|  |  |  | | | | | TE-RegenMed-StemCell feed | | | | | | | | | | | | | | | | | | | Alveolar epithelial cell therapy with human cord blood-derived hematopoietic progenitor cells. Am J Pathol. 2011 Mar;178(3):1329-39 Authors: De Paepe ME, Mao Q, Ghanta S, Hovanesian V, Padbury JF The role of umbilical cord blood (CB)-derived stem cell therapy in neonatal lung injury remains undetermined. We investigated the capacity of human CB-derived CD34(+) hematopoietic progenitor cells to regenerate injured alveolar epithelium in newborn mice. Double-transgenic mice with doxycycline (Dox)-dependent lung-specific Fas ligand (FasL) overexpression, treated with Dox between embryonal day 15 and postnatal day 3, served as a model of neonatal lung injury. Single-transgenic non-Dox-responsive littermates were controls. CD34(+) cells (1 × 10(5) to 5 × 10(5)) were administered at postnatal day 5 by intranasal inoculation. Engraftment, respiratory epithelial differentiation, proliferation, and cell fusion were studied at 8 weeks after inoculation. Engrafted cells were readily detected in all recipients and showed a higher incidence of surfactant immunoreactivity and proliferative activity in FasL-overexpressing animals compared with non-FasL-injured littermates. Cord blood-derived cells surrounding surfactant-immunoreactive type II-like cells frequently showed a transitional phenotype between type II and type I cells and/or type I cell-specific podoplanin immunoreactivity. Lack of nuclear colocalization of human and murine genomic material suggested the absence of fusion. In conclusion, human CB-derived CD34(+) cells are capable of long-term pulmonary engraftment, replication, clonal expansion, and reconstitution of injured respiratory epithelium by fusion-independent mechanisms. Cord blood-derived surfactant-positive epithelial cells appear to act as progenitors of the distal respiratory unit, analogous to resident type II cells. Graft proliferation and alveolar epithelial differentiation are promoted by lung injury. PMID: 21356383 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Autologous adipose tissue as a new source of progenitor cells for therapeutic angiogenesis. J Cardiol. 2009 Apr;53(2):155-63 Authors: Murohara T Therapeutic angiogenesis is an important means for salvaging tissues from severe ischemic diseases in patients with no option for other vascular interventions. A number of recent studies examined the possibilities of cell transplantation-mediated angiogenesis using autologous bone marrow mononuclear cells, CD34(+) cells, peripheral mononuclear cells, etc. Subcutaneous adipose tissue can be harvested by relatively easy technology. Recent studies indicate that adipose tissue contains progenitor cells that can give rise to several mesenchymal lineages. Moreover, these progenitor cells can release multiple angiogenic growth factors including vascular endothelial growth factor, hepatocyte growth factor, and chemokine stromal cell-derived factor. The combination of these biological properties of adipose-derived regenerative cells indicates that autologous adipose tissue will be a useful cell source for therapeutic angiogenesis. PMID: 19304117 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | MicroRNAs in adipogenesis and as therapeutic targets for obesity. Expert Opin Ther Targets. 2011 Feb 28; Authors: Alexander R, Lodish H, Sun L Introduction: Obesity and obesity-related disease have reached pandemic proportions and are prevalent even in developing countries. Adipose tissue is increasingly being recognized as a key regulator of whole-body energy homeostasis and consequently as a prime therapeutic target for metabolic syndrome. This review discusses the roles of miRNAs, small endogenously expressed RNAs that regulate gene expression at a post-transcriptional level, in the development and function of adipose tissue and other relevant metabolic tissues impacted by obesity. Several high-throughput studies have identified hundreds of miRNAs that are differentially expressed during the development of metabolic tissues or as an indication of pathophysiology. Further investigation has functionalized the regulatory capacity of individual miRNAs and revealed putative targets for these miRNAs. Therefore, as with several other pathologies, miRNAs are emerging as feasible therapeutic targets for metabolic syndrome. Areas covered: This review provides a comprehensive view of miRNAs involved in adipogenesis, from mesenchymal stem cell lineage determination through terminal adipocyte differentiation. We also discuss the differential expression of miRNAs among adipose depots and the dysregulation of miRNAs in other metabolic tissues during metabolic pathophysiology. Finally, we discuss the therapeutic potential of targeting miRNAs in obesity and give a perspective on the challenges and advantages of miRNA-based drugs. Expert opinion: miRNAs are extensive regulators of adipocyte development and function and are viable therapeutic targets for obesity. Despite the broad-spectrum