected to 12% strain, but the increase was significant only in the 14-day sample. This is the first report of the culture of bone marrow-derived hMSCs in 3D collagen matrices under cyclic strain, and the first demonstration that strain alone can induce osteogenic differentiation without the addition of osteogenic supplements. Induction of bone differentiation in 3D culture is a critical step in the creation of bioengineered bone constructs.     

Silica-based bioactive glasses modulate expression of bone morphogenetic protein-2 mRNA in Saos-2 osteoblasts in vitro

A chemical exchange of the silica gel layer forming on the surface of bioactive glasses is thought to be the principal reaction for bone-bioactive glass bonding. The contribution of biological molecules on cell-bioactive glass interaction is largely unknown. To further analyze the mechanisms involved in efficient bone bonding to bioactive glass, Saos-2 osteoblastic cells with proven osteogenic phenotype were cultured for 4, 7 and 14 days on two bioactive glasses with different Si contents. Culture plates and dishes made of bioactive (BAG, 53 % SiO2), biocompatible (BCG, 58% SiO2) and control (GO) glasses were extensively conditioned with phosphate buffer and DMEM medium before seeding the cells. Northern hybridization was used for analysis of mRNA levels of collagen type I (Col-I), alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP-2). A significant increase was observed in Col-I mRNA levels in cells grown on the two bioactive glasses when compared with those grown on controls at 4 and 7 days (p < 0.04). The mRNA level for ALP in the cultures of bioactive glasses-made plates and dishes was also increased over control at 7 days (p < 0.02) and remained this way between BAG and G0 at 14 days. Striking differences in BMP-2 mRNA levels existed between BAG and G0 plates and dishes at 7 days (p < 0.05). BMP-2 mRNA level in BAG group was higher than in BCG group at 4, 7 and 14 days, but without statistical significance. Saos-2 osteoblastic cells with strong ALP staining were mostly seen on BAG plates under a light microscope. In confocal microscopy, a bright FITC-stained F-actin ring was present in the cytoplasm of cells grown on BAG dish, demonstrating an active functional status. Stimulation of the expression of BMP-2 and other bone mRNAs by bioactive glasses in osteoblastic cells suggests biological involvement of bone related growth factors, peptides and cytokines in bone-bioactive glass bonding.