| Surface-directed assembly of cell-laden microgels.
September 25, 2009 at 10:31 am
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| | Surface-directed assembly of cell-laden microgels. Biotechnol Bioeng. 2009 Sep 23; Authors: Du Y, Ghodousi M, Lo E, Vidula MK, Emiroglu O, Khademhosseini A Cell-laden microscale hydrogels (microgels) can be used as tissue building blocks and assembled to create 3D tissue constructs with well-defined micro-architecture. In this paper, we present a bottom-up approach to achieve microgel assembly on a patterned surface. Driven by surface tension, the hydrophilic microgels can be assembled into well-defined shapes on a glass surface patterned with hydrophobic and hydrophilic regions. We found that the cuboidic microgels ( approximately 100-200 mum in width) could self-assemble into defined shapes with high fidelity to the surface patterns. The microgel assembly process was improved by increasing the hydrophilicity of the microgels and reducing the surface tension of the surrounding solution. The assembled microgels were stabilized by a secondary crosslinking step. Assembled microgels containing cells stained with different dyes were fabricated to demonstrate the application of this approach for engineering microscale tissue constructs containing multiple cell types. This bottom-up approach enables rapid fabrication of cell-laden microgel assemblies with pre-defined geometrical and biological features, which is easily scalable and can be potentially used in microscale tissue engineering applications. (c) 2009 Wiley Periodicals, Inc. PMID: 19777588 [PubMed - as supplied by publisher] |
| Serum-free, chemically defined medium with TGF- beta(3) enhances functional properties of nucleus pulposus cell-laden carboxymethylcellulose hydrogel constructs.
September 25, 2009 at 10:31 am
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| | Serum-free, chemically defined medium with TGF- beta(3) enhances functional properties of nucleus pulposus cell-laden carboxymethylcellulose hydrogel constructs. Biotechnol Bioeng. 2009 Sep 23; Authors: Reza AT, Nicoll SB Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies may provide a viable NP replacement therapy; however, culture conditions must be optimized to promote functional tissue development. In this study, a standard serum-containing medium formulation was compared to a chemically defined, serum-free medium to determine the effect on matrix elaboration and functional properties of NP cell-laden carboxymethylcellulose (CMC) hydrogels. Additionally, both media were further supplemented with transforming growth factor-beta 3 (TGF-beta(3)). Glycosaminoglycan (GAG) content increased in both TGF-beta(3)-treated groups and was highest for treated, serum-free constructs (9.46 +/- 1.51 microg GAG/mg wet weight), while there were no quantifiable GAGs in untreated serum-containing samples. Histology revealed uniform, interterritorial staining for chondroitin sulfate proteoglycan throughout the treated, serum-free constructs. Type II collagen content was greater in both serum-free groups and highest in treated, serum-free constructs. The equilibrium Young's modulus was highest in serum-free samples supplemented with TGF- beta(3) (18.54 +/- 1.92 kPa), and the equilibrium weight swelling ratio of these constructs approached that of the native NP tissue (22.19 +/- 0.46 vs. 19.94 +/- 3.09, respectively). Taken together, these results demonstrate enhanced functional matrix development by NP cells when cultured in CMC hydrogels maintained in serum-free, TGF-beta(3) supplemented medium, indicating the importance of medium formulation in NP construct development. (c) 2009 Wiley Periodicals, Inc. PMID: 19777586 [PubMed - as supplied by publisher] |
| Functionalization of chitosan/poly(lactic acid-glycolic acid) sintered microsphere scaffolds via surface heparinization for bone tissue engineering.