and redundancy of miRNA?target interactions, sophisticated bioinformatic approaches are making it possible to determine the most physiologically relevant miRNAs to target in disease. In vivo delivery of miRNAs for therapeutic purposes is rapidly developing and has been successful in other contexts. Additionally, miRNAs can be used as prognosis markers for disease onset and progression. Ultimately, miRNAs are prime therapeutic targets for obesity and its consequent pathologies in other metabolic tissues. PMID: 21355787 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Quantitative grafting of peptide onto the nontoxic biodegradable waterborne polyurethanes to fabricate peptide modified scaffold for soft tissue engineering. J Mater Sci Mater Med. 2011 Mar 1; Authors: Jiang X, Wang K, Ding M, Li J, Tan H, Wang Z, Fu Q Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide has frequently been used in the biomedical materials to enhance adhesion and proliferation of cells. In this work, we modified the nontoxic biodegradable waterborne polyurethanes (WBPU) with GRGDSP peptide and fabricated 3-D porous scaffold with the modified WBPU to investigate the effect of the immobilized GRGDSP peptide on human umbilical vein endothelial cells (HUVECs) adhesion and proliferation. A facile and reliable approach was first developed to quantitative grafting of GRGDSP onto the WBPU molecular backbone using ethylene glycol diglycidyl ether (EX810) as a connector. Then 3-D porous WBPU scaffolds with various GRGDSP content were fabricated by freeze-drying the emulsion. In both of the HUVECs adhesion and proliferation tests, enhanced cell performance was observed on the GRGDSP grafted scaffolds compared with the unmodified scaffolds and the tissue culture plate (TCP). The adhesion rate and proliferation rate increased with the increase of GRGDSP content in the scaffold and reached a maximum with peptide concentration of 0.85 μmol/g based on the weight of the polyurethanes. These results illustrate the necessity of the effective control of the GRGDSP content in the modified WBPU and support the potential utility of these 3-D porous modified WBPU scaffolds in the soft tissue engineering to guide cell adhesion, proliferation and tissue regeneration. PMID: 21360121 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | [Local treatment of chronic skin wounds in a Swiss out-patient wound centre 2010.] Ther Umsch. 2011 Mar;68(3):153-158 Authors: Baumgartner M, Tanner D, Hunziker T In Switzerland around 30'000 patients suffer from chronic skin wounds. Appropriate topical wound care along with treatment of the causes of the wounds enables to heal a lot of these patients and to avoid secondary disease such as infections. Thereby, the final goal of wound care is stable reepithelisation. Based on experience with chronic leg ulcers mainly in our out-patient wound centre, we give a survey of the wound dressings we actually use and discuss their wound-phase adapted application. Furthermore, we address the two tissue engineering products reimbursed in Switzerland, Apligraf and EpiDex, as well as the biological matrix product Oasis. The crucial question, which treatment options will be offered in future to the wound patients by our health regulatory and insurance systems, is open to debate. PMID: 21360461 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Composite thin film and electrospun biomaterials for urologic tissue reconstruction. Biotechnol Bioeng. 2011 Jan;108(1):207-15 Authors: Kundu AK, Gelman J, Tyson DR A replacement material for autologous grafts for urinary tract reconstruction would dramatically reduce the complications of surgery for these procedures. However, acellular materials have not proven to work sufficiently well, and cell-seeded materials are technically challenging and time consuming to generate. An important function of the urinary tract is to prevent urine leakage into the surrounding tissue--a function usually performed by the urothelium. We hypothesize that by providing an impermeable barrier in the acellular graft material, urine leakage would be minimized, as the urothelium forms in vivo. However, since urothelial cells require access to nutrients from the supporting vasculature, the impermeable barrier must degrade over time. Here we present the development of a novel biomaterial composed of the common degradable polymers, poly(ε-caprolactone) and poly(L-lactic acid) and generated by electrospinning directly onto spin-coated thin films. The composite scaffolds with thin films on the luminal surface were compared to their electrospun counterparts and commercially available small intestinal submucosa by surface analysis using scanning electron microscopy and by analysis of permeability to small molecules. In addition, the materials were examined for their ability to support urothelial cell adhesion, proliferation, and multilayered urothelium formation. We provide evidence that these unique composite scaffolds provide significant benefit over commonly used acellular materials in vitro and suggest that they be further examined in vivo. PMID: 20830673 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Regeneration of the Intervertebral Disc with Nucleus Pulposus Cell-seeded Collagenα/Hyaluronan/Chondroitin-6-sulfate tri-copolymer Constructs in a Rabbit Disc Degeneration Model. Spine (Phila Pa 1976). 