| Science and Research in Academic Plastic Surgery in Germany.
December 24, 2009 at 8:49 am
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| | Science and Research in Academic Plastic Surgery in Germany. Handchir Mikrochir Plast Chir. 2009 Dec;41(6):359-363 Authors: Giunta RE, Machens HG Plastic surgery has passed through a very positive evolution in the last decades on the solid fundament of constantly developing academic plastic surgery. Aim of this paper is an objective evaluation of the current status of academic plastic surgery regarding research topics, currently available ressources and scientific outcome based on a questionnaire. The return rate of the questionnaire in academic departments was 92%. Main topics in research besides wound healing were topics from regenerative medicine such as tissue engineering, biomaterials, genetherapy and angiogenesis with the main focus on skin and fat tissues. In the past five years a total of 25 million Euros of third party research grants were raised. Research relied mainly on interdisciplinary research facilities. Regarding the scientific outcome more than 200 scientific papers were published in basic science research journals having an impactfactor higher than two. These results clearly demonstrate that plastic surgery is scientifically highly productive in academic surroundings where independent departments are established. Considering that independent units of plastic surgery exist in a relatively small number of all 36 university hospitals in germany, it has to be claimed for further independent departments so to provide adequate research facilities for further evolution of academic plastic surgery. PMID: 20029742 [PubMed - as supplied by publisher] |
| On the Independent Origins of Complex Brains and Neurons.
December 24, 2009 at 8:49 am
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| | On the Independent Origins of Complex Brains and Neurons. Brain Behav Evol. 2009;74(3):177-190 Authors: Moroz LL Analysis of the origin and evolution of neurons is crucial for revealing principles of organization of neural circuits with unexpected implications for genomic sciences, biomedical applications and regenerative medicine. This article presents an overview of some controversial ideas about the origin and evolution of neurons and nervous systems, focusing on the independent origin of complex brains and possible independent origins of neurons. First, earlier hypotheses related to the origin of neurons are summarized. Second, the diversity of nervous systems and convergent evolution of complex brains in relation to current views about animal phylogeny is discussed. Third, the lineages of molluscs and basal metazoans are used as illustrated examples of multiple origins of complex brains and neurons. Finally, a hypothesis about the independent origin of complex brains, centralized nervous systems and neurons is outlined. Injury-associated mechanisms leading to secretion of signal peptides (and related molecules) can be considered as evolutionary predecessors of inter-neuronal signaling and the major factors in the appearance of neurons in the first place. PMID: 20029182 [PubMed - as supplied by publisher] |
| Receptor Channel TRPC6 Is a Key Mediator of Notch-Driven Glioblastoma Growth and Invasiveness.
December 24, 2009 at 8:49 am
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| | Receptor Channel TRPC6 Is a Key Mediator of Notch-Driven Glioblastoma Growth and Invasiveness. Cancer Res. 2009 Dec 22; Authors: Chigurupati S, Venkataraman R, Barrera D, Naganathan A, Madan M, Paul L, Pattisapu JV, Kyriazis GA, Sugaya K, Bushnev S, Lathia JD, Rich JN, Chan SL Glioblastoma multiforme (GBM) is the most frequent and incurable type of brain tumor of adults. Hypoxia has been shown to direct GBM toward a more aggressive and malignant state. Here we show that hypoxia increases Notch1 activation, which in turn induces the expression of transient receptor potential 6 (TRPC6) in primary samples and cell lines derived from GBM. TRPC6 is required for the development of the aggressive phenotype because knockdown of TRPC6 expression inhibits glioma growth, invasion, and angiogenesis. Functionally, TRPC6 causes a sustained elevation of intracellular calcium that is coupled to the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Pharmacologic inhibition of the calcineurin-NFAT pathway substantially reduces the development of the malignant GBM phenotypes under hypoxia. Clinically, expression of TRPC6 was elevated in GBM specimens in comparison with normal tissues. Collectively, our studies indicate that TRPC6 is a key mediator of tumor growth of GBM in vitro and in vivo and that TRPC6 may be a promising therapeutic target in the treatment of human GBM. Cancer Res; 70(1); 418-27. PMID: 20028870 [PubMed - as supplied by publisher] |
| Specific fibrinogen and thrombin concentrations promote neuronal rather than glial growth when primary neural cells are seeded within plasma-derived fibrin gels.
December 24, 2009 at 8:49 am
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| | Specific fibrinogen and thrombin concentrations promote neuronal rather than glial growth when primary neural cells are seeded within plasma-derived fibrin gels. Tissue Eng Part A. 2009 Dec 22; Authors: Mooney RG, Tawil BJ, Mahoney MJ Fibrin gels are attractive scaffolds useful for neural tissue engineering applications. The objective of this work was to investigate the apoptotic activity, survival, proliferation, and differentiation of a mixed population of primary neural cells composed of neurons and multipotent precursor cells when cultured in fibrin gels prepared with varying concentrations of fibrinogen (5 mg/ml - 25 mg/ml fibrinogen) and thrombin (1 U/ml - 125 U/ml thrombin). Within all fibrin gel formulations tested, the level of apoptosis on day 1 was low and cell survival was equivalent to levels in monolayer culture (67%). Proliferation in gels made from 5 mg/ml or 12.5 mg/ml fibrinogen was also similar to that observed in monolayer culture, though a lower proliferative response was observed in 25 mg/ml fibrinogen formulations. Relative to monolayer culture, cholinergic and dopaminergic neuronal presence was enhanced while glial cell growth was reduced in fibrin gel cultures. The extent to which levels were altered depended on fibrinogen and thrombin concentration. The findings here suggest the importance of fibrinogen and thrombin concentration in differentially regulating the growth and composition of neural cell populations and are of importance for neural tissue engineering strategies focused on the development of implantable scaffolds for treating neurodegenerative disorders. PMID: 20028220 [PubMed - as supplied by publisher] |
| Dynamic mechanical loading enhances functional properties of tissue engineered cartilage using mature canine chondrocytes.
December 24, 2009 at 8:49 am
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| | Dynamic mechanical loading enhances functional properties of tissue engineered cartilage using mature canine chondrocytes. Tissue Eng Part A. 2009 Dec 22; Authors: Bian L, Fong JV, Lima EG, Stoker AM, Ateshian GA, Cook JL, Hung CT Objective: The concept of cartilage functional tissue engineering (FTE) has promoted the use of physiologic loading bioreactor systems to cultivate engineered tissues with load-bearing properties. Prior studies have demonstrated that culturing agarose constructs seeded with primary bovine chondrocytes from immature joints, and subjected to dynamic deformation, produced equilibrium compressive properties and proteoglycan content matching the native tissue. In the process of translating these results to an adult canine animal model, it was found that protocols previously successful with immature bovine primary chondrocytes did not produce the same successful outcome when using adult canine primary chondrocytes. The objective of this study was to assess the efficacy of a modified FTE protocol using adult canine chondrocytes seeded in agarose hydrogel and subjected to dynamic loading. Method: Two modes of dynamic loading were applied to constructs using custom bioreactors: unconfined axial compressive deformational loading (DL, 1Hz, 10% deformation) or sliding contact loading (SLIDE, 0.5Hz, 10% deformation). Loading for 3 hours daily was initiated on day 0, 14 or 28 (DL0, DL14, DL28 and Slide14). Results: Constructs with applied loading (both DL and Slide) exhibited significant increases in Young's modulus compared to free-swelling (FS) control as early as day 28 in culture, p<0.05. However, glycosaminoglycan, collagen and DNA content were not statistically different among the various groups. The modulus values attained for engineered constructs compare favorably with (and exceed in some cases) those of native canine knee (patella groove and condyle) cartilage. Conclusion: Our findings successfully demonstrate a functional tissue engineering strategy incorporating clinically-relevant, adult chondrocytes and gel scaffold for engineering cartilage replacement tissue. These results, using continuous growth factor supplementation, are in contrast to our previously reported studies with immature chondrocytes where the sequential application of dynamic loading after transient TGF-beta3 application was found to be a superior culture protocol. Sliding which simulates aspects of joint articulation has shown promise in promoting engineered tissue development and provides an alternative option for functional tissue engineering of cartilage constructs to be further explored. PMID: 20028219 [PubMed - as supplied by publisher] |
| Effect of Nell-1 delivery on chondrocyte proliferation and cartilaginous extracellular matrix deposition.
