BACKGROUND

Interest in the potential application of adipose-derived stromal cells in cell-mediated tissue engineering of bone and other mesenchymal-derived tissues is growing. This study aimed to investigate the hypothesis that human adipose-derived stromal cells respond to and elaborate bone morphogenetic protein (BMP) 2, which could represent an important target of molecular manipulation to enhance the osteogenic potential of human adipose-derived stromal cells.

METHODS

Human adipose-derived stromal cells were differentiated for 10 days toward the osteogenic lineage in osteogenic differentiation media alone or supplemented with recombinant human BMP2 (rhBMP2). Alizarin red staining was quantified by spectrophotometry. Gene expression analyses were performed using quantitative real-time polymerase chain reaction. BMP2 levels in conditioned media were titered by enzyme-linked immunosorbent assay daily during osteogenic differentiation. Human adipose-derived stromal cells were cultured in complete or partially (50 percent) changed osteogenic differentiation media, or unchanged osteogenic differentiation media, to assay for pro-osteogenic secreted factors. In addition, human adipose-derived stromal cells were cultured in osteogenic differentiation media supplemented with BMP2/BMP4-neutralizing antibody.

RESULTS

Exogenous rhBMP2 significantly augmented the in vitro osteogenic potential of human adipose-derived stromal cells in a dose-dependent fashion, and significantly increased transcript levels of RUNX2 and osteocalcin. BMP2, BMP4, BMPR1B, and SMAD1/5 expression was significantly increased during differentiation. Enzyme-linked immunosorbent assay demonstrated significantly increased BMP2 elaboration during differentiation. Culture in conditioned osteogenic differentiation media led to significantly increased matrix mineralization. Mineralization was significantly decreased when osteogenic differentiation media was supplemented with a BMP2/BMP4-neutralizing antibody.

CONCLUSIONS

These data strongly support that BMP signaling is dynamic and important during normal in vitro osteogenic differentiation of human adipose-derived stromal cells. Thus, BMP2 may be used to enhance the osteogenic differentiation of human adipose-derived stromal cells for bone tissue engineering. Future studies will examine the effect of rhBMP2 on osteogenic differentiation of human adipose-derived stromal cells in vivo.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is characterized by heterotopic <em>bone</em> formation occurring in spinal ligament, causing severe compression myelopathy. In order to investigate the mechanism of OPLL development, we isolated spinal ligament cells from OPLL patients as well as non-OPLL patients, and established <em>10</em> OPLL cell lines and 7 non-OPLL cell lines, respectively. We analyzed the effects of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) on these cells with respect to alkaline phosphatase (AP) activity, DNA synthesis, and collagen production. BMP-2 caused a significant increase of AP activity in 4 OPLL cell lines, whereas the activity did not change in any non-OPLL cells. Among OPLL cells, BMP-2 stimulated DNA synthesis in four cell lines and procollagen type I carboxyl-terminal peptide (PICP) synthesis in five cell lines. Some non-OPLL cells also responded to BMP-2, as there was an increase of DNA synthesis in three cell lines and PICP synthesis in one cell line. These data collectively indicate that BMP-2 preferentially induces osteogenic differentiation in OPLL cells rather than in non-OPLL cells. OPLL cells, therefore, exhibit a different response to BMP-2 than non-OPLL cells, suggesting that the expression of BMP receptor(s) and/or the signal transduction initiated by BMP-2 in the spinal ligament cells of OPLL patients somewhat deviate from those in normal spinal ligament cells. Such abnormal characteristics of OPLL cells as described here provide some clues to the clarification of the pathogenesis of OPLL.

<em>Bone</em> marrow stromal cells (BMSCs) have been well established as an ideal source of cell-based therapy for <em>bone</em> tissue engineering applications. Boron (B) is a notable trace element in humans; so far, the effects of boron on the osteogenic differentiation of BMSCs have not been reported. The aim of this study was to evaluate the effects of boron (0, 1, <em>10</em>,<em>10</em>0, and 1,000 ng/ml) on osteogenic differentiation of human BMSCs. In this study, BMSCs proliferation was analyzed by cell counting kit-8 (CCK8) assay, and cell osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity assay, Von Kossa staining, and real-time PCR. The results indicated that the proliferation of BMSCs was no different from the control group when added with B at the concentration of 1, <em>10</em>, and <em>10</em>0 ng/ml respectively (P>> 0.05); in contrast, 1,000 ng/ml B inhibited the proliferation of BMSCs at days 4, 7, and 14 (P < 0.05). By ALP staining, we discovered that BMSCs treated with <em>10</em> and <em>10</em>0 ng/ml B presented a higher ALP activity compared with control (P < 0.05). By real-time PCR, we detected the messenger RNA expression of ALP, osteocalcin, collagen type I, and <em>bone</em> <em>morphogenetic</em> <em>proteins</em> 7 were also increased in <em>10</em> and <em>10</em>0 ng/ml B treatment groups (P < 0.05). The calcium depositions were increased in 1 and <em>10</em> ng/ml B treatment groups (P < 0.05). Taken all together, it was the first time to report that B could increase osteogenic effect by stimulating osteogenic differentiation-related marker gene synthesis during the proliferation and differentiation phase in human BMSCs and could be a promising approach for enhancing osteogenic capacity of cell-based construction in <em>bone</em> tissue engineering.