September 25, 2009 at 10:31 am
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| | Functionalization of chitosan/poly(lactic acid-glycolic acid) sintered microsphere scaffolds via surface heparinization for bone tissue engineering. J Biomed Mater Res A. 2009 Sep 23; Authors: Jiang T, Khan Y, Nair LS, Abdel-Fattah WI, Laurencin CT Scaffolds exhibiting biological recognition and specificity play an important role in tissue engineering and regenerative medicine. The bioactivity of scaffolds in turn influences, directs, or manipulates cellular responses. In this study, chitosan/poly(lactic acid-co-glycolic acid) (chitosan/PLAGA) sintered microsphere scaffolds were functionalized via heparin immobilization. Heparin was successfully immobilized on chitosan/PLAGA scaffolds with controllable loading efficiency. Mechanical testing showed that heparinization of chitosan/PLAGA scaffolds did not significantly alter the mechanical properties and porous structures. In addition, the heparinized chitosan/PLAGA scaffolds possessed a compressive modulus of 403.98 +/- 19.53 MPa and a compressive strength of 9.83 +/- 0.94 MPa, which are in the range of human trabecular bone. Furthermore, the heparinized chitosan/PLAGA scaffolds had an interconnected porous structure with a total pore volume of 30.93 +/- 0.90% and a median pore size of 172.33 +/- 5.89 mum. The effect of immobilized heparin on osteoblast-like MC3T3-E1 cell growth was investigated. MC3T3-E1 cells proliferated three dimensionally throughout the porous structure of the scaffolds. Heparinized chitosan/PLAGA scaffolds with low heparin loading (1.7 mug/scaffold) were shown to be capable of stimulating MC3T3-E1 cell proliferation by MTS assay and cell differentiation as evidenced by elevated osteocalcin expression when compared with nonheparinized chitosan/PLAGA scaffold and chitosan/PLAGA scaffold with high heparin loading (14.1 mug/scaffold). This study demonstrated the potential of functionalizing chitosan/PLAGA scaffolds via heparinization with improved cell functions for bone tissue engineering applications. (c) 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2009. PMID: 19777575 [PubMed - as supplied by publisher] |
| Successful repair of a critical-sized bone defect in the rat femur with a newly developed external fixator.
September 25, 2009 at 10:31 am
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| | Successful repair of a critical-sized bone defect in the rat femur with a newly developed external fixator. Tohoku J Exp Med. 2009 Oct;219(2):115-20 Authors: Zhao Z, Yang D, Ma X, Zhao H, Nie C, Si Z Regeneration of segmental bone defects has been a clinical challenge. Recent advances in the field of tissue engineering have developed new procedures enabling bone regeneration. Small animal models capable of supporting weight-bearing femoral defects are integral parts of orthopedic biomedical research. However, a drawback of bone healing research is the lack of stable and adaptable fixation devices for small animals. Therefore, we developed and evaluated an adjustable external fixation device in the maintenance of non-healable (ie critical-sized) segmental defects, and in the fixation of tissue-engineered bone grafts in a rat model. Male Sprague-Dawley rats (n = 24) underwent a femoral osteotomy to create a non-healing segmental defect (6 mm size), which was stabilized with the fixator. A treatment group (12 rats) received tissue-engineered bone graft implants consisting of biphasic calcium phosphate blocks seeded with bone mesenchymal stem cells, while other 12 animals received no bone graft (non-treatment group). The osteotomy gap remained unchanged in the non-treatment group over the 12-week period, indicating that the 6-mm bone defect is really non-healable in the rat femur and that the device has sufficient stability for the management of critical-sized femoral defects. At 12 weeks, the treatment group maintained the bone length throughout the study period and showed bridging of the defect, with remarkable new bone formation. In contrast, the non-treatment group showed marginal new bone formation, but no apparent healing. In conclusion, the novel device provides substantial benefits in the maintenance of critical-sized femoral defects and tissue-engineered bone grafts in a rat model. PMID: 19776528 [PubMed - in process] |
| Non-crystalline composite tissue engineering scaffolds using boron-containing bioactive glass and poly(d,l-lactic acid) coatings.