2011 Feb 24; Authors: Huang B, Zhuang Y, Li CQ, Liu LT, Zhou Y ABSTRACT: Study Design. Advancement in tissue engineering provides a promising approach to recover the functionality of the degenerated intervertebral disc. In our study, a nucleus pulposus (NP) cell-seeded Collagenα/Hyaluronan/Chondroitin-6-sulfate (Cα/HyA-CS) tri-copolymer construct was implanted into the disc space directly after nucleotomy in a rabbit model.Objective. The aim of this study was to investigate whether the NP cell-seeded Cα/HyA-CS tri-copolymer constructs could regenerate the degenerated disc in vivo after implantation into the rabbit nucleotomy model.Summary of Background Data. Nucleotomy is one of the most prevalent surgical modalities to treat degenerative disc disease (DDD), which could achieve good short term effects of pain relieve. Whereas removal of the entire or partial NP changes the biomechanical characteristics of the remaining disc and the adjacent vertebral segments, and a series of long-term complications such as accelerated annulus and the facet joints degeneration may ensue. Therefore, it is necessary to think about possible procedures immediately after the primary nucleotomy surgery to avoid these complications.Methods. NP cells isolated from thoracic and lumbar spines of New Zealand White rabbits, approximately 3 weeks of age and 1 kg in weight were labeled with a CFDA-SE fluorescent dye and seeded within the Cα/HyA-CS scaffold by a centrifugation method. After in vitro culture for 1 week, NP Cell-seeded Cα/HyA-CS tri-copolymer constructs were allografted into the disc defects of recipient rabbit immediately after nucleotomy of the lumbar spine. The Bradner disc index (BDI) and the T2-weighted signal intensity index were determined using lateral plane radiographs and magnetic resonance imaging (MRI) at 4, 12, 24 weeks after the operation. Finally, the operated discs were explanted for gross morphology observation, histological evaluation and cell viability assessment. Animals with nucleotomy only and cell-free Cα/HyA-CS scaffold implantation served as controls.Results. In our study, we could demonstrate that the T2-weighted signal intensity index of the operated discs decreased in all three groups 1 month after surgery, and the index of the cell-containing scaffold insertion group was significantly higher than the other two groups. After 24 weeks, the index of the cell-containing scaffold insertion group increased significantly. However, further decline was observed in both the non-insertion group and the scaffold insertion group. In radiographic analysis, the narrowing of the intervertebral disc space was significantly retarded by the cell-scaffold hybrids implantation up to 24 postoperative weeks. Furthermore, the gross morphology and histological evaluation indicated that the allografted NP cells were viable and showed extracellular matrix (ECM) production.Conclusions. In our study, we had constructed rabbit NP-cell seeded Cα/HyA-CS tri-copolymer implants in vitro. Immediately after nucleotomy of the recipient rabbit, we allografted the precultured cell-scaffold hybrids into the lacuna of the disc. Results documented survival of the allografted NP cells and ECM deposition, which finally resulted in maintenance of disc height and restoration of T2-weighted signal intensity on MRI. PMID: 21358466 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Prevention of muscle aging by myofiber-associated satellite cell transplantation. Sci Transl Med. 2010 Nov 10;2(57):57ra83 Authors: Hall JK, Banks GB, Chamberlain JS, Olwin BB Skeletal muscle is dynamic, adapting to environmental needs, continuously maintained, and capable of extensive regeneration. These hallmarks diminish with age, resulting in a loss of muscle mass, reduced regenerative capacity, and decreased functionality. Although the mechanisms responsible for this decline are unclear, complex changes within the local and systemic environment that lead to a reduction in regenerative capacity of skeletal muscle stem cells, termed satellite cells, are believed to be responsible. We demonstrate that engraftment of myofiber-associated satellite cells, coupled with an induced muscle injury, markedly alters the environment of young adult host muscle, eliciting a near-lifelong enhancement in muscle mass, stem cell number, and force generation. The abrogation of age-related atrophy appears to arise from an increased regenerative capacity of the donor stem cells, which expand to occupy both myonuclei in myofibers and the satellite cell niche. Further, these cells have extensive self-renewal capabilities, as demonstrated by serial transplantation. These near-lifelong, physiological