OBJECTIVE

To explore the mechanism of the protective effects of Panax notoginseng saponins (PNS) on kidney in diabetic rats.

METHODS

Diabetic rat model was obtained by intravenous injection of alloxan, and the rats were divided into model, PNS-<em>10</em>0 mg/(kg day) and PNS-200 mg/(kg day) groups, <em>10</em> each. Another <em>10</em> rats injected with saline were served as control. Periodic acid-Schiff staining and immunological histological chemistry were used to observe histomorphology and tissue expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7). Silent information regulator 1 (SIRT1) was silenced in rat mesangial cells by RNA interference. The mRNA expressions of SIRT-1, monocyte chemoattractant <em>protein</em>-1 (MCP-1), transforming growth factor β1 (TGF-β1) and plasminogen activator inhibitor-1 (PAI-1) were analyzed by reverse transcription polymerase chain reaction. The <em>protein</em> expressions of SIRT1 and the acetylation of nuclear factor κB (NF-κB) P65 were determined by western blotting. The concentration of MCP-1, TGF-β1 and malondialdehyde (MDA) in culture supernatant were detected by enzyme-linked immuno sorbent assay. The activity of superoxide dismutase (SOD) was detected by the classical method of nitrogen and blue four.

RESULTS

In diabetic model rats, PNS could not only reduce blood glucose and lipid (P<0.01), but also increase protein level of BMP-7 and inhibit PAI-1 expression for suppressing fibrosis of the kidney. In rat mesangial cells, PNS could up-regulate the expression of SIRT1 (P<0.01) and in turn suppress the transcription of TGF-β1 (P<0.05) and MCP-1 (P<0.05). PNS could also reverse the increased acetylation of NF-κB p65 by high glucose. In addition, redox regulation factor MDA was down-regulated (P<0.05) and SOD was up-regulated (P<0.01), which were both induced by SIRT1 up-regulation.

CONCLUSIONS

PNS could protect kidney from diabetes with the possible mechanism of up-regulating SIRT1, therefore inhibiting inflammation through decreasing the induction of inflammatory cytokines and TGF-β1, as well as activating antioxidant proteins.

Understanding the mechanisms that control the proliferation and commitment of human stem cells into cells of the osteogenic lineage for the preservation of skeletal structure is of basic importance in <em>bone</em> physiology. This study examines some aspects of the differentiation in vitro of human <em>bone</em> marrow fibroblastic cells cultured in the absence (basal media) or presence of 1nM dexamethasone and 50 micrograms/ml ascorbate for 6, <em>10</em>, 14, and 21 days. Northern blot analysis and in situ hybridisation with digoxygenin-labelled riboprobes for Type I collagen, osteocalcin, <em>bone</em> <em>morphogenetic</em> <em>proteins</em> 2 (BMP-2), and 4 (BMP-4) and the estrogen receptor alpha (ERalpha), together with immunocytochemical analysis of ERalpha expression and histochemical staining of alkaline phosphatase was performed. In basal media, alkaline phosphatase activity and collagen expressions were detected at day 6, ERalpha from day <em>10</em> and osteocalcin from day <em>10</em>. In the presence of dexamethasone and ascorbate, cell proliferation and alkaline phosphatase were markedly stimulated over <em>10</em> to 14 days with a dramatic increase in the temporal expression of Type I collagen, ERalpha, and osteocalcin mRNAs in these cultures. Northern blot analysis showed cells cultured in basal media, expressed the highest levels of the mRNA for each marker <em>protein</em> at day 14, whereas in the presence of ascorbate and dexamethasone, the highest levels for alkaline phosphatase, ERalpha, osteocalcin, BMP-2, and BMP-4 were observed at day 21. ERalpha, BMP-2, and BMP-4 expression were found to correlate temporally with induction of the osteoblast phenotype as determined by alkaline phosphatase, collagen, and osteocalcin expression. These results give additional information on the development of the osteoblast phenotype from early fibroblastic stem cells and on the biological factors involved in this process. These studies suggest a role for estrogen and BMP-2 and -4 in the differentiation of osteoprogenitor cells.

The signaling domain of Sonic hedgehog (Shh), a potent upstream regulator of cell fate that has been implicated in osteoblast differentiation from undifferentiated mesenchymal cells in its endogenous form, was investigated in an immobilized form as a means for accelerating differentiation of uncommitted cells to the osteoblast phenotype. A recombinant cysteine-modified N-terminal Shh (mShh) was synthesized, purified, and immobilized onto interpenetrating polymer network (IPN) surfaces also grafted with a <em>bone</em> sialo<em>protein</em>-derived peptide containing the Arg-Gly-Asp (RGD) sequence (bsp-RGD (15)), at calculated densities of 2.42 and <em>10</em> pmol/cm2, respectively. The mitogenic effect of mShh was dependent on the mode of presentation, as surfaces with immobilized mShh and bsp-RGD (15) had no effect on the growth rate of rat <em>bone</em> marrow-derived mesenchymal stem cells (BMSCs), while soluble mShh enhanced cell growth compared to similar surface without mShh supplementation. In conjunction with media supplemented with <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 and -4, mShh and bsp-RGD (15)-grafted IPN surfaces enhanced the alkaline phosphatase activity of BMSCs compared with tissue culture polystyrene and bsp-RGD (15)-grafted IPN surfaces supplemented with soluble mShh, indicating enhanced osteoblast differentiation. The adhesive peptide bsp-RGD (15) was necessary for cell attachment and proliferation, as well as differentiation in response to immobilized mShh. The addition of immobilized Shh substantially improved the differentiation of uncommitted BMSCs to the osteoblast lineage, and therefore warrants further testing in vivo to examine the effect of the stated biomimetic system on peri-implant <em>bone</em> formation and implant fixation.