December 24, 2009 at 8:49 am
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| | Effect of Nell-1 delivery on chondrocyte proliferation and cartilaginous extracellular matrix deposition. Tissue Eng Part A. 2009 Dec 22; Authors: Lee M, Siu RK, Ting K, Wu B Cartilage tissue engineering using chondrogenic growth factors is an attractive strategy to promote cartilage repair. Bone morphogenetic proteins (BMPs) have been widely studied for their application in cartilage repair. However, functional heterogeneity of BMPs and unpredictable effects such as cyst formation may limit their therapeutic use. Thus, the use of alternative growth factors with greater osteochondral specificity may be advantageous for cartilage regeneration. Nell-1 [Nel-like molecule-1; Nel (a protein strongly expressed in neural tissue encoding epidermal growth factor like domain)] is a novel growth factor believed to specifically target cells committed to the osteochondral lineage. Mutation of the Nell-1gene has been shown to disrupt normal cartilage growth and development in rodents. This study investigates the chondrogenic potential of recombinant human Nell-1 protein in a three-dimensional (3D) alginate hydrogel microenvironment containing rabbit chondrocytes. To provide controlled delivery and maximize biological efficiency, Nell-1 was incorporated in chitosan microparticles. Over 42 days of culture, chondrocyte proliferation and cluster formation was significantly enhanced by Nell-1 in a dose dependent manner. Furthermore, the clusters formed in the presence of Nell-1 contained more type II collagen and glycosaminoglycans than clusters formed within Nell-free control gels. These findings demonstrate the ability of Nell-1 to promote chondrocyte proliferation and deposition of cartilage specific extracellular matrix materials. PMID: 20028218 [PubMed - as supplied by publisher] |
| California stem-cell grants awarded.
December 24, 2009 at 8:49 am
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| | California stem-cell grants awarded. Nature. 2009 Nov 5;462(7269):22 Authors: Check Hayden E PMID: 19890299 [PubMed - indexed for MEDLINE] |
| Stem cells may improve the adaptability of dental implants.
December 24, 2009 at 8:49 am
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| | Stem cells may improve the adaptability of dental implants. J Mich Dent Assoc. 2009 Sep;91(9):26 Authors: Foe D PMID: 19835211 [PubMed - indexed for MEDLINE] |
| Treatment of human infrabony periodontal defects by grafting human cultured periosteum sheets combined with platelet-rich plasma and porous hydroxyapatite granules: case series.
December 24, 2009 at 8:49 am
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| | Treatment of human infrabony periodontal defects by grafting human cultured periosteum sheets combined with platelet-rich plasma and porous hydroxyapatite granules: case series. J Int Acad Periodontol. 2009 Jul;11(3):206-13 Authors: Okuda K, Yamamiya K, Kawase T, Mizuno H, Ueda M, Yoshie H In a previous publication from our research group we reported that a combination of platelet-rich plasma (PRP) and hydroxyapatite (HA) granules resulted in a significantly favorable clinical result in treating osseous defects. The aim of this study is to evaluate the clinical response of human cultured periosteum (HCP) sheets in combination with autologous PRP and HA granules used as a tissue-engineered grafting material for the treatment of infrabony defects in three female patients. Periosteum cell specimens were harvested from the mandible of each patient and cultured for six weeks until HCP sheets were formed. The periodontal surgery fully exposed the osseous defects and debridement and root planing were carried out. The coagulated PRP and HA mixture were placed into the osseous defects, and then they were covered with autologous HCP sheets. Six months post-surgery there were gains in clinical attachment and radiographic evidence of infrabony osseous fill. It may be concluded that HCP sheets combined with PRP and HA granules showed significant promise for treating human periodontal infrabony defects. A factor contributing to these favorable clinical results would be the presence of osteogenic cells in the HCP sheets, which provided greater regeneration potential. PMID: 19753798 [PubMed - indexed for MEDLINE] |
| Planar biaxial behavior of fibrin-based tissue-engineered heart valve leaflets.
December 24, 2009 at 8:49 am
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| | Planar biaxial behavior of fibrin-based tissue-engineered heart valve leaflets. Tissue Eng Part A. 2009 Oct;15(10):2763-72 Authors: Robinson PS, Tranquillo RT To design more effective tissue-engineered heart valve replacements, the replacement tissue may need to mimic the biaxial stress-strain behavior of native heart valve tissue. This study characterized the planar biaxial properties of tissue-engineered valve leaflets and native aortic valve leaflets. Fibrin-based valve equivalent (VE) and porcine aortic valve (PAV) leaflets were subjected to incremental biaxial stress relaxation testing, during which fiber alignments were measured, over a range of strain ratios. Results showed that VE leaflets exhibited a modulus and fiber reorientation behavior that correlated with strain ratio. In contrast, PAV leaflets maintained their relaxed modulus and fiber alignment when exposed to nonequibiaxial strain, but exhibited changes in stress relaxation. In uniaxial and equi-biaxial tension, there were few observed differences in relaxation behavior between VE and PAV leaflets, despite differences in the modulus and fiber reorientation. Likewise, in both tissues there was similar relaxation response in the circumferential and radial directions in biaxial tension, despite different moduli in these two directions. This study presents some fundamental differences in the mechanical response to biaxial tension of fibrin-based tissue-engineered constructs and native valve tissue. It also highlights the importance of using a range of strain ratios when generating mechanical property data for valvular and engineered tissues. The data presented on the stress-strain, relaxation, and fiber reorientation of VE tissue will be useful in future efforts to mathematically model and improve fibrin-based tissue-engineered constructs. PMID: 19368523 [PubMed - indexed for MEDLINE] |
| Electrostatic crosslinked in situ-forming in vivo scaffold for rat bone marrow mesenchymal stem cells.
December 24, 2009 at 8:49 am
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| | Electrostatic crosslinked in situ-forming in vivo scaffold for rat bone marrow mesenchymal stem cells. Tissue Eng Part A. 2009 Oct;15(10):3201-9 Authors: Kim KS, Lee JY, Kang YM, Kim ES, Lee B, Chun HJ, Kim JH, Min BH, Lee HB, Kim MS We herein formulated and characterized an in situ-forming gel consisting of sodium carboxymethylcellulose (CMC) and poly(ethyleneimine) (PEI) and examined its use as an in vivo scaffold for rat bone marrow stem cells (rBMSCs). The changes of zeta potential, size, and viscosity of CMC solutions with 0 to 30 wt% PEI confirmed the electrostatic interaction and temperature-dependence between anionic CMC and cationic PEI. The CMC/PEI solution produced an electrostatically crosslinked gel with a three-dimensional network structure. The CMC solution containing 10 wt% PEI transformed to a gel at temperatures greater than 35 degrees C and was chosen for subcutaneous injection into rats. The CMC/PEI (90/10) gel with pore structure acted as a suitable biocompatible substrate for the in vitro and in vivo attachment and proliferation of rBMSCs. As the CMC/PEI (90/10) solution with and without rBMSCs was injected into Fisher rats, it became a gel in the subcutaneous dorsum of the rats and maintained its shape even after 28 days in vivo. The injected rBMSCs survived in the CMC gel for 28 days. Injection of CMC/PEI gel alone induced macrophage accumulation in the host tissue and at the edge of the gel, whereas injection of CMC/PEI gel containing rBMSCs was associated with less macrophage accumulation, indicating immunosuppression by the transplanted rBMSCs. Our results collectively show that CMC/PEI gel could serve as an in situ-forming gel scaffold for entrapped rBMSCs in vivo. PMID: 19366343 [PubMed - indexed for MEDLINE] |
| Effect of polymer molecular weight on the bone biological activity of biodegradable polymer/calcium phosphate cement composites.