changes suggest an approach for the amelioration of muscle atrophy and diminished function that arise with aging through myofiber-associated satellite cell transplantation. PMID: 21068442 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Cell-based Meniscal Tissue Engineering: A Case for Synoviocytes. Clin Orthop Relat Res. 2011 Mar 1; Authors: Fox DB, Warnock JJ BACKGROUND: Avascular meniscal injuries are largely incapable of healing; the most common treatment remains partial meniscectomy despite the risk of subsequent osteoarthritis. Meniscal responses to injury are partially mediated through synovial activity and strategies have been investigated to encourage healing through stimulating or transplanting adjacent synovial lining. However, with their potential for chondrogenesis, synovial fibroblast-like stem cells hold promise for meniscal cartilage tissue engineering. QUESTIONS/PURPOSES: Thus, specific purposes of this review were to (1) examine how the synovial intima and synoviomeniscal junction affect current meniscal treatment modalities; and (2) examine the components of tissue engineering (cells, scaffolds, bioactive agents, and bioreactors) in the specific context of how cells of synovial origin may be used for meniscal healing or regeneration. METHODS: An online bibliographic search through PubMed was performed in March 2010. Studies were subjectively evaluated and reviewed if they addressed the question posed. Fifty-four resources were initially retrieved, which offered information on the chondrogenic potential of synovial-based cells that could prove valuable for meniscal fibrocartilage engineering. RESULTS: Based on the positive effects of adjoining synovium on meniscal healing as used in some current treatment modalities, the chondrogenic potential of fibroblast-like stem cells of synovial origin make this cell source a promising candidate for cell-based tissue engineering strategies. CONCLUSIONS: The abundance of autologous synovial lining, its ability to regenerate, and the potential of synovial-derived stem cells to produce a wide spectrum of chondral matrix components make it an ideal candidate for future meniscal engineering investigations. PMID: 21360210 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Transforming growth factor-beta increases the expression of vascular smooth muscle cell Markers in human multi-lineage progenitor cells. Med Sci Monit. 2011 Feb 25;17(3):BR55-61 Authors: Yang H, Zhang L, Weakley SM, Lin PH, Yao Q, Chen C Background: Vascular smooth muscle cell (SMC) differentiation is an essential component of vascular repair and tissue engineering. However, currently used cell models for the study of SMC differentiation have several limitations. Multi-lineage progenitor cells (MLPCs) originate from human umbilical cord blood and are cloned from a single cell. The object of this study was to investigate whether MLPCs could differentiate into SMCs in vitro with induction by transforming growth factor beta1 (TGF-beta1).<br /> Material/Methods: MLPCs were treated without or with TGF-beta1 (1 and 5 ng/mL) in mesenchymal stem cell media plus 1% FBS for 7 days. Total RNA was isolated from the MLPCs, and semi-quantitative real-time PCR was performed to test the following mRNA levels: early and late phase SMC-specific markers, two endothelial cell (EC)-specific markers, endothelial progenitor cell (EPC) marker CD34, TGF-beta1 accessory protein CD105, and adhesion molecule CD146.<br /> Results: TGF-beta1 (1 ng/mL) significantly increased the mRNA levels of SMC-specific markers SM22α, calponin-1, SM α-actin, caldesmon, tropomyosin and MLCK as well as adhesion molecule CD146. The mRNA levels of EC-specific markers VE-cadherin and VEGFR-2, EPC marker CD34 and TGF-beta1 accessory protein CD105 were decreased significantly, after MLPC were treated with TGF-beta1 (1 ng/mL). TGF-beta1 at 5 ng/mL showed similar effect on the expression of these genes.<br /> Conclusions: This study demonstrates that in the presence of TGF-beta1, MLPCs undergo SMC lineage differentiation indicating that MLPCs are a promising cell model for SMC lineage differentiation studies, which may contribute to advances in vascular repair and tissue engineering.<br /> PMID: 21358594 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Application of finite element analysis to the design of tissue leaflets for a percutaneous aortic valve. J Mech Behav Biomed Mater. 