September 25, 2009 at 10:31 am
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| | Non-crystalline composite tissue engineering scaffolds using boron-containing bioactive glass and poly(d,l-lactic acid) coatings. Biomed Mater. 2009 Sep 23;4(5):55002 Authors: Mantsos T, Chatzistavrou X, Roether JA, Hupa L, Arstila H, Boccaccini AR The aim of this study was the fabrication of three-dimensional, highly porous, bioactive scaffolds using a recently developed bioactive glass powder, denominated '0106', with nominal composition (in wt%): 50 SiO(2), 22.6 CaO, 5.9 Na(2)O, 4 P(2)O(5), 12 K(2)O, 5.3 MgO and 0.2 B(2)O(3). The optimum sintering conditions for the fabrication of scaffolds by the foam-replica method were identified (sintering temperature: 670 degrees C and dwell time: 5 h). Composite samples were also fabricated by applying a biopolymer coating of poly((D,L)-lactic acid) (PDLLA) using a dip coating process. The average compressive strength values were 0.4 MPa for uncoated and 0.6 MPa for coated scaffolds. In vitro bioactivity studies in simulated body fluid (SBF) showed that a carbonate hydroxyapatite (HCAp) layer was deposited on uncoated and coated scaffolds after only 4 days of immersion in SBF, demonstrating the high in vitro bioactivity of the scaffolds. It was also confirmed that the scaffold structure remained amorphous (no crystallization) after the specific heat treatment used, with scaffolds exhibiting mechanical properties and bioactivity suitable for use in bone tissue engineering applications. PMID: 19776493 [PubMed - as supplied by publisher] |
| The remodeling of cardiovascular bioprostheses under influence of stem cell homing signal pathways.
September 25, 2009 at 10:31 am
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| | The remodeling of cardiovascular bioprostheses under influence of stem cell homing signal pathways. Biomaterials. 2009 Sep 21; Authors: De Visscher G, Lebacq A, Mesure L, Blockx H, Vranken I, Plusquin R, Meuris B, Herregods MC, Van Oosterwyck H, Flameng W Optimizing current heart valve replacement strategies by creating living prostheses is a necessity to alleviate complications with current bioprosthetic devices such as calcification and degeneration. Regenerative medicine, mostly in vitro tissue engineering, is the forerunner of this optimization search, yet here we show the functionality of an in vivo alternative making use of 2 homing axes for stem cells. In rats we studied the signaling pathways of stem cells on implanted bioprosthetic tissue (photooxidized bovine pericardium (POP)), by gene and protein expression analysis. We found that SDF-1alpha/CXCR4 and FN/VLA4 homing axes play a role. When we implanted vascular grafts impregnated with SDF-1alpha and/or FN as carotid artery interpositions, primitive cells were attracted from the circulation. Next, bioprosthetic heart valves, constructed from POP impregnated with SDF-1alpha and/or FN, were implanted in pulmonary position. As shown by CD90, CD34 and CD117 immunofluorescent staining they became completely recellularized after 5 months, had a normal function and biomechanical properties and specifically the combination of SDF-1alpha and FN had an optimal valve-cell phenotype. PMID: 19775751 [PubMed - as supplied by publisher] |
| Gene delivery via DNA incorporation within a biomimetic apatite coating.
September 25, 2009 at 10:31 am
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| | Gene delivery via DNA incorporation within a biomimetic apatite coating. Biomaterials. 2009 Sep 21; Authors: Luong LN, McFalls KM, Kohn DH Integrating inductivity with conductivity in a material may advance tissue engineering. An organic/inorganic hybrid was developed by incorporating plasmid DNA encoding for the beta-gal gene complexed with Lipofectamine 2000((R)) (DNA-Lipoplex) within apatite via coprecipitation. It was hypothesized that this system will result in enhanced transfection efficiency compared to DNA-Lipoplexes adsorbed to the mineral surface and DNA coprecipitated without Lipofectamine 2000((R)). PLGA films were cast onto glass slips and apatite and DNA were coprecipitated in modified simulated body fluid (mSBF). DNA-Lipoplex presence in mineral, DNA-Lipoplex stability (vs. coprecipitation time), and transfection efficiency (determined with C3H10T1/2 cells) as a function of coprecipitation time, DNA-Lipoplex concentration, and DNA incorporation method were studied. DNA-Lipoplex presence and spatial distribution on apatite were confirmed through fluorescence. Transfection efficiency was highest for 6h of DNA-Lipoplex coprecipitation. Differences in transfection efficiency were found between the DNA concentrations, with the highest efficiency for coprecipitation being 40mug/ml (p</=0.009 relative to other coprecipitation concentrations). Significant differences in transfection efficiency existed between incorporation methods (p<0.05) with the highest efficiency for DNA-Lipoplex coprecipitation. This hybrid material system not only integrates inductivity provided by the DNA and conductivity provided by the apatite, but it also has significant implications in non-viral gene delivery due to its ability to increase transfection efficiency. PMID: 19775750 [PubMed - as supplied by publisher] |
| Recombinant sendai virus-mediated gene transfer to mouse pancreatic stem cells.