December 24, 2009 at 8:49 am
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| | Effect of polymer molecular weight on the bone biological activity of biodegradable polymer/calcium phosphate cement composites. Tissue Eng Part A. 2009 Oct;15(10):3183-91 Authors: Bodde EW, Habraken WJ, Mikos AG, Spauwen PH, Jansen JA Previous studies demonstrated that the addition of biodegradable polymer microparticles to calcium phosphate (CaP) cement improves the cement's degradative behavior without affecting its handling characteristics, especially its injectability and moldability. We investigated the influence of molecular weight of polymeric microparticles included in CaP cement on implant degradation and bone formation in critical-sized defects. Forty rats received cranial defects filled with formulations of CaP cement and poly(DL-lactic-co-glycolic acid) (PLGA) microparticles. Microparticles consisted of 100% high- (HMW) or low-molecular-weight (LMW) PLGA or mixtures of these (25%, 50%, or 75%). Implantation time was 12 weeks. Porosity measurements showed that the 100% HMW group was significantly less porous than the other groups. Histology and histomorphometry revealed significantly greater implant degradation in the 100% LMW group. Defect bridging was mainly seen in the 75% and 100% LMW groups, with the highest amount of bone in the 100% LMW formulation. These results suggest that LMW PLGA microparticles are associated with better bone formation than HMW PLGA, which is most likely explained by the greater degradation of LMW PLGA microparticles. In conclusion, CaP cement composites with high percentages of LMW PLGA microparticles show good bone transductive behavior, with complete defect bridging. The 100% LMW group turned out to be the best formulation. PMID: 19364281 [PubMed - indexed for MEDLINE] |
| Enhancement of embryonic stem cell differentiation promoted by avian chorioallantoic membranes.
December 24, 2009 at 8:49 am
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| | Enhancement of embryonic stem cell differentiation promoted by avian chorioallantoic membranes. Tissue Eng Part A. 2009 Oct;15(10):3193-200 Authors: Talavera-Adame D, Dafoe DC, Ng TT, Wachsmann-Hogiu S, Castillo-Henkel C, Farkas DL Avian chorioallantoic membrane (CAM) has been used as a model to explore angiogenesis and to study the microvasculature of transplanted tissues. Because CAM provides a vascular bed, cells can be implanted, and their development can be monitored and modified. We used the CAM model to study the differentiation process of embryoid bodies (EBs) derived from mouse embryonic stem cells (ESCs) influenced by the CAM vascular bed. After EBs were incubated in CAM for 5 days, they underwent further differentiation and became tissue masses (TMs) of different morphologies from those that grew outside CAM. Immunohistochemical analysis of TMs demonstrated tissue-specific markers such as neurofilament light, CD34, collagen IV, cardiac myosin heavy chain (MHC), and cardiotin. Differentiated mouse blood vessels stained with anti-CD31 were found within the TMs, as well as blood vessels stained positive for QH1 and QCPN, markers for quail endothelial cells and perinuclear quail antigen, respectively. Quail erythrocytes inside mouse blood vessels suggested a connection between existing quail vessels and blood vessels growing inside the TMs as a result of EB differentiation. Therefore, CAM could be a suitable model to trigger and study the differentiation of EBs in close interaction with surrogate quail blood vessels. PMID: 19364272 [PubMed - indexed for MEDLINE] |
| Engineered extracellular matrices modulate the expression profile and feeder properties of bone marrow-derived human multipotent mesenchymal stromal cells.
December 24, 2009 at 8:49 am
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| | Engineered extracellular matrices modulate the expression profile and feeder properties of bone marrow-derived human multipotent mesenchymal stromal cells. Tissue Eng Part A. 2009 Oct;15(10):3161-71 Authors: Seib FP, Müller K, Franke M, Grimmer M, Bornhäuser M, Werner C The bone marrow harbors multipotent mesenchymal stromal cells (MSCs) that nurture hematopoietic stem cells (HSCs). The extracellular matrix (ECM) is an integral part of the bone marrow, and the aim of this study was therefore to examine the effect of engineered ECM substrates on MSC gene expression over time and to determine quantitatively the functional ability of ECM-cultured MSCs to support HSCs. ECMs were surface immobilized using thin films of maleic anhydride to covalently immobilize tropocollagen or fibrillar collagen type I to the substrate. Where indicated, collagen type I fibrils were supplemented with heparin or hyaluronic acid. All surfaces maintained MSC viability and supported cell expansion. Microarray analysis of MSCs cultured on engineered ECM substrates revealed that culture time, as well as substrate composition, significantly affected expression levels. Based on these studies, it was possible to predict the effect of these substrates on in vitro HSC clonogenicity and self-renewal. The ability to regulate the expression of stromal factors using engineered ECM is exciting and warrants further studies to identify the ECM components and combinations that maximize the expansion of clonogenic HSCs. PMID: 19358630 [PubMed - indexed for MEDLINE] |
| CD133+ endothelial progenitor cells as a potential cell source for a bioartificial glomerulus.
December 24, 2009 at 8:49 am
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| | CD133+ endothelial progenitor cells as a potential cell source for a bioartificial glomerulus. Tissue Eng Part A. 2009 Oct;15(10):3173-82 Authors: Vu DM, Masuda H, Yokoyama TA, Fujimura S, Kobori M, Ito R, Sawada K, Saito A, Asahara T Development of a bioartificial glomerulus, a hemofilter in which the inner surface of hollow fibers is endothelialized, requires expandable, nonimmunogenic, antithrombogenic, and highly permeable endothelial cells. We used human umbilical cord blood CD133(+) endothelial progenitor cells (EPCs) to evaluate the feasibility of application of EPCs for bioartificial glomerulus. Numbers of adhered CD133(+) EPCs (adhered EPCs) was approximately 25 to 30 times as great in the expansion culture group as in the non-expansion group. Adhered EPCs had endothelial cell features, including the expression of CD31, Kinase domain region, von Willebrand factor, vascular endothelial-cadherin, positive for Ulex europeus agglutinin I staining, and up-take of acetylated low-density lipoprotein. Adhered EPCs secreted 6-keto-prostaglandin F(1alpha) identically to that secreted by human umbilical vein endothelial cells (HUVECs). The cells also expressed messenger RNA for phospholipase A(2), cyclooxygenase (COX)-1, COX-2, prostaglandin I(2) synthase, tissue plasminogen activator, and thrombomodulin (TM). TM protein in adhered EPCs properly activated protein C. Scanning electron microscopy revealed the suppression of platelet adhesion and aggregation on the surface of cell monolayer. Adhered EPCs treated with 50 microg/mL of cytochalasin B induced a larger diameter and a greater number of fenestrae, subsequently producing significantly more ultrafiltration than the non-treated cell. These results suggest that CD133(+) EPCs would potentially be applicable in bioartificial glomerulus. PMID: 19358628 [PubMed - indexed for MEDLINE] |
| Amniotic fluid stem cells produce robust mineral deposits on biodegradable scaffolds.