2011 Jan;4(1):85-98 Authors: Smuts AN, Blaine DC, Scheffer C, Weich H, Doubell AF, Dellimore KH Percutaneous Aortic Valve (PAV) replacement is an attractive alternative to open heart surgery, especially for patients considered to be poor surgical candidates. Despite this, PAV replacement still has its limitations and associated risks. Bioprosthetic heart valves still have poor long-term durability due to calcification and mechanical failure. In addition, the implantation procedure often presents novel challenges, including damage to the expandable stents and bioprosthetic leaflets. In this study, a simplified version of Fung's elastic constitutive model for skin, developed by Sun and Sacks, was implemented using finite element analysis (FEA) and applied to the modelling of bovine and kangaroo pericardium. The FEA implementation was validated by simulating biaxial tests and by comparing the results with experimental data. Concepts for different PAV geometries were developed by incorporating valve design and performance parameters, along with stent constraints. The influence of effects such as different leaflet material, material orientation and abnormal valve dilation on the valve function was investigated. The stress distribution across the valve leaflet was also examined to determine the appropriate fibre direction for the leaflet. The simulated attachment forces were compared with suture tearing tests performed on the pericardium to evaluate suture density. It is concluded that kangaroo pericardium is suitable for PAV applications, and superior to bovine pericardium, due to its lower thickness and greater extensibility. PMID: 21094482 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Tissue-engineered vascularized bone grafts: Basic science and clinical relevance to trauma and reconstructive microsurgery. Microsurgery. 2011 Feb 25; Authors: Johnson EO, Troupis T, Soucacos PN Bone grafts are an important part of orthopaedic surgeon's armamentarium. Despite well-established bone-grafting techniques, large bone defects still represent a challenge. Efforts have therefore been made to develop osteoconductive, osteoinductive, and osteogenic bone-replacement systems. The long-term clinical goal in bone tissue engineering is to reconstruct bony tissue in an anatomically functional three-dimensional morphology. Current bone tissue engineering strategies take into account that bone is known for its ability to regenerate following injury, and for its intrinsic capability to re-establish a complex hierarchical structure during regeneration. Although the tissue engineering of bone for the reconstruction of small to moderate sized bone defects technically feasible, the reconstruction of large defects remains a daunting challenge. The essential steps towards optimized clinical application of tissue-engineered bone are dependent upon recent advances in the area of neovascularization of the engineered construct. Despite these recent advances, however, a gap from bench to bedside remains; this may ultimately be bridged by a closer collaboration between basic scientists and reconstructive surgeons. The aim of this review is to introduce the basic principles of tissue engineering of bone, outline the relevant bone physiology, and discuss the recent concepts for the induction of vascularization in engineered bone tissue. © 2011 Wiley-Liss, Inc. Microsurgery, 2011. PMID: 21360585 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Incorporation of a Decorin Biomimetic Enhances the Mechanical Properties of Electrochemically Aligned Collagen Threads. Acta Biomater. 2011 Feb 25; Authors: Kishore V, Paderi JE, Akkus A, Smith KM, Balachandran D, Beaudoin S, Panitch A, Akkus O Orientational anisotropy of collagen molecules is integral for the mechanical strength of collagen-rich tissues. We have previously reported a novel methodology to synthesize highly oriented electrochemically aligned collagen (ELAC) threads with mechanical properties converging upon those of native tendon. Decorin, a small leucine rich proteoglycan (SLRP), binds to fibrillar collagen and has been suggested to enhance the mechanical properties of tendon. Based on the structure of natural decorin, we have previously designed and synthesized a peptidoglycan (DS-SILY) that mimics decorin both structurally and functionally. In this study, we investigated the effect of the incorporation of DS-SILY on the mechanical properties and structural organization of ELAC threads. The results indicated that the addition of DS-SILY at a molar ratio of 30:1 (Collagen:DS-SILY) significantly enhanced the ultimate stress and ultimate strain of the ELAC threads. Furthermore, differential scanning calorimetry revealed that the addition of DS-SILY at a molar ratio of 30:1 resulted in a more thermally stable collagen structure. However, addition of DS-SILY at a higher concentration (10:1 Collagen:DS-SILY) yielded weaker threads with mechanical properties comparable to collagen control threads. Transmission emission microscopy revealed that the addition of DS-SILY at a higher concentration (10:1) resulted in pronounced aggregation of collagen fibrils. More importantly, these aggregates were not aligned along the long axis of the ELAC thereby compromising on the overall tensile properties of the material. We conclude that incorporation of an optimal amount of DS-SILY is a promising approach to synthesize mechanically competent collagen based biomaterials for tendon tissue engineering applications. PMID: 21356334 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Evaluation of tumorigenic risk of tissue-engineered oral mucosal epithelial cells by using combinational examinations. Cell Transplant. 