METHODS

Efficacy of a bovine-derived osteoinductive growth factor was studied in a rabbit model and in a nonhuman primate model of posterolateral lumbar spinal fusion.

OBJECTIVE

To determine the minimum effective dose of growth factor and the influence of different carrier material on the outcome of intertransverse process lumbar fusion.

BACKGROUND

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> and related growth factors are becoming increasingly available in purified extract or genetically engineered forms and are capable of inducing new <em>bone</em> formation in vivo. Osteoinductive growth factors to enhance lumbar spinal infusion have not been well studied in models of posterolateral intertransverse process fusion. Because of the diminished potential of <em>bone</em> regeneration in primates (including humans) compared with phylogenetically lower animals, extrapolations regarding dose and efficacy cannot be made directly from results obtained in experiments performed on phylogenetically lower animals. Experiments on non-human primates are a critical step before attempting to use these growth factors on humans. METHODS. One hundred fifteen adult New Zealand white rabbits and <em>10</em> adult rhesus macaques underwent single level posterolateral intertransverse process lumbar spinal arthrodesis to evaluate different doses and carrier materials for a bovine-derived osteoinductive <em>bone</em> <em>protein</em> extract. Rabbit fusion masses were evaluated 5 weeks after arthrodesis by manual palpation, radiography, biomechanical testing, and light microscopy. Monkey fusion masses were evaluated 12 weeks after arthrodesis by radiography and light microscopy.

RESULTS

Successful posterolateral intertransverse process spinal fusions were achieved in the rabbit models using an osteoinductive growth factor with three different carriers (autogenous iliac bone, demineralized allogeneic bone matrix, and natural coral). There was a dose-dependent response to the osteoinductive growth factor in the rabbit model, indicating that a threshold must be overcome before bone formation is induced. The methodology for biologic enhancement of spinal fusion developed in the rabbit model transferred successfully to the rhesus monkey, where the use of the osteoinductive growth factor with a demineralized bone matrix carrier resulted in spinal fusion in 12 weeks.

CONCLUSIONS

These experiments provide an essential building block in the understanding of the biology of spinal fusion and the use of osteoinductive growth factors to enhance a posterolateral intertransverse process spinal fusion. The achievement of posterolateral spinal fusion in the rhesus monkey using an osteoinductive growth factor is a significant step toward the biologic enhancement of spinal fusion in humans.

The use of poly(ethylene glycol) (PEG) hydrogels in tissue engineering is limited by their persistence in the site of regeneration. In an attempt to produce inert hydrolytically degradable PEG-based hydrogels, star (SPELA) poly(ethylene glycol-co-lactide) acrylate macromonomers with short lactide segments (<15 lactides per macromonomer) were synthesized. The SPELA hydrogel was characterized with respect to gelation time, modulus, water content, sol fraction, degradation, and osteogenic differentiation of encapsulated marrow stromal cells (MSCs). The properties of SPELA hydrogel were compared with those of the linear poly(ethylene glycol-co-lactide) acrylate (LPELA). The SPELA hydrogel had higher modulus, lower water content, and lower sol fraction than the LPELA. The shear modulus of SPELA hydrogel was 2.2 times higher than LPELA, whereas the sol fraction of SPELA hydrogel was 5 times lower than LPELA. The degradation of SPELA hydrogel depended strongly on the number of lactide monomers per macromonomer (nL) and showed a biphasic behavior. For example, as nL increased from 0 to 3.4, 6.4, 11.6, and 14.8, mass loss increased from 7 to 37, 80, <em>10</em>0% and then deceased to 87%, respectively, after 6 weeks of incubation. The addition of 3.4 lactides per macromonomer ((<em>10</em> wt % dry macromonomer or <2 wt % swollen hydrogel) increased mass loss to 50% after 6 weeks. Molecular dynamic simulations demonstrated that the biphasic degradation behavior was related to aggregation and micelle formation of lactide monomers in the macromonomer in aqueous solution. MSCs encapsulated in SPELA hydrogel expressed osteogenic markers Dlx5, Runx2, osteopontin, and osteocalcin and formed a mineralized matrix. The expression of osteogenic markers and extent of mineralization was significantly higher when MSCs were encapsulated in SPELA hydrogel with the addition of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP2). Results demonstrate that hydrolytically degradable PEG-based hydrogels are potentially useful as a delivery matrix for stem cells in regenerative medicine.

BACKGROUND

Although enamel matrix derivative (EMD) has demonstrated the ability to promote angiogenesis and osteogenesis both in vitro and in vivo, the specific elements within the EMD compound responsible for these effects remain unknown.

METHODS

Nine different protein pools from a commercially produced EMD were collected based on molecular weight. Six of these pools, along with the complete EMD unfractionated compound and positive and negative controls, were tested for their ability to induce bone formation in a calvarial induction assay. Immunocytochemistry of phosphorylated SMAD1/5/8 (phospho-SMAD), osterix, and vascular endothelial growth factor A (VEGF-A) was carried out at selected time points. Finally, proteomic analysis was completed to determine the specific protein-peptide content of the various osteoinductive pools.

