| Effects of individual control of pH and hypoxia in chondrocyte culture.
October 9, 2009 at 10:09 am
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| | Effects of individual control of pH and hypoxia in chondrocyte culture. J Orthop Res. 2009 Oct 7; Authors: Das RH, van Osch GJ, Kreukniet M, Oostra J, Weinans H, Jahr H Effects of oxygen tension (pO(2)) and pH on gene and protein expression and metabolic activity of human chondrocytes were independently assessed. Chondrocytes were cultured under a range of pH (6.4-7.4) and different pO(2) (5 and 20%) during 5 days in a bioreactor. Effects on gene expression, DNA content, protein expression, and metabolic activity were determined. Linear regression analysis showed that gene expression of type I collagen (COL1), SOX9, and VEGF is significantly lower at acidic pH, while expression of aggrecan, type II collagen, and HIF1A is pH-independent. Higher protein levels of VEGF were found under low pO(2). Acidic pH severely lowered VEGF release into medium, glucose consumption, and lactate production. Extracellular pH proved to more potently influence cell function than oxygen tension, the latter showing down-regulation of COL1 gene expression and up-regulation of VEGF protein under hypoxia. Hypoxic culture inhibits COL1 mRNA expression pH-dependently, while expression of SOX9 is largely hypoxia independent, but pH dependent. Expression of HIF1A and VEGF revealed divergent pH dependencies. Subtle fluctuations in extracellular pH and oxygen tension clearly influence chondrocyte metabolism and marker expression. Sophisticated pH and oxygen control not only allows study of (patho)physiological changes, but also opens new venues in cartilage tissue engineering. (c) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res. PMID: 19813243 [PubMed - as supplied by publisher] |
| Preliminary Investigation of Seeding Mesenchymal Stem Cells on Biodegradable Scaffolds for Vascular Tissue Engineering In Vitro.
October 9, 2009 at 10:09 am
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| | Preliminary Investigation of Seeding Mesenchymal Stem Cells on Biodegradable Scaffolds for Vascular Tissue Engineering In Vitro. ASAIO J. 2009 Oct 6; Authors: Li CM, Wang ZG, Gu YQ, Dong JD, Qiu RX, Bian C, Liu XF, Feng ZG We used epsilon-caprolactone/l-lactide (PCLA) as a biodegradable scaffold and bone marrow (BM) mesenchymal stem cells (MSCs) as seeding cells for vascular tissue engineering: we expected MSCs to grow in the scaffolds in a bioreactor. The MSCs we used were from the BM of dogs, and vascular scaffolds were carried out on the electrospinning process of PCLA copolymers. MSCs expressed CD44 and CD105 but did not express CD34 or CD14 at an identical time point. Scaffolds were nontoxic to cells and were favorable for the growth and migration of MSCs. After culture in a bioreactor with mechanical stimulation, cells completely covered the surfaces of PCLA scaffolds and penetrated or infiltrated into the inside of the scaffold structure. PMID: 19812476 [PubMed - as supplied by publisher] |
| Blood compatibility evaluation of poly(d,l-lactide-co-beta-malic acid) modified with the GRGDS sequence.
October 9, 2009 at 10:09 am
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| | Blood compatibility evaluation of poly(d,l-lactide-co-beta-malic acid) modified with the GRGDS sequence. Colloids Surf B Biointerfaces. 2009 Sep 19; Authors: Liu Y, Wang W, Wang J, Wang Y, Yuan Z, Tang S, Liu M, Tang H Endothelialization is an ideal approach to improve the blood compatibility of synthetic polymers. However, cell detachment is inevitable under shear flow conditions. Therefore, the issue of blood compatibility needs to be addressed for both the bare and the endothelialized polymer. RGD-containing polymer P-GS5 was synthesized by modification of poly(d,l-lactide-co-beta-malic acid) (PLMA) with the peptide GRGDS. The compositions, molecular weights and hydrophilicities of poly(d,l-lactide) (PDLLA), PLMA, and P-GS5 were characterized by nuclear magnetic resonance (NMR), X-ray photoelectron spectroscopy (XPS), gel-permeation chromatography (GPC) and water contact angle measurements, respectively. The blood compatibilities of the bare and the endothelialized polymers were evaluated by clotting time and platelet adhesion tests. The results showed that the coagulation pathways were not influenced before and after cell culture; the bare P-GS5 attracted less platelet adhesion and induced lower pseudopodia extension compared with PDLLA and PLMA, and the platelet adhesion on P-GS5 was almost completely eliminated after cell seeding. The results suggest that P-GS5 could be a potentially useful material in vascular tissue engineering. PMID: 19811897 [PubMed - as supplied by publisher] |
| Deterministic Lateral Displacement as a Means to Enrich Large Cells for Tissue Engineering.