2010;19(8):999-1006 Authors: Thépot A, Morel AP, Justin V, Desanlis A, Thivillier L, Hoffman E, Till M, Accardi R, Tommasino M, Breton P, Hainaut P, Damour O Recently, oral mucosal epithelial cells were proposed as a cell source of the autologous cell transplant therapy for corneal trauma or disease. The question addressed is to know if the biological conditions of grafting could induce certain cellular, molecular, and genetic alterations that might increase the risk of mutations and possibly of cellular transformation. Recent progress in cancer research enables us to depict the generation mechanisms and basic characteristics of human cancer cells from molecular, cytological, and biological aspects. The aim of this study is to evaluate the risk of tumorigenicity of the oral mucosal epithelial culture process in order to mitigate that risk, if any, before clinical application. Oral mucosal epithelial cells from three different human donors were investigated by combinational examinations to detect possible tumorigenic transformation. We investigated (i) clonogenic and karyology types, (ii) the validation of proliferation rate, (iii) the epithelial-mesenchymal transition, (iv) anchorage-independent growth potential, and (v) tumorigenicity on nude mice. Results show that the culture process used in this study presents no risk of tumorigenicity. PMID: 20977830 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Chondrogenic induction of human mesenchymal stem cells using combined growth factors for cartilage tissue engineering. J Tissue Eng Regen Med. 2011 Feb 28; Authors: Bosetti M, Boccafoschi F, Leigheb M, Bianchi AE, Cannas M The objective of this study was to evaluate whether growth factors (FGF-2, FGF-4 and FGF-6) used alone or in combination with TGFβ2 are able to increase the proliferation and induce the differentiation of human bone marrow mesenchymal stem cells (hMSCs) to chondrocytes, with a view to using them in cartilage tissue engineering. Cells cultured in monolayer, used to test the activity of the growth factors on cell proliferation, showed that a combination of FGFs with TGFβ2 increases cell proliferation compared to cells cultured in control medium or in the presence of growth factors alone. The chondrogenic potential, evaluated in three-dimensional (3D) cell aggregates, showed that FGF-2 and FGF-6, when used in combination with TGFβ2 increased the size and glycosaminoglycan content of the cell aggregates without increasing cell number. Extracellular matrix (ECM) also showed higher collagen type II immunoreactivity, which was particularly evident in an area similar to a germinative pole that was observed only in pellets cultured with FGF-2 and FGF-6 combined with TGFβ2, or in pellets cultured with FGF-2 alone. Moreover, the RT-PCR assay has highlighted an increased expression of collagen type II and Sox9, used as gene markers for chondrogenesis. We can conclude that combinations of FGF-2 or FGF-6 with TGFβ2 may provide a novel tool to induce the differentiation of adult human mesenchymal stem cells for applications in cartilage tissue engineering. Copyright © 2011 John Wiley & Sons, Ltd. PMID: 21360690 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Effects of FGF2 and FGF9 on osteogenic differentiation of bone marrow-derived progenitors. In Vitro Cell Dev Biol Anim. 2011 Feb 27; Authors: Kizhner T, Ben-David D, Rom E, Yayon A, Livne E Bone repair is a major concern in reconstructive surgery. Transplants containing osteogenically committed mesenchymal stem cells (MSCs) provide an alternative source to the currently used autologous bone transplants which have limited supply and require additional surgery to the patient. A major drawback, however is the lack of a critical mass of cells needed for successful transplantation. The purpose of the present study was to test the effects of FGF2 and FGF9 on expansion and differentiation of MSCs in order to establish an optimal culture protocol resulting in sufficient committed osteogenic cells required for successful in vivo transplantation. Bone marrow-derived MSCs cultured in αMEM medium supplemented with osteogenic supplements for up to three passages (control medium), were additionally treated with FGF2 and FGF9 in various combinations. Cultures were evaluated for viability, calcium deposition and in vivo osteogenic capacity by testing subcutaneous transplants in nude mice. FGF2 had a positive effect on the proliferative capacity of cultured MSCs compared to FGF9 and control medium treated cultures. Cultures treated with FGF2 followed by FGF9 showed an increased amount of extracted Alizarin red indicating greater osteogenic differentiation. Moreover, the osteogenic capacity of cultured cells transplanted in immunodeficient mice revealed that cells that were subjected to treatment with FGF2 in the first two passages and subsequently to FGF9 in the last passage only, were more successful in forming new bone. It is concluded that the