December 24, 2009 at 8:49 am
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| | Amniotic fluid stem cells produce robust mineral deposits on biodegradable scaffolds. Tissue Eng Part A. 2009 Oct;15(10):3129-38 Authors: Peister A, Deutsch ER, Kolambkar Y, Hutmacher DW, Guldberg RE Insufficient availability of osteogenic cells limits bone regeneration through cell-based therapies. This study investigated the potential of amniotic fluid-derived stem (AFS) cells to synthesize mineralized extracellular matrix within porous medical-grade poly-varepsilon-caprolactone (mPCL) scaffolds. The AFS cells were initially differentiated in two-dimensional (2D) culture to determine appropriate osteogenic culture conditions and verify physiologic mineral production by the AFS cells. The AFS cells were then cultured on 3D mPCL scaffolds (6-mm diameter x 9-mm height) and analyzed for their ability to differentiate to osteoblastic cells in this environment. The amount and distribution of mineralized matrix production was quantified throughout the mPCL scaffold using nondestructive micro computed tomography (microCT) analysis and confirmed through biochemical assays. Sterile microCT scanning provided longitudinal analysis of long-term cultured mPCL constructs to determine the rate and distribution of mineral matrix within the scaffolds. The AFS cells deposited mineralized matrix throughout the mPCL scaffolds and remained viable after 15 weeks of 3D culture. The effect of pre-differentiation of the AFS cells on the subsequent bone formation in vivo was determined in a rat subcutaneous model. Cells that were pre-differentiated for 28 days in vitro produced seven times more mineralized matrix when implanted subcutaneously in vivo. This study demonstrated the potential of AFS cells to produce 3D mineralized bioengineered constructs in vitro and in vivo and suggests that AFS cells may be an effective cell source for functional repair of large bone defects. PMID: 19344289 [PubMed - indexed for MEDLINE] |
| Preadipocytes stimulate ductal morphogenesis and functional differentiation of human mammary epithelial cells on 3D silk scaffolds.
December 24, 2009 at 8:49 am
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| | Preadipocytes stimulate ductal morphogenesis and functional differentiation of human mammary epithelial cells on 3D silk scaffolds. Tissue Eng Part A. 2009 Oct;15(10):3087-98 Authors: Wang X, Zhang X, Sun L, Subramanian B, Maffini MV, Soto A, Sonnenschein C, Kaplan DL Epithelial-mesenchymal interactions play an important role in regulating normal tissue development as well as tumor development for the mammary gland, but much is yet to uncover to reach a full understanding of their complexity. To address this issue, the establishment of relevant, surrogate, three-dimensional (3D) human tissue culture models is essential. In the present study, a novel 3D coculture system was developed to study the interactions between human mammary epithelial cells (MCF10A) and adipocytes, a prominent stromal cell type in native breast tissue. The MCF10A cells were cultured within a mixture of Matrigel and collagen in 3D porous silk scaffolds with or without predifferentiated human adipose-derived stem cells (hASCs). The presence of hASCs inhibited MCF10A cell proliferation, induced both alveolar and ductal morphogenesis, and enhanced their functional differentiation as evidenced by histology and functional analysis. The alveolar structures formed by cocultures exhibited proper, immature polarity when compared with native breast tissue. In contrast, only alveolar structures with reverted polarity were observed in the MCF10A monocultures. The effect of ductal morphogenesis in cocultures may correlate to hepatocyte growth factor secreted by the predifferentiated hASCs, based on results from a cytokine blocking assay. Taken together, this in vitro coculture model on silk scaffolds effectively reconstitutes a physiologically relevant 3D microenvironment for epithelial cells and stromal cells and provides a useful system to study tissue organization and epithelial morphogenesis in normal or diseased breast development. PMID: 19338449 [PubMed - indexed for MEDLINE] |
| Surface-phosphorylated copolymer promotes direct bone bonding.
December 24, 2009 at 8:49 am
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| | Surface-phosphorylated copolymer promotes direct bone bonding. Tissue Eng Part A. 2009 Oct;15(10):3061-9 Authors: Gopalakrishnanchettiyar SS, Mohanty M, Kumary TV, Valappil MP, Parameshwaran R, Varma HK The bone bonding potential of surface-phosphorylated poly (2-hydroxyethyl methacrylate-co-methyl methacrylate) [poly (HEMA-co-MMA)] has been investigated and compared with commercially available poly (methyl methacrylate) bone cement (CMW1 radiopaque, Depuy; Johnson & Johnson, Blackpool, Lancashire, England, United Kingdom) as control. Poly (HEMA-co-MMA) is synthesized by free radical-initiated copolymerization and surface functionalized by phosphorylation. The X-ray photoelectron spectroscopy confirms the presence of surface-bound phosphate groups on poly (HEMA-co-MMA). The surface-phosphorylated poly (HEMA-co-MMA) promotes in vitro biomineralization, cell viability, cell adhesion, and expression of bone-specific markers such as osteocalcin and alkaline phosphatase. The bone implantation study performed in rabbits as per ISO 10993-6; 1994 (E) shows that surface-phosphorylated poly (HEMA-co-MMA) elicits bone bonding and new bone formation. New woven bone trabeculae are formed at the defect site of surface-phosphorylated poly (HEMA-co-MMA) within 1 week, while for control sample, inflammatory cells--predominantly, macrophages, fibroblasts, and fibrocytes--are present at the cortical margins around the defect. The 4 and 12 weeks postimplantation results show that the major part of the defects around the surface-phosphorylated poly (HEMA-co-MMA) implant is bridged with new woven bone, with significant remodeling (evident from resorption bays) along both the margins of the defect, but for control implants, the defects are only partially closed, with slight remodeling along the margins, but most of them are separated by fibrous tissue. PMID: 19338435 [PubMed - indexed for MEDLINE] |
| Cell-induced alignment augments twitch force in fibrin gel-based engineered myocardium via gap junction modification.
December 24, 2009 at 8:49 am
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| | Cell-induced alignment augments twitch force in fibrin gel-based engineered myocardium via gap junction modification. Tissue Eng Part A. 2009 Oct;15(10):3099-108 Authors: Black LD, Meyers JD, Weinbaum JS, Shvelidze YA, Tranquillo RT A high-potential therapy for repairing the heart post-myocardial infarction is the implantation of tissue-engineered myocardium. While several groups have developed constructs that mimic the aligned structure of the native myocardium, to date no one has investigated the particular functional benefits conferred by alignment. In this study we created myocardial constructs in both aligned and isotropic configurations by entrapping neonatal rat cardiac cells in fibrin gel. Constructs were cultured statically for 2 weeks, and then characterized. Histological staining showed spread cells that express typical cardiac cell markers in both configurations. Isotropic constructs had higher final cell and collagen densities, but lower passive mechanical properties than aligned constructs. Twitch force associated with electrical pacing, however, was 181% higher in aligned constructs, and this improvement was greater than what would be expected from merely aligning the cells in the isotropic constructs in the force measurement direction. Our hypothesis was that this was due to improved gap junction formation/function facilitated by cell alignment, and further analyses of the twitch force data, as well as Western blot results of connexin 43 expression and phosphorylation state, support this hypothesis. Regardless of the specific mechanism, the results presented in this study underscore the importance of recapitulating the anisotropy of the native tissue in engineered myocardium. PMID: 19338433 [PubMed - indexed for MEDLINE] |
| Neurotrophic factor-expressing mesenchymal stem cells survive transplantation into the contused spinal cord without differentiating into neural cells.