October 9, 2009 at 10:09 am
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| | Deterministic Lateral Displacement as a Means to Enrich Large Cells for Tissue Engineering. Anal Chem. 2009 Oct 7; Authors: Green JV, Radisic M, Murthy SK The enrichment or isolation of selected cell types from heterogeneous suspensions is required in the area of tissue engineering. State of the art techniques utilized for this separation include preplating and sieve-based approaches that have limited ranges of purity and variable yield. Here, we present a deterministic lateral displacement (DLD) microfluidic device that is capable of separating large epithelial cells (17.3 +/- 2.7 in diameter) from smaller fibroblast cells (13.7 +/- 3.0 mum in diameter) as a potential alternative approach. The mixed suspension examined is intended to represent the content of digested rat cardiac tissue, which contains equal proportions of cardiomyocyte (17.0 +/- 4.0 mum diameter) and nonmyocyte populations (12.0 +/- 3.0 mum diameter). High purity separation (>97%) of the larger cell type is achieved with 90% yield in a rapid and single-pass process. The significance of this work lies in the recognition that DLD design principles can be applied for the microfluidic enrichment of large cells, up to the 40 mum diameter level examined in this work. PMID: 19810716 [PubMed - as supplied by publisher] |
| Newly developed tissue-engineered material for reconstruction of vascular wall without cell seeding.
October 9, 2009 at 10:09 am
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| | Newly developed tissue-engineered material for reconstruction of vascular wall without cell seeding. Ann Thorac Surg. 2009 Oct;88(4):1269-76 Authors: Takahashi H, Yokota T, Uchimura E, Miyagawa S, Ota T, Torikai K, Saito A, Hirakawa K, Kitabayashi K, Okada K, Sawa Y, Okita Y BACKGROUND: We have developed a tissue-engineered patch for cardiovascular repair. Tissue-engineered patches facilitated site-specific in situ recellularization and required no pretreatment with cell seeding. This study evaluated the patches implanted into canine pulmonary arteries. METHODS: Tissue-engineered patches are biodegradable sheets woven with double-layer fibers. The fiber is composed of polyglycolic acid and poly-L-lactic acid, and compounding collagen microsponges. The patches (20- x 25-mm) were implanted into the canine pulmonary arterial trunks. At 1, 2, and 6 months after implantation (n = 4), they were explanted and characterized by histologic and biochemical analyses. Commercially available patches served as the control. No anticoagulant therapy was administered postoperatively. RESULTS: No aneurysm or thrombus was present within the patch area in all groups. The remodeled tissue predominantly consisted of elastic and collagen fibers, and the endoluminal surface was covered with a monolayer of endothelial cells and multilayers of smooth muscle cells beneath the endothelial layer. The elastic and collagen fibers and smooth muscle cells kept increasing with a maximum at 6 months, while a monolayer of endothelial cells was preserved. The expression levels of messenger RNA of several growth factors in the tissue-engineered patches were higher than those of native tissue at 1 and 2 months and decreased to normal level at 6 months. No regenerated tissue was found on the endoluminal surface in the control group. CONCLUSIONS: The novel tissue-engineered patches showed in situ repopulation of host cells without prior ex vivo cell seeding. This is promising material for repair of the cardiovascular system. PMID: 19766820 [PubMed - indexed for MEDLINE] |
| Synergistic effects of autologous cell and hepatocyte growth factor gene therapy for neovascularization in a murine model of hindlimb ischemia.
October 9, 2009 at 10:09 am
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| | Synergistic effects of autologous cell and hepatocyte growth factor gene therapy for neovascularization in a murine model of hindlimb ischemia. Am J Physiol Heart Circ Physiol. 2009 Oct;297(4):H1329-36 Authors: Yamamoto Y, Matsuura T, Narazaki G, Sugitani M, Tanaka K, Maeda A, Shiota G, Sato K, Yoshida A, Hisatome I Autologous cell implantation and angiogenic gene therapy have been evaluated in critical limb ischemic patients. Here, we compared the features of these strategies individually and in combination. C57BL/6J mice with ischemic hindlimbs were injected with adherent mononuclear cells (aMNCs) from bone marrow or adenovirus encoding the hepatocyte growth factor (HGF) gene (Ad-HGF). Under comparable angiogenic conditions, 10 x 10(5) aMNCs produced significantly higher amounts of VEGF and FGF-2 and stimulated the number of arterioles in ischemic muscle compared with 1 x 10(8) plaque-forming units (pfu) of Ad-HGF. Ad-HGF produced 10 times more HGF in ischemic muscle compared with aMNCs. Injection of 0.3 x 10(5) aMNCs previously transfected with Ad-HGF (aMNC/Ad-HGF) increased blood flow and elevated the numbers of capillaries and arterioles to levels comparable with that seen with 10 x 10(5) aMNCs or 1 x 10(8) pfu of Ad-HGF. Hypoxic conditions induced the apoptotic death of aMNCs. However, coincubation with HGF or aMNC/Ad-HGF protected cells against apoptosis. HGF stimulated the migration of aMNCs, and the migration capacity of the aMNC/Ad-HGF group was significantly higher than that in the aMNC/Ad-LacZ group. In conclusion, cell-based HGF gene therapy decreased the number of cells required for neovascularization. This strategy can be an effective angiogenic therapy. PMID: 19666845 [PubMed - indexed for MEDLINE] |
| Inkjet printing of growth factor concentration gradients and combinatorial arrays immobilized on biologically-relevant substrates.