RESULTS

One of the lower-molecular-weight pools tested, pool 7, showed bone induction responses significantly greater than those of the other pools and the complete EMD compound and was concentration dependent. Dynamic bone formation rate analysis demonstrated that pool 7 was optimally active at the 5- to 10-μg concentration. It was demonstrated that EMD and pool 7 induced phospho-SMAD, osterix, and VEGF-A, which is indicative of increased bone morphogenetic protein (BMP) signaling. Proteomic composition analysis demonstrated that pool 7 had the highest concentration of the biologically active amelogenin-leucine-rich amelogenin peptide and ameloblastin 17-kDa peptides.

CONCLUSIONS

These studies demonstrate that the low-molecular-weight protein pools (7 to 17 kDa) within EMD have greater osteoinductive potential than the commercially available complete EMD compound and that the mechanism of action, in part, is through increased BMP signaling and increased osterix and VEGF-A. With this information, selected components of EMD can now be formulated for optimal osteo- and angio-genesis.

To investigate the effects of Genistein on the osteogenic related gene expression profiles during osteoblastic differentiation of human <em>bone</em> marrow mesenchymal stem cell (hBMSC) cultures, the hBMSCs were cultured under osteogenic differentiation medium with the addition of Genistein (<em>10</em>(-8)∼<em>10</em>(-5) M) for 12 days. The cell proliferation was measured by BrdU incorporation, while the osteoblastic differentiation in hBMSC cultures was assessed by cellular alkaline phosphatase (ALP) activity. The cell apoptosis was determined by caspase 3/7 activation. GEArray Q series human osteogenesis gene array was used to analyze large-scale gene expression in Genistein-treated hBMSC cultures compared to the control group. Quantitative real-time RT-PCR, small interfering RNA (siRNA), and western blot analysis were used to confirm the microarray data in five representative transcripts. Genistein (<em>10</em>(-8)∼<em>10</em>(-6) M) dose- and time-dependently increased cell proliferation and cellular ALP activity, but had no significant effect on cell apoptosis in hBMSC cultures. The 96-gene array analysis indicated that 22 genes were upregulated more than 2-fold and 7 genes were downregulated at least 1.5-fold. The expressions of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs), small mothers against decapentaplegic homologs (SMADs), and Runt-related transcription factor 2 (RUNX2) were concomitantly increased under Genistein treatment while insulin-like growth factor 2 and inhibitory SMADs 6 and 7 expressions were significantly decreased. The results of the real-time RT-PCR had a correlation with the results of microarray analysis and were estrogen-receptor dependent. Specific gene siRNAs knock-down further confirmed the osteogenic effects of Genistein on BMP2, SMAD5 and RUNX2 <em>protein</em> expression. Genistein enhanced osteogenic differentiation in cultured hBMSCs mainly through the BMP-dependent SMADs and RUNX2 signaling.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2), a member of the transforming growth factor superfamily, has been shown to have inhibitory effect on many tumor types. However, the effect of BMP-2 on human renal cell carcinoma (RCC) is still unknown. We previously showed that BMP-2 inhibits tumorigenicity of cancer stem cells in human osteosarcoma OS99-1 cells. Our study investigates the effect of BMP-2 on human RCC using ACHN and Caki-2 cell lines. Three types of BMP receptors were found to be expressed in ACHN and Caki-2 cells. In vitro, BMP-2 was found to inhibit the growth of ACHN and Caki-2 cells. The antiproliferative effect seems to be due to cell cycle arrest in the G1 phase, which was revealed by flow cytometry analysis. Using reverse transcriptase polymerase chain reaction analysis, we demonstrated BMP-2 upregulated osteogenic markers Runx-2 and Collagen Type I gene expression in ACHN and Caki-2 cells. Treatment of ACHN and Caki-2 cells with BMP-2 induced a rapid phosphorylation of Smad1/5/8. In vivo, all animals receiving low number of ACHN (1 × <em>10</em>(4)) and Caki-2 (5 × <em>10</em>(4)) cells treated with 30 μg of BMP-2 per animal showed limited tumor growth with significant <em>bone</em> formation, whereas untreated cells developed large tumor masses without <em>bone</em> formation in immunodeficient non-obese diabetic (NOD)/severe combined immunodeficient (SCID) mice. These results suggest that BMP-2 inhibits growth of RCC as well as causes induction of osseous <em>bone</em> formation. Further research is needed to determine the relationship between inhibition of cell proliferation and <em>bone</em> induction.

METHODS

An in vitro biologic study of the effects of adenovirus expressing bone morphogenetic proteins (BMPs) and adenovirus expressing Sox9 on extracellular matrix metabolism by bovine nucleus pulposus cells.

OBJECTIVE

To compare the effects of recombinant adenoviral vectors expressing various BMPs (2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, and 15) and Sox9 on extracellular matrix accumulation by bovine nucleus pulposus cells.

BACKGROUND

Nucleus pulposus matrix production may be promoted by transducing the cells with genes that permit the sustained expression of growth factors. The choice of the particular factors or BMPs to be studied for these applications has been largely based on the commercial availability of such products. To our knowledge, this study is the first effort to evaluate systematically the relative effectiveness of the various members of the BMP family in promoting intervertebral disc matrix repair.