December 24, 2009 at 8:49 am
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| | Neurotrophic factor-expressing mesenchymal stem cells survive transplantation into the contused spinal cord without differentiating into neural cells. Tissue Eng Part A. 2009 Oct;15(10):3049-59 Authors: Rooney GE, McMahon SS, Ritter T, Garcia Y, Moran C, Madigan NN, Flügel A, Dockery P, O'Brien T, Howard L, Windebank AJ, Barry FP The aim of this study was to assess the feasibility of transplanting mesenchymal stem cells (MSCs), genetically modified to express glial-derived neurotrophic factor (GDNF), to the contused rat spinal cord, and to subsequently assess their neural differentiation potential. MSCs expressing green fluorescent protein were transduced with a retroviral vector to express the neurotrophin GDNF. The transduction protocol was optimized by using green fluorescent protein-expressing retroviral constructs; approximately 90% of MSCs were transduced successfully after G418 selection. GDNF-transduced MSCs expressed the transgene and secreted growth factor into the media (approximately 12 ng/500,000 cells secreted into the supernatant 2 weeks after transduction). Injuries were established using an impactor device, which applied a given, reproducible force to the exposed spinal cord. GDNF-expressing MSCs were transplanted rostral and caudal to the site of injury. Spinal cord sections were analyzed 2 and 6 weeks after transplantation. We demonstrate that GDNF-transduced MSCs engraft, survive, and express the therapeutic gene up to 6 weeks posttransplantation, while maintaining an undifferentiated phenotype. In conclusion, transplanted MSCs have limited capacity for the replacement of neural cells lost as a result of a spinal cord trauma. However, they provide excellent opportunities for local delivery of neurotrophic factors into the injured tissue. This study underlines the therapeutic benefits associated with cell transplantation and provides a good example of the use of MSCs for gene delivery. PMID: 19335061 [PubMed - indexed for MEDLINE] |
| Medium osmolarity and pericellular matrix development improves chondrocyte survival when photoencapsulated in poly(ethylene glycol) hydrogels at low densities.
December 24, 2009 at 8:49 am
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| | Medium osmolarity and pericellular matrix development improves chondrocyte survival when photoencapsulated in poly(ethylene glycol) hydrogels at low densities. Tissue Eng Part A. 2009 Oct;15(10):3037-48 Authors: Villanueva I, Bishop NL, Bryant SJ The ability to encapsulate cells over a range of cell densities is important toward mimicking cell densities of native tissues and rationally designing strategies where cell source and/or cell numbers are clinically limited. Our preliminary findings demonstrate that survival of freshly isolated adult bovine chondrocytes dramatically decreases when photoencapsulated in poly(ethylene glycol) hydrogels at low densities (4 million cells/mL). During enzymatic digestion of cartilage, chondrocytes undergo a harsh change in their microenvironment. We hypothesize that the absence of exogenous antioxidants, the hyposmotic environment, and the loss of a protective pericellular matrix (PCM) increase chondrocytes' susceptibility to free radical damage during photoencapsulation. Incorporation of antioxidants and serum into the encapsulation medium improved cell survival twofold compared to phosphate-buffered saline. Increasing medium osmolarity from 330 to 400 mOsm (physiological) improved cell survival by 40% and resulted in approximately 2-fold increase in adenosine triphosphate (ATP) production 24 h postencapsulation. However, cell survival was only temporary. Allowing cells to reproduce some PCM before photoencapsulation in 400 mOsm medium resulted in superior cell survival during and postencapsulation for up to 15 days. In summary, the combination of antioxidants, physiological osmolarity, and the development of some PCM result in an improved robustness against free radical damage during photoencapsulation. PMID: 19331581 [PubMed - indexed for MEDLINE] |
| Engineered polyelectrolyte multilayer substrates for adhesion, proliferation, and differentiation of human mesenchymal stem cells.
December 24, 2009 at 8:49 am
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| | Engineered polyelectrolyte multilayer substrates for adhesion, proliferation, and differentiation of human mesenchymal stem cells. Tissue Eng Part A. 2009 Oct;15(10):2977-90 Authors: Semenov OV, Malek A, Bittermann AG, Vörös J, Zisch AH Polyelectrolyte multilayer coatings have emerged as substrates to control cellular behavior, but interactions with human multipotent mesenchymal stromal cells (MSCs) have not been studied. We looked at layer-by-layer coatings of cationic poly-L-lysine (PLL) and anionic hyaluronic acid (HA) as substrates for MSCs of placenta and adipose tissue. This system allows for modulation of thickness (number of deposition cycles), stiffness (chemical cross-linking of bulk layer), and adhesiveness (fibronectin (FN) interface). Native, as-built PLL/HA multilayer coatings were poorly adhesive for MSCs despite spectroscopy-confirmed high surface density of pre-adsorbed FN. Stratification of cross-linked PLL/HA multilayers of different stiffnesses revealed that multilayers modified with a high cross-linking regimen became efficient substrates for MSC adhesion and proliferation. MSCs on cross-linked multilayers grew to confluence. Using comparative confocal microscopy analysis of PLL/HA multilayers with physically adsorbed versus chemically coupled FN, we demonstrated that cross-linking strongly influenced FN surface distribution, leading to denser presentation of adhesion sites for cells. The covalent affixation of FN promoted focal adhesion formation and was critical to maintaining densely grown MSC cultures over weeks for their differentiation. Multilayer-bound MSCs were capable of differentiating into osteocytes and chondrocytes upon culture with induction factors. Together, cross-linked, FN-terminated PLL/HA multilayers provide a versatile platform for studies of human MSCs for biotechnological or therapeutic applications. PMID: 19320572 [PubMed - indexed for MEDLINE] |
| In vivo remodeling and structural characterization of fibrin-based tissue-engineered heart valves in the adult sheep model.
December 24, 2009 at 8:49 am
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| | In vivo remodeling and structural characterization of fibrin-based tissue-engineered heart valves in the adult sheep model. Tissue Eng Part A. 2009 Oct;15(10):2965-76 Authors: Flanagan TC, Sachweh JS, Frese J, Schnöring H, Gronloh N, Koch S, Tolba RH, Schmitz-Rode T, Jockenhoevel S Autologous fibrin-based tissue-engineered heart valves have demonstrated excellent potential as patient-derived valve replacements. The present pilot study aims to evaluate the structure and mechanical durability of fibrin-based heart valves after implantation in a large-animal model (sheep). Tissue-engineered heart valves were molded using a fibrin scaffold and autologous arterial-derived cells before 28 days of mechanical conditioning. Conditioned valves were subsequently implanted in the pulmonary trunk of the same animals from which the cells were harvested. After 3 months in vivo, explanted valve conduits (n = 4) had remained intact and exhibited native tissue consistency, although leaflets demonstrated insufficiency because of tissue contraction. Routine histology showed remarkable tissue development and cell distribution, along with functional blood vessel ingrowth. A confluent monolayer of endothelial cells was present on the valve surface, as evidenced by scanning electron microscopy and positive von Willebrand factor staining. Immunohistochemistry and extracellular matrix (ECM) assay demonstrated complete resorption of the fibrin scaffold and replacement with ECM proteins. Transmission electron microscopy revealed mature collagen formation and viable, active resident tissue cells. The preliminary findings of implanted fibrin-based tissue-engineered heart valves are encouraging, with excellent tissue remodeling and structural durability after 3 months in vivo. The results from this pilot study highlight the potential for construction of completely "autologous" customized tissue-engineered heart valves based on a patient-derived fibrin scaffold. PMID: 19320544 [PubMed - indexed for MEDLINE] |
| The effect of hydrostatic pressure on three-dimensional chondroinduction of human adipose-derived stem cells.