September 25, 2009 at 10:31 am
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| | Recombinant sendai virus-mediated gene transfer to mouse pancreatic stem cells. Cell Transplant. 2009;18(5):573-80 Authors: Oishi K, Noguchi H, Yukawa H, Inoue M, Takagi S, Iwata H, Hasegawa M, Hayashi S Efficient gene transfer into stem cells is essential for the basic research and for therapeutic applications in gene-modified regenerative medicine. Adenovirus (AdV) vectors, one of the most commonly used types of vectors, can mediate high, albeit transient, levels of expression of the transgene in pancreatic stem/progenitor cells. However, high multiplicity of infection (MOI) with AdV vectors can result in cellular toxicity. Therefore, AdV vectors have been of limited usefulness in clinical applications. In this study, we investigated the in vitro gene transfer efficiency of Sendai virus (SeV) vectors, a paramyxovirus vector that can efficiently introduce foreign genes without toxicity into several cell types, including pancreatic stem cells. The dose-dependent GFP expression of pancreatic stem cells transfected with SeV vectors after 48 h of culture at 37 degrees C was observed. The transfection of pancreatic stem cells with SeV vectors and AdV vectors results in equal expression of the transgene (GFP expression) in the cells after 48 h of culture at 37 degrees C. Although the transfection of pancreatic stem cells with AdV vectors at high MOIs was cytotoxic, transfection with SeV vectors at high MOIs was rarely cytotoxic. In addition, pancreatic stem cells transfected with SeV maintained their differentiation ability. These data suggest that SeV could provide advantages with respect to safety issues in gene-modified regenerative medicine. PMID: 19775519 [PubMed - in process] |
| Regenerative medicine for diabetes mellitus.
September 25, 2009 at 10:31 am
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| | Regenerative medicine for diabetes mellitus. Cell Transplant. 2009;18(5):491-6 Authors: Kobayashi N, Yuasa T, Okitsu T In diabetes, a loss of pancreatic beta-cells causes insulin dependency. When insulin dependency is caused by type 1 diabetes or pancreatic diabetes, for example, pancreatic beta-cells need to be regenerated for definitive treatment. The methods for generating pancreatic beta-cells include a method of creating pancreatic beta-cells in vitro and implanting them into the body and a method of regenerating pancreatic beta-cells in the body via gene introduction or the administration of differential proliferation factors to the body. Moreover, the number of pancreatic beta-cells is also low in type 2 diabetes, caused by the compounding factors of insulin secretory failure and insulin resistance; therefore, if pancreatic beta-cells can be regenerated in a living body, then a further amelioration of the pathology can be expected. The development of pancreatic beta-cell-targeting regenerative medicine can lead to the next generation of diabetes treatment. PMID: 19775508 [PubMed - in process] |
| Reduced Differentiation Efficiency of Murine Embryonic Stem Cells in Stirred Suspension Bioreactors.