protocol using FGF2 prior to FGF9 is beneficial to cell expansion and commitment, resulting in higher in vivo bone formation for successful bone tissue engineering. PMID: 21359818 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Smart Approach To Evaluate Drug Diffusivity in Injectable Agar-Carbomer Hydrogels for Drug Delivery. J Phys Chem B. 2011 Feb 28; Authors: Santoro M, Marchetti P, Rossi F, Perale G, Castiglione F, Mele A, Masi M Hydrogels are commonly studied for tissue engineering applications and controlled drug delivery. In order to evaluate their reliability as scaffolds and delivery devices, literature describes many release studies performed involving different analytical techniques. However, these experiments can be expensive, time-consuming, and often not reproducible. In this study, two injectable agar-carbomer-based hydrogels were studied, both being loaded with sodium fluorescein, a harmless fluorophore with a steric hindrance similar to many small drugs, such as for example steroids and other neuroprotecting agents. Starting from simple, traditional, and inexpensive release experiments, it was possible to indirectly estimate the self-diffusion coefficient (D) of loaded sodium fluorescein. Such a parameter was also directly measured in the gel matrix by means of high resolution magic angle spinning (HRMAS) diffusion-ordered spectroscopy NMR. Because of the agreement between the calculated values and those measured by HRMAS-NMR spectroscopy, the latter approach can be considered as a simple and fast alternative to long analytic procedures. PMID: 21355616 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Modern concepts in mandibular reconstruction in oral and oropharyngeal cancer. Curr Opin Otolaryngol Head Neck Surg. 2011 Feb 25; Authors: Wang KH, Inman JC, Hayden RE PURPOSE OF REVIEW: This article reviews literature pertaining to advances in oromandibular reconstruction in the context of a 30-year background of microvascular free tissue transfer, which still represents the current standard of care. RECENT FINDINGS: Most literature reiterates established patterns of reconstruction. Notable exceptions address these key areas: the use of computer-assisted modeling to more closely and efficiently design the excision of the mandible, to produce the template and to contour the fibula bone to fit the defect; the evolution of plating techniques to maximize the bony repair; distraction osteogenesis; tissue engineering; and optimal techniques for dealing with osteonecrosis, from both radiation and bisphosphonate use. SUMMARY: Osteocutaneous free flaps remain the standard of care. The fibula flap has emerged as the accepted favorite among these flaps. Evolution of fibula flap reconstruction constitutes a major portion of the current literature. Improvements upon free tissue transfer are currently elusive, largely due to associated radiation. Tissue engineering holds promise as the next plateau but is not yet readily applicable. PMID: 21358547 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Polyrotaxanes for applications in life science and biotechnology. Appl Microbiol Biotechnol. 2011 Mar 1; Authors: Li JJ, Zhao F, Li J Due to their low cytotoxicity, controllable size, and unique architecture, cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes have inspired interesting exploitation as novel biomaterials. This review will update the recent progress in the studies on the structures of polyrotaxanes and polypseudorotaxanes based on different CDs and polymers, followed by summarizing their potential applications in life science and biotechnology, such as drug delivery, gene delivery, and tissue engineering. CD-based biodegradable polypseudorotaxane hydrogels could be used as promising injectable drug delivery systems for sustained and controlled drug release. Polyrotaxanes with drug or ligand-conjugated CDs threaded on polymer chain with biodegradable end group could be useful for controlled and multivalent targeting delivery. Cationic polyrotaxanes consisting of multiple oligoethylenimine-grafted CDs threaded on a block copolymer chain were attractive non-viral gene carries due to the strong DNA-binding ability, low cytotoxicity, and high gene transfection efficiency. Cytocleavable end caps were also introduced in the polyrotaxane systems in order to ensure efficient endosomal escape for intracellular trafficking of DNA. Finally, hydrolyzable polyrotaxane hydrogels with cross-linked α-CDs could be a desirable scaffold for cartilage and bone tissue engineering. PMID: 21360153 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Case study: first implantation of a frozen, devitalized tissue-engineered vascular graft for urgent hemodialysis access. J Vasc Access. 