December 24, 2009 at 8:49 am
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| | The effect of hydrostatic pressure on three-dimensional chondroinduction of human adipose-derived stem cells. Tissue Eng Part A. 2009 Oct;15(10):2937-45 Authors: Ogawa R, Mizuno S, Murphy GF, Orgill DP BACKGROUND: The optimal production of three-dimensional cartilage in vitro requires both inductive factors and specified culture conditions (e.g., hydrostatic pressure [HP], gas concentration, and nutrient supply) to promote cell viability and maintain phenotype. In this study, we optimized the conditions for human cartilage induction using human adipose-derived stem cells (ASCs), collagen scaffolds, and cyclic HP treatment. METHODS: Human ASCs underwent primary culture and three passages before being seeded into collagen scaffolds. These constructs were incubated for 1 week in an automated bioreactor using cyclic HP at 0-0.5 MPa, 0.5 Hz, and compared to constructs exposed to atmospheric pressure. In both groups, chondrogenic differentiation medium including transforming growth factor-beta1 was employed. One, 2, 3, and 4 weeks after incubation, the cell constructs were harvested for histological, immunohistochemical, and gene expression evaluation. RESULTS: In histological and immunohistochemical analyzes, pericellular and extracellular metachromatic matrix was observed in both groups and increased over 4 weeks, but accumulated at a higher rate in the HP group. Cell number was maintained in the HP group over 4 weeks but decreased after 2 weeks in the atmospheric pressure group. Chondrogenic-specific gene expression of type II and X collagen, aggrecan, and SRY-box9 was increased in the HP group especially after 2 weeks. CONCLUSION: Our results demonstrate chondrogenic differentiation of ASCs in a three-dimensional collagen scaffolds with treatment of a cyclic HP. Cyclic HP was effective in enhancing accumulation of extracellular matrix and expression of genes indicative of chondrogenic differentiation. PMID: 19290804 [PubMed - indexed for MEDLINE] |
| Human Vitronectin-Derived Peptide Covalently Grafted onto Titanium Surface Improves Osteogenic Activity: A Pilot In Vivo Study on Rabbits.
December 24, 2009 at 8:49 am
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| | Human Vitronectin-Derived Peptide Covalently Grafted onto Titanium Surface Improves Osteogenic Activity: A Pilot In Vivo Study on Rabbits. Tissue Eng Part A. 2009 Oct;15(10):2917-26 Authors: Cacchioli A, Ravanetti F, Bagno A, Dettin M, Gabbi C Peptide and protein exploitation for the biochemical functionalization of biomaterial surfaces allowed fabricating biomimetic devices able to evoke and promote specific and advantageous cell functions in vitro and in vivo. In particular, cell adhesion improvement to support the osseointegration of implantable devices has been thoroughly investigated. This study was aimed at checking the biological activity of the (351-359) human vitronectin precursor (HVP) sequence, mapped on the human vitronectin protein; the peptide was covalently linked to the surface of titanium cylinders, surgically inserted in the femurs of New Zealand white rabbits and analyzed at short experimental time points (4, 9, and 16 days after surgery). To assess the osteogenic activity of the peptide, three vital fluorochromic bone markers were used (calcein green, xylenol orange, and calcein blue) to stain the areas of newly grown bone. Static and dynamic histomorphometric parameters were measured at the bone-implant interface and at different distances from the surface. The biological role of the (351-359)HVP sequence was checked by comparing peptide-grafted samples and controls, analyzing how and how much its effects change with time across the bone regions surrounding the implant surface. The results obtained reveal a major activity of the investigated peptide 4 days after surgery, within the bone region closest to the implant surface, and larger bone to implant contact 9 and 16 days after surgery. Thus, improved primary fixation of endosseous devices can be foreseen, resulting in an increased osteointegration. PMID: 19290802 [PubMed - indexed for MEDLINE] |
| The effectiveness of a novel cartridge-based bioreactor design in supporting liver cells.
December 24, 2009 at 8:49 am
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| | The effectiveness of a novel cartridge-based bioreactor design in supporting liver cells. Tissue Eng Part A. 2009 Oct;15(10):2903-16 Authors: Niu M, Hammond P, Coger RN There are a number of applications--ranging from temporary strategies for organ failure to pharmaceutical testing--that rely on effective bioreactor designs. The significance of these devices is that they provide an environment for maintaining cells in a way that allows them to perform key cellular and tissue functions. In the current study, a novel cartridge-based bioreactor was developed and evaluated. Its unique features include its capacity for cell support and the adaptable design of its cellular space. Specifically, it is able to accommodate functional and reasonably sized tissue (>2.0 x 10(8) cells), and can be easily modified to support a range of anchorage-dependent cells. To evaluate its efficacy, it was applied to liver support in the current study. This involved evaluating the performance of rat primary hepatocytes within the unique cartridges in culture--sans bioreactor--and after being loaded within the novel bioreactor. Compared to collagen sandwich culture functional controls, hepatocytes within the unique cartridge design demonstrated significantly higher albumin production and urea secretion rates when cultured under dynamic flow conditions--reaching peak values of 170 +/- 22 microg/10(6) cells/day and 195 +/- 18 microg/10(6) cells/day, respectively. The bioreactor's effectiveness in supporting live and functioning primary hepatocytes is also presented. Cell viability at the end of 15 days of culture in the new bioreactor was 84 +/- 18%, suggesting that the new design is effective in maintaining primary hepatocytes for at least 2 weeks in culture. Liver-specific functions of urea secretion, albumin synthesis, and cytochrome P450 activity were also assessed. The results indicate that hepatocytes are able to achieve good functional performance when cultured within the novel bioreactor. This is especially true in the case of cytochrome P450 activity, where by day 15 of culture, hepatocytes within the bioreactor reached values that were 56.6% higher than achieved by the collagen sandwich functional control cultures. The success of the novel cartridge-based bioreactor in supporting hepatocytes with good viability and functional performance suggests that it is an effective design for supporting anchorage-dependent cells. PMID: 19271993 [PubMed - indexed for MEDLINE] |
| Polyphenol-stabilized tubular elastin scaffolds for tissue engineered vascular grafts.
December 24, 2009 at 8:49 am
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| | Polyphenol-stabilized tubular elastin scaffolds for tissue engineered vascular grafts. Tissue Eng Part A. 2009 Oct;15(10):2837-51 Authors: Chuang TH, Stabler C, Simionescu A, Simionescu DT Tissue-engineered vascular grafts require elastic, acellular porous scaffolds with controlled biodegradability and properties matching those of natural arteries. Elastin would be a desirable component for such applications, but elastin does not easily regenerate experimentally. Our approach is to develop tubular elastin scaffolds using decellularization and removal of collagen from porcine carotid arteries ( approximately 5 mm diameter) using alkaline extraction. Because elastin is susceptible to rapid degeneration after implantation, scaffolds were further treated with penta-galloyl glucose (PGG), an established polyphenolic elastin-stabilizing agent. Scaffolds were compared in vitro with detergent-decellularized arteries for structure, composition, resistance to degradation, mechanical properties, and cytotoxicity and in vivo for cell infiltration and remodeling potential. Results showed effective decellularization and almost complete collagen removal by alkaline extraction. PGG-treated elastin scaffolds proved to be resistant to elastase digestion in vitro, maintained their cylindrical shapes, showed high resistance to burst pressures, and supported growth of endothelial cells and fibroblasts. In vivo results showed that PGG treatment reduced the rate of elastin biodegradation and controlled cell infiltration but did not hamper new collagen and proteoglycan deposition and secretion of matrix-degrading proteases. Alkali-purified, PGG-treated tubular arterial elastin scaffolds exhibit many desirable properties to be recommended for clinical applications as vascular grafts. PMID: 19254115 [PubMed - indexed for MEDLINE] |
| ERK1/2 activation induced by inflammatory cytokines compromises effective host tissue integration of engineered cartilage.