September 25, 2009 at 10:31 am
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| | Reduced Differentiation Efficiency of Murine Embryonic Stem Cells in Stirred Suspension Bioreactors. Stem Cells Dev. 2009 Sep 23; Authors: Taiani J, Krawetz RJ, Nieden NZ, Wu YE, Kallos MS, Matyas JR, Rancourt DE The use of embryonic stem cells (ESCs) for regenerative medicine has generated increased attention due to the favorable attributes of these cells; namely, they are pluripotent and possess long-term self-renewal capacity. The initial aims of the present study were: (i) to use stirred-suspension bioreactors to expand and differentiate ESCs into osteogenic and chondrogenic cell types, and (ii) to explore if these ESC-derived cells influenced skeletal healing in an in vivo fracture model. We show that differentiation protocols used in static culture are insufficient when applied directly to suspension culture bioreactors. Moreover, when bioreactor-differentiated cells are transplanted into a burr-hole defect in bone, severe disruption of the bone architecture was noted at the fracture site, as determined by microcomputed tomography (microCT) imaging and histopathology. Further characterization of the bioreactor-differentiated cultures revealed that a subpopulation of cells in the resulting aggregates expressed the pluripotency marker Oct-4 in the nucleus. Nuclear Oct-4 expression persisted even after 30 days of culture in the absence of LIF. Remarkably, and unlike ESCs differentiated into skeletal cell types in static cultures, bioreactor-differentiated aggregates implanted subcutaneously into SCID mice formed teratomas. The development of effective ESC differentiation protocols for suspension bioreactors will require a more complete understanding of the environmental conditions within these culture systems and the influence that these conditions have on the regulation of pluripotency and differentiation in ESCs. PMID: 19775198 [PubMed - as supplied by publisher] |
| Reciprocal regulation of IL-6 and IL-10 balance by HGF via recruitment of heme oxygenase-1 in macrophages for attenuation of liver injury in a mouse model of endotoxemia.
September 25, 2009 at 10:31 am
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| | Reciprocal regulation of IL-6 and IL-10 balance by HGF via recruitment of heme oxygenase-1 in macrophages for attenuation of liver injury in a mouse model of endotoxemia. Int J Mol Med. 2009 Aug;24(2):161-70 Authors: Kamimoto M, Mizuno S, Nakamura T Acute liver injury is a clinical hallmark of endotoxemia regarding the features of septic organ failure. In this process, interleukin (IL)-6 and IL-10 are key contributors for eliciting pro- and anti-inflammatory responses, respectively. In contrast, heme oxygenase-1 (HO-1) provides a defense mechanism against endotoxemia by controlling the IL-6/IL-10 balance, but how higher levels of HO-1 are sustained under pathological conditions remains unknown. Using a mouse model of endotoxemia, we provide evidence to show that hepatocyte growth factor (HGF) enhances HO-1 expression in macrophages, thereby up-regulating IL-10 and down-regulating IL-6 productions. Lipopolysaccharide (LPS)-treated mice manifested acute liver injury similar to that observed in septic patients, while administration of recombinant HGF enhanced expression of HO-1 by hepatic macrophages in vivo. As a result, HGF blocked the onset of hepatic injuries in LPS-treated mice. More importantly, when an HO-1 inhibitor (Sn-PP) was administered with HGF into LPS-treated mice, the protective effects of HGF against hepatic injury were attenuated. Furthermore, Sn-PP partially restored the HGF-mediated decrease in plasma IL-6 levels, while it inhibited the HGF-stimulated increase in plasma IL-10 levels. In the culture of macrophages (Raw264.7), HGF enhanced the LPS-mediated HO-1 induction, and this effect was abolished by cycloheximide, but not by actinomycin-D, thus suggesting that a post-transcriptional pathway is involved in HGF-mediated up-regulation of HO-1. Based on the current data, we conclude that up-regulation of HO-1 plays an important role in HGF-mediated hepatoprotection during endotoxemia, by favoring production of IL-10 over IL-6. PMID: 19578789 [PubMed - indexed for MEDLINE] |
| The transcriptional foundation of pluripotency.