METHODS

Adult bovine nucleus pulposus cells cultured in monolayer were transduced with adenoviruses expressing human BMP-2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, and 15, and adenovirus expressing Sox9. Proteoglycan and collagen accumulation, and cell proliferation were measured 6 days after viral transduction. As a positive control, cells were cultured without any exogenous gene in the presence of recombinant human (rh)BMP-7.

RESULTS

Nucleus pulposus cells transduced with adenoviruses expressing BMP-2, 3, 4, 5, 7, 8, 10, 13, 15, and Sox9 accumulated more proteoglycans than nucleus pulposus cells transduced with adenovirus expressing green fluorescent protein (control). It is noteworthy that nucleus pulposus cells transduced with adenoviruses expressing BMP-2 and 7 resulted in essentially as great a stimulation of proteoglycan accumulation as nucleus pulposus cells maintained in the presence of rhBMP-7 (adenoviruses expressing BMP-2: 104% increase; adenoviruses expressing BMP-7: 162% increase; and rhBMP-7: 120% increase). Nucleus pulposus cells transduced with BMP-2, 4, 5, 7, 8, 10, 14, 15, and Sox9 accumulated significantly more collagen compared to nucleus pulposus cells transduced with adenovirus expressing green fluorescent protein; adenoviruses expressing BMP-4 and 14 were the most effective (552% and 661% increase, respectively). Nucleus pulposus cells also proliferated, as measured by deoxyribonucleic acid content, when transduced with adenoviruses expressing BMP-2 and 8.

CONCLUSIONS

To our knowledge, for the first time, we have shown the relative effectiveness of 12 different BMPs and Sox9 in stimulating proteoglycan and collagen production by nucleus pulposus cells. Adenoviruses expressing BMP-2 and 7 were the most effective in stimulating proteoglycan accumulation, while adenoviruses expressing BMP-4 and 14 were the most effective in stimulating collagen accumulation. To our knowledge, this study is the first to compare the relative effectiveness of various BMPs and Sox9 on extracellular matrix accumulation by nucleus pulposus cells, and could help to develop more efficacious approaches to the treatment of degenerating intervertebral discs.

Hesperidin found in citrus fruits has been reported to be a promising bioactive compound for maintaining an optimal <em>bone</em> status in ovariectomized rodent models. In this study, we examined the capacity of hesperetin (Hp) to affect the proliferation, differentiation and mineralization of rodent primary osteoblasts. Then, the impact of Hp on signalling pathways known to be implicated in <em>bone</em> formation was explored. We exposed osteoblasts to physiological concentrations of 1 microM Hp (Hp1) and <em>10</em> microM Hp (Hp<em>10</em>). Neither proliferation nor mineralization was affected by Hp at either dose during 19 days of exposure. Hp at both doses enhanced differentiation by significantly increasing alkaline phosphatase (ALP) activity from Day 14 of exposure (Day 19: Hp1: +9%, Hp<em>10</em>: +14.8% vs. control; P<.05). However, Hp did not induce an obvious formation of calcium nodules. The effect of Hp<em>10</em> on ALP was inhibited by addition of noggin <em>protein</em>, suggesting a possible action of this flavanone through the <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) pathway. Indeed, Hp<em>10</em> significantly induced (1.2- to 1.4-fold) mRNA expression of genes involved in this signalling pathway (i.e., BMP2, BMP4, Runx2 and Osterix) after 48 h of exposure. This was strengthened by enhanced phosphorylation of the complex Smad1/5/8. Osteocalcin mRNA level was up-regulated by Hp only at <em>10</em> microM (2.2 fold vs. control). The same dose of Hp significantly decreased osteopontin (OPN) <em>protein</em> level (50% vs. control) after 14 days of culture. Our findings suggest that Hp may regulate osteoblast differentiation through BMP signalling and may influence the mineralization process by modulating OPN expression.

The potential of adult human adipose tissue stem cells (hASCs) to differentiate into hepatocytes has generated much excitement over the possible use of hASCs in therapeutic applications. An understanding of the molecular mechanisms that underlie the plasticity of hASCs toward hepatocytes will help to make this possibility a reality. Herein, we show that a homogenous population of hASCs characterized by a high level of CD73, CD90, and CD<em>10</em>5 express the pluripotent transcription factors OCT4, SOX2, NANOG, and SALL4 under proliferation conditions. A high level of activin A allows for hASCs acquiring the fate of definitive endoderm (DE) cells and expressing the specific transcription factors HEX, FOXA2, SOX17, and GATA4 synchronously. Using a reproducible three-stage method by mimicking liver embryogenesis, hASCs were directed to differentiate into functional hepatocytes. In the first stage, hASCs were induced to become DE cells by 2 days cultured in serum-free medium and 3 days of activin A treatment. Next, the presence of fibroblast growth factor (FGF) 4 and <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) 2 in the medium for 5 days induced efficient hepatic differentiation from DE cells. After <em>10</em> days of further maturated by the sequential exposure to hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone (DEX), the hASC-derived hepatocytes expressed mature hepatocytes marker and exhibited functional characterization, including albumin secretion, glycogen storage, urea production, activity of drug transporters, and cytochrome P450 activity. These findings will be useful for the implementation of hASC-derived hepatocytes in therapeutic purposes, metabolic analyses, drug toxicity screening, and studies of hepatocyte function.