December 24, 2009 at 8:49 am
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| | ERK1/2 activation induced by inflammatory cytokines compromises effective host tissue integration of engineered cartilage. Tissue Eng Part A. 2009 Oct;15(10):2825-35 Authors: Djouad F, Rackwitz L, Song Y, Janjanin S, Tuan RS OBJECTIVE: Proinflammatory cytokines are known to provoke degradative signaling cascades that promote extracellular matrix disintegration in articular cartilage. Because integration of the repair tissue into the surrounding native cartilage to produce a mechanically stable interface has a profound impact on the viability and functionality of the restored joint surface, this study examined the effects of proinflammatory cytokines on the properties of tissue-engineered cartilage in the context of integration. METHODS: Using an established in vitro cartilage defect model, we examined the integration of chondrocyte-laden agarose constructs into native articular cartilage and the biochemical and biomechanical alterations of these implants upon treatment with interleukin 1-beta (IL1-beta) and tumor necrosis factor-alpha (TNF-alpha). Additionally, we probed extracellular regulated kinase (ERK) signaling involvement in response to proinflammatory cytokines. RESULTS: The time-dependent accumulation of extracellular matrix and concomitant increase in Young's modulus observed in the absence of cytokines was significantly decreased upon IL1-beta and TNF-alpha treatment. Push-out test showed the highest interface strength in hybrid cultures maintained without cytokines, which was significantly lowered with IL1-beta and TNF-alpha treatment. Histological characteristics of the interface region are consistent with the biochemical findings. Treatment with an inhibitor of ERK pathway antagonized the deleterious effects caused by both cytokines. CONCLUSION: This study is the first to show the functional catastrophic effects of IL1-beta and TNF-alpha on the biochemical, structural, and integrative properties of tissue-engineered cartilage and their significant counteraction by the blockade of ERK signaling pathway. With the discovery of new potential chemical entities, ERK inhibitor may emerge as a new therapeutic approach for functional integration and mechanical integrity of an engineered cartilage to the host tissue and, therefore, enhance long-term viability and functionality of the restored joint surface. PMID: 19243242 [PubMed - indexed for MEDLINE] |
| Dynamic compression stimulates proteoglycan synthesis by mesenchymal stem cells in the absence of chondrogenic cytokines.
December 24, 2009 at 8:49 am
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| | Dynamic compression stimulates proteoglycan synthesis by mesenchymal stem cells in the absence of chondrogenic cytokines. Tissue Eng Part A. 2009 Oct;15(10):2817-24 Authors: Kisiday JD, Frisbie DD, McIlwraith CW, Grodzinsky AJ The objective of this study was to evaluate the effect of dynamic compression on mesenchymal stem cell (MSC) chondrogenesis. Dynamic compression was applied to agarose hydrogels seeded with bone marrow-derived adult equine MSCs. In the absence of the chondrogenic cytokine transforming growth factor beta (TGFbeta), dynamic compression applied for 12 h per day led to significantly greater proteoglycan synthesis than in unloaded TGFbeta-free cultures, although at a rate that was approximately 20% to 35% of unloaded TGFbeta cultures. These data suggest that the emergence of aggrecan dominated a chondrogenic response to loading as increases in proteoglycan synthesis. Cross-sectional analyses were conducted to subjectively identify potential spatial distributions of heterogeneous differentiation. In loaded samples, cell viability and metachromatic staining was low near the porous compression platen interface but increased with depth, reaching levels in the lower portion of the hydrogel that resembled unloaded TGFbeta cultures. These results suggest that the combination of high hydrostatic pressure and low dynamic strain and fluid flow had a stronger effect on chondrogenesis than did low hydrostatic pressure coupled with high dynamic strain and fluid flow. Next, the 12-h per day loading protocol was applied in the presence of TGFbeta. Biosynthesis in loaded cultures was less than in unloaded TGFbeta samples. Taken together, these data suggest that the duration of loading necessary to stimulate mechanoinduction of MSCs may not be optimal for neo-tissue accumulation in the presence of chondrogenic cytokines. PMID: 19243241 [PubMed - indexed for MEDLINE] |
| Collagen I-matrigel scaffolds for enhanced Schwann cell survival and control of three-dimensional cell morphology.
December 24, 2009 at 8:49 am
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| | Collagen I-matrigel scaffolds for enhanced Schwann cell survival and control of three-dimensional cell morphology. Tissue Eng Part A. 2009 Oct;15(10):2785-93 Authors: Dewitt DD, Kaszuba SN, Thompson DM, Stegemann JP We report on the ability to control three-dimensional Schwann cell (SC) morphology using collagen I-Matrigel composite scaffolds for neural engineering applications. SCs are supportive of nerve regeneration after injury, and it has recently been reported that SCs embedded in collagen I, a material frequently used in guidance channel studies, do not readily extend processes, instead adopting a spherical morphology indicative of little interaction with the matrix. We have modified collagen I matrices by adding Matrigel to make them more supportive of SCs and characterized these matrices and SC morphology in vitro. Incorporation of 10%, 20%, 35%, and 50% Matrigel by volume resulted in 2.4, 3.5, 3.7, and 4.2 times longer average SC process length after 14 days in culture than with collagen I-only controls. Additionally, only 35% and 50% Matrigel constructs were able to maintain SC number over 14 days, whereas an 88% decrease in cells from initial seeding density was observed in collagen-only constructs over the same time period. Mechanical testing revealed that the addition of 50% Matrigel increased matrix stiffness from 6.4 kPa in collagen I-only constructs to 9.8 kPa. Furthermore, second harmonic generation imaging showed that the addition of Matrigel resulted in non-uniform distribution of collagen I, and scanning electron microscope imaging illustrated distinct differences in the fibrillar structure of the different constructs. Collectively, this work lays a foundation for developing scaffolding materials that are concurrently supportive of neurons and SCs for future neural engineering applications. PMID: 19231925 [PubMed - indexed for MEDLINE] |
| Requirement of IFN-gamma-mediated indoleamine 2,3-dioxygenase expression in the modulation of lymphocyte proliferation by human adipose-derived stem cells.