September 25, 2009 at 10:31 am
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| | The transcriptional foundation of pluripotency. Development. 2009 Jul;136(14):2311-22 Authors: Chambers I, Tomlinson SR A fundamental goal in biology is to understand the molecular basis of cell identity. Pluripotent embryonic stem (ES) cell identity is governed by a set of transcription factors centred on the triumvirate of Oct4, Sox2 and Nanog. These proteins often bind to closely localised genomic sites. Recent studies have identified additional transcriptional modulators that bind to chromatin near sites occupied by Oct4, Sox2 and Nanog. This suggests that the combinatorial control of gene transcription might be fundamental to the ES cell state. Here we discuss how these observations advance our understanding of the transcription factor network that controls pluripotent identity and highlight unresolved issues that arise from these studies. PMID: 19542351 [PubMed - indexed for MEDLINE] |
| Estimation of relationship between the structure of 1,2,3,4-tetrahydroisoquinoline derivatives determined by a semiempirical molecular-orbital method and their cytotoxicity.
September 25, 2009 at 10:31 am
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| | Estimation of relationship between the structure of 1,2,3,4-tetrahydroisoquinoline derivatives determined by a semiempirical molecular-orbital method and their cytotoxicity. Anticancer Res. 2009 Jun;29(6):2265-71 Authors: Ishihara M, Hatano H, Kawase M, Sakagami H A semiempirical molecular-orbital method (CAChe 4.9, PM5) was applied to delineate the relationship between the cytotoxicity (evaluated by 50% cytotoxic concentration, CC(50)) of nineteen 1,2,3,4-tetrahydroisoquinoline derivatives, their molecular weight and the sixteen chemical parameters (descriptors) determined by CONFLEX/PM5 method. There was little or no correlation between the CC(50) in HL-60 cells and the heat of formation, stability of hydration (DeltaH), dipole moment, electron affinity, ionization potential, highest occupied molecular orbital energy (E(HOMO)), lowest unoccupied molecular orbital energy (E(LUMO)), absolute hardness (eta, softness and hardness of the molecule) or molecular weight (r(2)<0.312). On the other hand, there was a good correlation between the CC(50) and the hydrophobicity (log P) (r(2)=0.503), and the descriptors for the molecular size such as surface area (r(2)=0.771), volume (r(2)=0.805) and width (r(2)=0.757). Similar, but not so clear-cut correlation was found in HSC-2, HSC-3 and HSC-4 human oral squamous cell carcinoma cell lines. The present study demonstrates that the cytotoxicity of 1,2,3,4-tetrahydroisoquinoline derivatives depends more on the descriptors for molecular size rather than the physicochemical descriptors. PMID: 19528491 [PubMed - indexed for MEDLINE] |
| Distinct roles for isoforms of the catalytic subunit of class-IA PI3K in the regulation of behaviour of murine embryonic stem cells.
September 25, 2009 at 10:31 am
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| | Distinct roles for isoforms of the catalytic subunit of class-IA PI3K in the regulation of behaviour of murine embryonic stem cells. J Cell Sci. 2009 Jul 1;122(Pt 13):2311-21 Authors: Kingham E, Welham M Self-renewal of embryonic stem cells (ESCs) is essential for maintenance of pluripotency, which is defined as the ability to differentiate into any specialised cell type comprising the adult organism. Understanding the mechanisms that regulate ESC self-renewal and proliferation is required before ESCs can fulfil their potential in regenerative therapies, and murine ESCs (mESCs) have been widely used as a model. Members of the class-IA phosphoinositide 3-kinase (PI3K) family of lipid kinases regulate a variety of physiological responses, including cell migration, proliferation and survival. PI3Ks have been reported to regulate both proliferation and self-renewal of mESCs. Here we investigate the contribution of specific class-IA PI3K isoforms to the regulation of mESC fate using small-molecule inhibitors with selectivity for particular class-IA PI3K catalytic isoforms, and siRNA-mediated knockdown. Pharmacological inhibition or knockdown of p110beta promoted mESC differentiation, accompanied by a decrease in expression of Nanog. By comparison, pharmacological inhibition or siRNA-mediated knockdown of p110alpha had no effect on mESC self-renewal per se, but instead appeared to reduce proliferation, which was accompanied by inhibition of leukaemia inhibitory factor (LIF) and insulin-induced PI3K signalling. Our results suggest that PI3Ks contribute to the regulation of both mESC pluripotency and proliferation by differential coupling to selected p110 catalytic isoforms. PMID: 19509054 [PubMed - indexed for MEDLINE] | 
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