October 9, 2009 at 10:09 am
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| | Inkjet printing of growth factor concentration gradients and combinatorial arrays immobilized on biologically-relevant substrates. Comb Chem High Throughput Screen. 2009 Jul;12(6):604-18 Authors: Miller ED, Phillippi JA, Fisher GW, Campbell PG, Walker LM, Weiss LE Current methods for engineering immobilized, 'solid-phase' growth factor patterns have not addressed the need for presentation of the growth factors in a biologically-relevant context. We developed an inkjet printing methodology for creating solid-phase patterns of unmodified growth factors on native biological material substrates. We demonstrate this approach by printing gradients of fluorescently labeled bone morphogenetic protein-2 (BMP-2) and insulin-like growth factor-II (IGF-II) bio-inks on fibrin-coated surfaces. Concentration gradients were created by overprinting individual substrate locations using a dilute bio-ink to modulate the surface concentration of deposited growth factor. Persistence studies using fluorescently-labeled BMP-2 verified that the gradients retained their shape for up to 7 days. Desorption experiments performed with (125)I-BMP-2 and (125)I-IGF-II were used to quantify the surface concentration of growth factor retained on the substrate for up to 10 days in serum containing media after rinsing of the unbound growth factor. The inkjet method is programmable so the gradient shape can be easily modified as demonstrated by printed linear gradients with varying slopes and exponential gradients. In addition, the versatility of this method enabled combinatorial arrays of multiple growth factors to be created by printing overlapping patterns. The overlapping printing method was used to create a combinatorial square pattern array consisting of various surface concentrations of BMP-2 and fibroblast growth factor-2 (FGF-2). C2C12 myogenic precursor cells were seeded on the arrays and alkaline phosphatase staining was performed to determine the effect of FGF-2 and BMP-2 surface concentration on guiding C2C12 cells towards an osteogenic lineage. These results demonstrate the utility of inkjet printing for creating orthogonal growth factor gradients to investigate how combinations of immobilized growth factors influence cell fate. PMID: 19601758 [PubMed - indexed for MEDLINE] |
| Propensity of human embryonic stem cell lines during early stage of lineage specification controls their terminal differentiation into mature cell types.
October 9, 2009 at 10:09 am
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| | Propensity of human embryonic stem cell lines during early stage of lineage specification controls their terminal differentiation into mature cell types. Exp Biol Med (Maywood). 2009 Oct;234(10):1230-43 Authors: Pal R, Totey S, Mamidi MK, Bhat VS, Totey S Human embryonic stem cells (hESCs) are able to stably maintain their characteristics for an unlimited period; nevertheless, substantial differences among cell lines in gene and protein expression not manifested during the undifferentiated state may appear when cells differentiate. It is widely accepted that developing an efficient protocol to control the differentiation of hESCs will enable us to produce adequate numbers of desired cell types with relative ease for diverse applications ranging from basic research to cell therapy and drug screening. Hence of late, there has been considerable interest in understanding whether and how hESC lines are equivalent or different to each other in their in vitro developmental tendencies. In this study, we compared the developmental competences of two hESC lines (HUES-9 and HUES-7) at molecular, cellular and functional levels, upon spontaneous differentiation without any added inducing agents. Both cell lines generated the three embryonic germ layers, extra-embryonic tissues and primordial germ cells during embryoid body (EB) formation. However HUES-9 showed a stronger propensity towards formation of neuroectodermal lineages, whereas HUES-7 differentiated preferentially into mesoderm and endoderm. Upon further differentiation, HUES-9 generated largely neural cells (neurons, oligodendrocytes, astrocytes and gangliosides) whereas HUES-7 formed mesendodermal derivatives, including cardiomyocytes, skeletal myocytes, endothelial cells, hepatocytes and pancreatic cells. Overall, our findings endorse the hypothesis that independently-derived hESCs biologically differ among themselves, thereby displaying varying differentiation propensity. These subtle differences not only highlight the importance of screening and deriving lines for lineage-specific differentiation but also indicate that individual lines may possess a repertoire of capabilities that is unique. PMID: 19546356 [PubMed - indexed for MEDLINE] |
| Detection and identification of specific bacteria in wound biofilms using peptide nucleic acid fluorescent in situ hybridization (PNA FISH).