December 24, 2009 at 8:49 am
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| | Requirement of IFN-gamma-mediated indoleamine 2,3-dioxygenase expression in the modulation of lymphocyte proliferation by human adipose-derived stem cells. Tissue Eng Part A. 2009 Oct;15(10):2795-806 Authors: DelaRosa O, Lombardo E, Beraza A, Mancheño-Corvo P, Ramirez C, Menta R, Rico L, Camarillo E, García L, Abad JL, Trigueros C, Delgado M, Büscher D Human adipose-derived mesenchymal stem cells (hASCs) are mesenchymal stem cells (MSCs) with reduced immunogenicity and capability to modulate immune responses. Whereas the immunosuppressive activity of bone marrow-MSCs has received considerable attention during the last few years, the specific mechanisms underlying hASC-mediated immunosuppression have been poorly studied. Recent studies comparing both cell types have reported differences at transcriptional and proteomic levels, suggesting that hASCs and bone marrow-MSCs, while having similarities, are quite different. This suggests that different mechanisms of immunosuppression may apply. Here, we report that hASCs inhibit peripheral blood mononuclear cells (PBMCs), and CD4(+) and CD8(+) T cell proliferation in both cell-cell contact and transwell conditions, which is accompanied by a reduction of proinflammatory cytokines. We demonstrate that hASCs do not constitutively express immunomodulatory factors. Conditioned supernatants from hASCs stimulated by IFN-gamma, PBMCs, or activated PBMCs highly inhibited PBMC proliferation, indicating that inhibitory factors are released upon hASC activation. Many factors have been involved in MSC-mediated immunosuppression, including IFN-gamma, IL-10, hepatocyte growth factor, prostaglandin E2, transforming growth factor-beta1, indoleamine 2,3-dioxygenase (IDO), nitric oxide, and IL-10. Using pharmacological inhibitors, neutralizing antibodies, and genetically modified hASCs that constitutively express or silence IDO enzyme, we demonstrate that, in the case of hASCs, the IFN-gamma/IDO axis is essential. Taken together, our data support the key role of IDO in the therapeutic use of hASC on immunomediated diseases. PMID: 19231921 [PubMed - indexed for MEDLINE] |
| Three-dimensional in vitro effects of compression and time in culture on aggregate modulus and on gene expression and protein content of collagen type II in murine chondrocytes.
December 24, 2009 at 8:49 am
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| | Three-dimensional in vitro effects of compression and time in culture on aggregate modulus and on gene expression and protein content of collagen type II in murine chondrocytes. Tissue Eng Part A. 2009 Oct;15(10):2807-16 Authors: Chokalingam K, Hunter S, Gooch C, Frede C, Florer J, Wenstrup R, Butler D The objectives of this study were to determine how culture time and dynamic compression, applied to murine chondrocyte-agarose constructs, influence construct stiffness, expression of col2 and type II collagen. Chondrocytes were harvested from the ribs of six newborn double transgenic mice carrying transgenes that use enhanced cyan fluorescent protein (ECFP) and green fluorescent protein (GFP-T) as reporters for expression from the col2a1 and col1a1 promoters, respectively. Sixty-three constructs (8 mm diameter x 3 mm thick) per animal were created by seeding chondrocytes (10 x 10(6) per mL) in agarose gel (2% w/v). Twenty-eight constructs from each animal were stimulated for 7, 14, 21, or 28 days in a custom bioreactor housed in an electromagnetic system. Twenty-eight constructs exposed to identical culture conditions but without mechanical stimulation served as nonstimulated controls for 7, 14, 21, and 28 days. The remaining seven constructs served as day 0 controls. Fluorescing cells with rounded morphology were present in all constructs at all five time points. Seven, 14, 21, and 28 days of stimulation significantly increased col2 expression according to ECFP fluorescence and messenger RNA expression according to quantitative reverse transcriptase polymerase chain reaction. Col2 gene expression in stimulated and nonstimulated constructs showed initial increases up to day 14 and then showed decreases by day 28. Stimulation significantly increased type II collagen content at 21 and 28 days and aggregate modulus only at 28 days. There was a significant increase in aggregate modulus in stimulated constructs between day 0 and 7 and between day 21 and day 28. This study reveals that compressive mechanical stimulation is a potent stimulator of col2 gene expression that leads to measurable but delayed increases in protein (type II collagen) and then biomechanical stiffness. Future studies will examine the effects of components of the mechanical signal in culture and address the question of whether such in vitro improvements in tissue-engineered constructs enhance repair outcomes after surgery. PMID: 19231914 [PubMed - indexed for MEDLINE] |
| Effects of flow shear stress and mass transport on the construction of a large-scale tissue-engineered bone in a perfusion bioreactor.
December 24, 2009 at 8:49 am
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| | Effects of flow shear stress and mass transport on the construction of a large-scale tissue-engineered bone in a perfusion bioreactor. Tissue Eng Part A. 2009 Oct;15(10):2773-83 Authors: Li D, Tang T, Lu J, Dai K Currently, a tissue-engineered bone is usually constructed using a perfusion bioreactor in vitro. In the perfusion culture, fluid flow can exert shear stress on the cells seeded on scaffold, improving the mass transport of the cells. This experiment studied the effects of flow shear stress and mass transport, respectively, on the construction of a large-scale tissue-engineered bone using the critical-sized beta-tricalcium phosphate scaffold seeded with human bone marrow-derived mesenchymal stem cells (hBMSCs). This was done by changing flow rate and adding dextran into the media, thus changing the media's viscosity. The cells were seeded onto the scaffolds and were cultured in a perfusion bioreactor for up to 28 days with different fluid flow shear stress or different mass transport. When the mass transport was 3 mL/min, the flow shear stress was, respectively, 0.005 Pa (0.004-0.007 Pa), 0.011 Pa (0.009-0.013 Pa), or 0.015 Pa (0.013-0.018 Pa) in different experiment group obtained by simulation and calculation using fluid dynamics. When the flow shear stress was 0.015 Pa (0.013-0.018 Pa), the mass transport was, respectively, 3, 6, or 9 mL/min. After 28 days of culture, the construction of the tissue-engineered bone was assessed by osteogenic differentiation of hBMSCs and histological assay of the constructs. Extracellular matrix (ECM) was distributed throughout the entire scaffold and was mineralized in the perfusion culture after 28 days. Increasing flow shear stress accelerated the osteogenic differentiation of hBMSCs and improved the mineralization of ECM. However, increasing mass transport inhibited the formation of mineralized ECM. So, both flow shear stress and transport affected the construction of the large-scale tissue-engineered bone. Moreover, the large-scale tissue-engineered bone could be better produced in the perfusion bioreactor with 0.015 Pa (0.013-0.018 Pa) of fluid flow shear stress and 3 mL/min of mass transport. PMID: 19226211 [PubMed - indexed for MEDLINE] |
| Bioengineered tissue substitutes in implant extrusion.
December 24, 2009 at 8:49 am
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| | Bioengineered tissue substitutes in implant extrusion. J Plast Reconstr Aesthet Surg. 2009 Dec;62(12):e658-60 Authors: Murphy KD, McGoldrick C, Khan K PMID: 19046935 [PubMed - indexed for MEDLINE] |
| The pi-helix formation between Asp369 and Thr375 as a key factor in E1-E2 conformational change of Na+/K+-ATPase.
December 24, 2009 at 8:49 am
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| | The pi-helix formation between Asp369 and Thr375 as a key factor in E1-E2 conformational change of Na+/K+-ATPase. Physiol Res. 2009;58(4):583-9 Authors: Tejral G, Kolácná L, Schoner W, Amler E Molecular modeling of the H4-H5-loop of the alpha2 isoform of Na+/K+-ATPase in the E1 and E2 conformations revealed that twisting of the nucleotide (N) domain toward the phosphorylation (P) domain is connected with the formation of a short pi-helix between Asp369 and Thr375. This conformational change close to the hinge region between the N-domain and the P-domain could be an important event leading to a bending of the N-domain by 64.7 degrees and to a shortening of the distance between the ATP binding site and the phosphorylation site (Asp369) by 1.22 nm from 3.22 nm to 2.00 nm. It is hypothesized that this shortening mechanism is involved in the Na+-dependent formation of the Asp369 phospho-intermediate as part of the overall Na+/K+-ATPase activity. PMID: 18657006 [PubMed - indexed for MEDLINE] | | | 
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