LIM mineralization <em>protein</em>-1 (LMP-1) is a novel intracellular osteogenic factor associated with <em>bone</em> development that has been implicated in the <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) pathway. This preliminary study evaluated the possibility of LMP-1-based retroviral gene therapy to stimulate osteoblast differentiation in vitro and fracture repair in vivo. A Moloney leukemia virus (MLV)-based retroviral vector to express LMP-1 with a hemagglutinin (HA) tag was developed, and its effects were evaluated on MC3T3-E1 cell differentiation and in the rat femur fracture model. MC3T3-E1 osteoblasts transduced with the MLV-HA-LMP-1 vector demonstrated significantly increased osteoblast marker gene expression (P < 0.05) and mineral deposition compared to control transduced cells. Femoral midshaft fractures were produced in Fischer 344 rats by the three-point bending technique. The MLV-HA-LMP-1 or control vector was applied at the fracture site through percutaneous injections 1 day postfracture. Analysis of fracture healing of <em>10</em> MLV-HA-LMP-1-treated and <em>10</em> control MLV-beta-galactosidase (beta-gal)-treated animals was completed at 3 weeks by X-ray, peripheral quantitative computed tomography, and histology. MLV-HA-LMP-1-treated animals had 63% more <em>bone</em> mineral content at the fracture site (P < 0.01), 34% greater total hard callus area (P < 0.05), and 45% less cartilage in the fracture callus (P < 0.05) compared to MLV-beta-gal-treated animals. There was no effect of LMP-1 treatment on the density of the hard callus. Immunohistochemistry revealed expression of the LMP-1 transgene in the fracture callus at 21 days postfracture. Immunohistochemistry also revealed that LMP-1 transgene expression did not result in an increase in BMP-4 expression in the fracture callus. Compared to MLV-BMP-4 gene therapy studies, MLV-HA-LMP-1 gene therapy improved bony union of the fracture gap to a greater extent and did not cause heterotopic <em>bone</em> formation. This suggests that LMP-1 may be a better potential candidate for gene therapy for fracture repair than BMP-4. These exciting, albeit preliminary, findings indicate that LMP-1-based gene therapy may potentially be a simple and effective means to enhance fracture repair that warrants further investigation.

OBJECTIVE

The purpose of this study was to investigate bone morphogenetic protein (BMP) 2 expression after implantation of a statin and recombinant human BMP-2 (rhBMP-2) and to compare the bone regeneration capability of these substances in the rabbit nasal bone using immunohistologic methods.

METHODS

Twelve adult male Japanese white rabbits (n = 12; age 12-16 weeks, weight 2.5-3.0 kg) were divided into 3 experimental groups and 1 control group. A total of 48 bone defects, 4 per rabbit, were created in the nasal bone while preserving the nasal membrane. In the experimental groups, 1 group was implanted with 10 mg of a statin dissolved in 0.2 mL water with an atelocollagen sponge (ACS); the second group was implanted with 5 microg rhBMP-2 with an ACS; and in the third group only the ACS was implanted. No material was implanted in the control group. Animals were killed at 1, 2, and 4 weeks after surgery. The parts that had been operated on were removed and prepared for histologic assessment. The expression of BMP-2 was evaluated using immunohistochemistry, and double-immunostaining for BMP-2 and Ki-67 was observed by fluorescent microscopy.

RESULTS

No significant differences were observed between the statin/ACS group and rhBMP-2/ACS group at 1, 2, and 4 weeks after surgery. The number of cells which stained positively for BMP-2 increased significantly in both of the implanted groups compared with the control group (P < .0001). The positive fluorescent double-immunostaining for BMP-2 and Ki-67 was similar in both implanted groups.

CONCLUSIONS

This study suggests that statin/ACS implants show BMP-2 expression and osteoinductive activity that is similar to those of rhBMP-2/ACS implants.

Surgical treatment of critical-size posttraumatic <em>bone</em> defects is still a challenging problem, even in modern <em>bone</em> and joint surgery. Progress in cellular and molecular biology during the last decade now permits novel approaches in <em>bone</em> engineering. Recent conceptual and technical advances have enabled the use of mitotically expanded, <em>bone</em>-derived cells as a therapeutic approach for tissue repair. Using three different tissue carrier systems, we successfully cultivated human osteoblasts in a newly developed perfusion chamber. We studied cell proliferation and the expression of osteocalcin, osteopontin, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2A, alkaline phosphatase, and vascular endothelial growth factor as parameters for osteoblast function and viability. Adherence of highly enriched human osteoblasts had already started after 1 h and resulted in completely overgrown human <em>bone</em> pieces after <em>10</em> days. Expression analysis of <em>bone</em>-specific alkaline phosphatase indicated differentiating osteoblasts, whereas the high mRNA expression of osteocalcin and osteopontin revealed terminally differentiated osteoblasts and the process of mineralization. Additionally, gene expression was significantly higher when demineralized <em>bone</em> was used as biomatrix, compared to autoclaved <em>bone</em> and hydroxyapatite ceramics. We conclude that with our newly developed perfusion culture system, vital autogenous <em>bone</em> implants of clinically applicable size can be generated within 17 days in order to manage critical-size <em>bone</em> defects.