October 9, 2009 at 10:09 am
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| | Detection and identification of specific bacteria in wound biofilms using peptide nucleic acid fluorescent in situ hybridization (PNA FISH). Microbiology. 2009 Aug;155(Pt 8):2603-11 Authors: Malic S, Hill KE, Hayes A, Percival SL, Thomas DW, Williams DW Biofilms provide a reservoir of potentially infectious micro-organisms that are resistant to antimicrobial agents, and their importance in the failure of medical devices and chronic inflammatory conditions is increasingly being recognized. Particular research interest exists in the association of biofilms with wound infection and non-healing, i.e. chronic wounds. In this study, fluorescent in situ hybridization (FISH) was used in combination with confocal laser scanning microscopy (CLSM) to detect and characterize the spatial distribution of biofilm-forming bacteria which predominate within human chronic skin wounds (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sp. and Micrococcus sp.). In vitro biofilms were prepared using a constant-depth film fermenter and a reconstituted human epidermis model. In vivo biofilms were also studied using biopsy samples from non-infected chronic venous leg ulcers. The specificity of peptide nucleic acid (PNA) probes for the target organisms was confirmed using mixed preparations of planktonic bacteria and multiplex PNA probing. Identification and location of individual bacterial species within multi-species biofilms demonstrated that P. aeruginosa was predominant. CLSM revealed clustering of individual species within mixed-species biofilms. FISH analysis of archive chronic wound biopsy sections showed bacterial presence and allowed bacterial load to be determined. The application of this standardized procedure makes available an assay for identification of single- or multi-species bacterial populations in tissue biopsies. The technique provides a reliable tool to study bacterial biofilm formation and offers an approach to assess targeted biofilm disruption strategies in vivo. PMID: 19477903 [PubMed - indexed for MEDLINE] |
| Microengineered platforms for cell mechanobiology.
October 9, 2009 at 10:09 am
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| | Microengineered platforms for cell mechanobiology. Annu Rev Biomed Eng. 2009;11:203-33 Authors: Kim DH, Wong PK, Park J, Levchenko A, Sun Y Mechanical forces play important roles in the regulation of various biological processes at the molecular and cellular level, such as gene expression, adhesion, migration, and cell fate, which are essential to the maintenance of tissue homeostasis. In this review, we discuss emerging bioengineered tools enabled by microscale technologies for studying the roles of mechanical forces in cell biology. In addition to traditional mechanobiology experimental techniques, we review recent advances of microelectromechanical systems (MEMS)-based approaches for cell mechanobiology and discuss how microengineered platforms can be used to generate in vivo-like micromechanical environment in in vitro settings for investigating cellular processes in normal and pathophysiological contexts. These capabilities also have significant implications for mechanical control of cell and tissue development and cell-based regenerative therapies. PMID: 19400708 [PubMed - indexed for MEDLINE] |
| Minimally invasive surgical approach for three-vessel occlusion as a model of vascular dementia in the rat-brain bioenergetics assay.
October 9, 2009 at 10:09 am
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| | Minimally invasive surgical approach for three-vessel occlusion as a model of vascular dementia in the rat-brain bioenergetics assay. J Neurol Sci. 2009 Aug 15;283(1-2):178-81 Authors: Horecký J, Baciak L, Kasparová S, Pacheco G, Aliev G, Vancová O Brain energy disorders and oxidative stress due to chronic hypoperfusion are considered to be major risk factors in the pathogenesis of dementia. The aim of our study was to evaluate changes of the brain creatine kinase (BB-CK) reaction and mitochondrial respiratory chain function in male Wistar rats exposed to chronic cerebral hypoperfusion. Three-vessel occlusion (3-VO) was accomplished without thoracotomy using a minimally-invasive surgical approach for the occlusion of the brachiocephalic trunk and the left common carotid artery (CCA). The forward rate constant of creatine kinase (k(for)) was measured in vivo by saturation transfer of (31)P magnetic resonance spectroscopy (MRS) at 2 and 10 weeks of permanent 3-VO. The function of the mitochondrial respiratory chain in vitro was assessed polarographically at 10 weeks after 3-VO. When compared to the controls, the significant 42% reduction of k(for) at 2 resp. 10 weeks indicated disorders in brain energy metabolism, which is in agreement with the 12% decrease of the oxidative phosphorylation coefficient (ADP:O) and with the 14% decrease of the oxidative phosphorylation rate (OPR) measured in isolated mitochondria. Oxidative modification of the creatine kinase system (inactivation of enzymes) and metabolic disorders due to chronic 3-VO, thus, may participate in vascular cognitive impairment and neuronal degeneration. PMID: 19272617 [PubMed - indexed for MEDLINE] | | | 
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