OBJECTIVE

Periodontal ligament (PDL) cell differentiation into osteoblasts is important in bone formation. Bone formation is a complex biological process and involves several tightly regulated gene expression patterns of bone-related proteins. The expression patterns of bone related proteins are regulated in a temporal manner both in vivo and in vitro. The aim of this study was to observe the gene expression profile in PDL cell proliferation, differentiation, and mineralization in vitro.

METHODS

PDL cells were grown until confluence, which were then designated as day 0, and nodule formation was induced by the addition of 50 µg/mL ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone to the medium. The dishes were stained with Alizarin Red S on days 1, 7, 14, and 21. Real-time polymerase chain reaction was performed for the detection of various genes on days 0, 1, 7, 14, and 21.

RESULTS

On day 0 with a confluent monolayer, in the active proliferative stage, c-myc gene expression was observed at its maximal level. On day 7 with a multilayer, alkaline phosphatase, bone morphogenetic protein (BMP)-2, and BMP-4 gene expression had increased and this was followed by maximal expression of osteocalcin on day 14 with the initiation of nodule mineralization. In relationship to apoptosis, c-fos gene expression peaked on day 21 and was characterized by the post-mineralization stage. Here, various genes were regulated in a temporal manner during PDL fibroblast proliferation, extracellular matrix maturation, and mineralization. The gene expression pattern was similar.

CONCLUSIONS

We can speculate that the gene expression pattern occurs during PDL cell proliferation, differentiation, and mineralization. On the basis of these results, it might be possible to understand the various factors that influence PDL cell proliferation, extracellular matrix maturation, and mineralization with regard to gene expression patterns.

BACKGROUND

Human embryonic stem cells (hESCs) are a potentially inexhaustible source of cells for replacement therapy. However, successful preclinical and clinical progress requires efficient and controlled differentiation towards the specific differentiated cell fate.

METHODS

We previously developed a strategy to generate blast cells (BCs) from hESCs that were capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Although the BCs were shown to repair damaged vasculature in multiple animal models, the large-scale generation of cells under these conditions was challenging. Here we report a simpler and more efficient method for robust generation of hemangioblastic progenitors.

RESULTS

In addition to eliminating several expensive factors that are unnecessary, we demonstrate that <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-4 and VEGF are necessary and sufficient to induce hemangioblastic commitment and development from hESCs during early stages of differentiation. BMP-4 and VEGF significantly upregulate T-brachyury, KDR, CD31 and Lmo2 gene expression, while dramatically downregulating Oct-4 expression. The addition of basic FGF during growth and expansion was found to further enhance BC development, consistently generating approximately 1 x <em>10</em>(8) BCs from one six well plate of hESCs.

CONCLUSIONS

This new method represents a significantly improved system for generating hemangioblasts from hESCs, and although simplified, results in an eightfold increase in cell yield.

The immobilization of biomolecules on biomaterial surfaces allows for the control of their localization and retention. In numerous studies, <em>proteins</em> have been simply adsorbed to enhance the biological performance of various materials in vivo. We investigated the potential of surface modification techniques on hydroxyapatite (HA) ceramic discs in an in vitro approach. A novel method for <em>protein</em> immobilization was evaluated using the aminobisphosphonates pamidronate and alendronate, which are strong Ca chelating agents, and was compared with the established silanization technique. Lysozyme and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) were used to assess the suitability of the two surface modification methods with regard to the enzymatic activity of lysozyme and to the capacity of BMP-2 to stimulate the osteoblastic differentiation of C2C12 mouse myoblasts. After immobilization, a 2.5-fold increase in enzymatic activity of lysozyme was observed compared with the control. The alkaline phosphatase activity per cell stimulated by immobilized BMP-2 was 2.5-fold higher [9 x <em>10</em>(-6) I.U.] than the growth factor on unmodified surfaces [2-4 x <em>10</em>(-6) I.U.]. With regard to the increase in <em>protein</em> activity, both procedures lead to equivalent results. Thus, the bisphosphonate-based surface modification represents a safe and easy alternative for the attachment of <em>proteins</em> to HA surfaces.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs), potential regulators of cellular growth and metastasis that signal through an interaction with plasma membrane receptors, have been suggested to be important regulators of malignant cells. The present study was carried out to evaluate the potential role of BMP receptor (BMP-R) types IA, IB, and II in bladder transitional cell carcinoma (TCC) cells. Initially, we investigated the expression of these BMP-Rs in 30 archival tissues of human bladder TCC using immunohistochemistry; <em>10</em> benign bladder specimens were used for comparison. The results demonstrated that the expression of BMP-Rs is localized preferentially to the transitional epithelium and that there was a significant association between loss of BMP-RII expression and tumor grade. To find a cell line that can serve as a model system for clinical observation, we subsequently examined sensitivity to BMP-4 and expression of BMP-RII, BMP-RIA, and BMP-RIB in three human bladder cancer cell lines, TCC-Sup, RT4, and TSU-Pr1. Of the three cell lines, TSU-Pr1 exhibited a decreased level of BMP-RII expression and was resistant to the growth-inhibitory effect of BMP-4. Overexpression of BMP-RII in TSU-Pr1 cells not only restored BMP-4 responsiveness but also significantly decreased tumorigenicity in vivo. Taken together, these results demonstrate that human bladder TCC tissues have a frequent loss of BMP-RII expression and that overexpression of BMP-RII leads to restoration of BMP signaling and decreased tumor growth in the human bladder TCC cell line TSU-Pr1.

OBJECTIVE

To determine the role of Notch1 and Hes1 in regulating the activation of hepatic stellate cells (HSCs) and whether Hes1 is regulated by transforming growth factor (TGF)/bone morphogenetic protein (BMP) signaling.

METHODS

Immunofluorescence staining was used to detect the expression of desmin, glial fibrillary acidic protein and the myofibroblastic marker α-smooth muscle actin (α-SMA) after freshly isolated, normal rat HSCs had been activated in culture for different numbers of days (0, 1, 3, 7 and 10 d). The expression of α-SMA, collagen1α2 (COL1α2), Notch receptors (Notch1-4), and the Notch target genes Hes1 and Hey1 were analyzed by reverse transcriptase-polymerase chain reaction. Luciferase reporter assays and Western blot were used to study the regulation of α-SMA, COL1α1, COL1α2 and Hes1 by NICD1, Hes1, CA-ALK3, and CA-ALK5 in HSC-T6 cells. Moreover, the effects of inhibiting Hes1 function in HSC-T6 cells using a Hes1 decoy were also investigated.

RESULTS

The expression of Notch1 and Hes1 mRNAs was significantly down-regulated during the culture of freshly isolated HSCs. In HSC-T6 cells, Notch1 inhibited the promoter activities of α-SMA, COL1α1 and COL1α2. On the other hand, Hes1 enhanced the promoter activities of α-SMA and COL1α2, and this effect could be blocked by inhibiting Hes1 function with a Hes1 decoy. Furthermore, co-transfection of pcDNA3-CA-ALK3 (BMP signaling activin receptor-like kinase 3) and pcDNA3.1-NICD1 further increased the expression of Hes1 compared with transfection of either vector alone in HSC-T6 cells, while pcDNA3-CA-ALK5 (TGF-β signaling activin receptor-like kinase 5) reduced the effect of NICD1 on Hes1 expression.

CONCLUSIONS

Selective interruption of Hes1 or maintenance of Hes1 at a reasonable level decreases the promoter activities of α-SMA and COL1α2, and these conditions may provide an anti-fibrotic strategy against hepatic fibrosis.

Osteogenic differentiation of vascular smooth muscle cells (VSMCs) results in medial artery calcification, which is common in diabetes, but the pathogenesis is poorly understood. We aimed to explore the pathophysiological roles of insulin resistance (IR) on medial artery calcification in rats with <em>10</em>% fructose in drinking water. After 12 weeks of fructose feeding, rats showed severe IR, with increased levels of fasting blood glucose, serum insulin and oral glucose tolerance test (OGTT). Fructose-fed rats showed aortic calcification, increased aortic calcium deposition and irregular elastic fibers in the medial layer of the vessel wall. Moreover, plasma phosphorus concentration, calcium × phosphorus product and alkaline phosphatase (ALP) activity, and aortic calcium content and ALP activity were significantly increased. Fructose feeding increased mRNA levels of osteopontin, type III sodium-dependent phosphate co-transporter, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 and the key transcription factor core binding factor alpha 1 in aortic tissue and downregulated mRNA levels of osteoprotegerin and matrix γ-carboxyglutamic acid <em>protein</em>. Fructose feeding decreased <em>protein</em> levels of smooth-muscle lineage markers and induced severe lipid peroxidation injury. IR induced by high fructose feeding could evoke osteogenic transdifferentiation of VSMCs and promote vascular calcification.

Background -Hereditary Hemorrhagic Telangiectasia (HHT) is an inherited vascular disorder that causes arterial-venous malformations (AVMs). Mutations in the genes encoding Endoglin (ENG) and Activin-receptor-like kinase 1 (AVCRL1 encoding ALK1) cause HHT type 1 and 2, respectively. Mutations in the SMAD4 gene are present in families with Juvenile Polyposis/HHT syndrome that involves AVMs. SMAD4 is a downstream effector of Transforming growth factor-β (TGFβ)/<em>Bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) family ligands that signal via Activin like kinase receptors (ALKs). Ligand-neutralizing antibodies or inducible, endothelial-specific Alk1 deletion induce AVMs in mouse models as a result of increased PI3K/AKT signaling. Here we addressed if SMAD4 was required for BMP9-ALK1 effects on PI3K/AKT pathway activation. Methods -We generated a tamoxifen-inducible, postnatal endothelial-specific Smad4 mutant mice (Smad4iΔEC). Results -We found that loss of endothelial Smad4 resulted in AVM formation and lethality. AVMs formed in regions with high blood flow in developing retinas and other tissues. Mechanistically, BMP9 signaling antagonized flow-induced AKT activation in an ALK1 and SMAD4 dependent manner. Smad4iΔEC endothelial cells in AVMs displayed increased PI3K/AKT signaling, and pharmacological PI3K inhibitors or endothelial Akt1 deletion both rescued AVM formation in Smad4iΔEC mice. BMP9-induced SMAD4 inhibited Casein Kinase 2 (CK2) transcription, in turn limiting PTEN phosphorylation and AKT activation. Consequently, CK2 inhibition prevented AVM formation in Smad4iΔEC mice. Conclusions -Our study reveals SMAD4 as an essential effector of BMP9-<em>10</em>/ALK1 signaling that affects AVM pathogenesis via regulation of CK2 expression and PI3K/AKT1 activation.