2011 Jan-Mar;12(1):67-70 Authors: Wystrychowski W, Cierpka L, Zagalski K, Garrido S, Dusserre N, Radochonski S, McAllister TN, L'heureux N Previously we reported on the mid- to long-term follow-up in the first clinical trial to use a completely autologous tissue-engineered graft in the high pressure circulation. In these early studies, living grafts were built from autologous fibroblasts and endothelial cells obtained from small skin and vein biopsies. The graft was assembled using a technique called tissue-engineering by self-assembly (TESA), where robust conduits were grown without support from exogenous biomaterials or synthetic scaffolding. One limitation with this earlier work was the long lead times required to build the completely autologous vascular graft. Here we report the first implant of a frozen, devitalized, completely autologous Lifeline™ vascular graft. In a departure from previous studies, the entire fibroblast layer, which provides the mechanical backbone of the graft, was air-dried then stored at -80°C until shortly before implant. Five days prior to implant, the devitalized conduit was rehydrated, and its lumen was seeded with living autologous endothelial cells to provide an antithrombogenic lining. The graft was implanted as an arteriovenous shunt between the brachial artery and the axillary vein in a patient who was dependent upon a semipermanent dialysis catheter placed in the femoral vein. Eight weeks postoperatively, the graft functions without complication. This strategy of preemptive skin and vein biopsy and cold-preserving autologous tissue allows the immediate availability of an autologous arteriovenous fistula, and is an important step forward in our strategy to provide allogeneic tissue-engineered grafts available "off-the-shelf". PMID: 21360466 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Vascularization of prevascularized and non-prevascularized fibrin-based human adipose tissue constructs after implantation in nude mice. J Tissue Eng Regen Med. 2011 Feb 24; Authors: Verseijden F, Posthumus-van Sluijs SJ, van Neck JW, Hofer SO, Hovius SE, van Osch GJ Adipose regeneration strategies have been hampered by the inability to supply an adequate vascular supply following implantation. Vascularization in vitro, also called prevascularization, is a promising method that could promote the vascularization of engineered adipose tissue constructs upon implantation. In this study we compared the ability of prevascularized-to-non-prevascularized fibrin-based human adipose tissue to promote vascularization. Human adipose tissue-derived stromal cells (ASCs) and different mixtures (1:1, 1:2 and 1:5) of ASCs with human umbilical vein endothelial cells (HUVECs) were cultured in fibrin at two different densities (1.0 × 10(6) and 10 × 10(6) cells/ml) for 7 days. Histological analysis revealed that prevascular structures formed in 1:5 ASC/HUVEC fibrin-based constructs seeded with a total of 10 × 10(6) cells/ml. These constructs and ASC-only constructs were implanted subcutaneously in athymic mice for 7 days and generated lipid-containing grafts. The numbers and densities of blood vessels within the ASC/HUVEC constructs were similar to those of ASC-only constructs. Furthermore, immunostaining studies demonstrated human-derived vasculature within a few of the ASC/HUVEC and ASC-only constructs. A subset of this human-derived vasculature contained erythrocytes, indicating integration with the host vasculature. In conclusion, our study indicated no difference in the rate of vascularization of prevascularized ASC/HUVEC and non-prevascularized ASC-only fibrin-based constructs, suggesting that prevascularization of these fibrin-based constructs does not promote vascularization. Our results further indicated that not only endothelial cells, but also ASCs may contribute to the formation of vascular lumina upon implantation. This finding is interesting, since it demonstrates the possibility of vascularized adipose tissue engineering from a single cell source. Copyright © 2011 John Wiley & Sons, Ltd. PMID: 21360688 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Expression profile of the stem cell markers in human Hertwig's epithelial root sheath/Epithelial rests of Malassez cells. Mol Cells. 2011 Feb 22; Authors: Nam H, Kim J, Park J, Park JC, Kim JW, Seo BM, Lee JC, Lee G Hertwig's epithelial root sheath/Epithelial rests of Malassez (HERS/ERM) cells are unique epithelial cells in the periodontal ligament. They remain in periodontal tissues through-out the adult life, and it is expected that their functional role is to maintain the homeostasis of the periodontium through reciprocal interactions with other periodontal cells. In this study, we investigated whether HERS/ERM cells have primitive stem cell characteristics: those of embryonic stem cells as well as of epithelial stem cells. Primary HERS/ERM cells had typical epithelial cell morphology and characteristics and they maintained for more than five passages. They expressed epithelial stem cell-related genes: ABCG2, ANp63, p75, EpCAM, and Bmi-1. Moreover, the expression of embryonic stem cell markers such as Oct-4, Nanog, and SSEA-4 were detected. Next, we investigated whether the expression of these stem cell markers was maintained during the sub-culture process. HERS/ERM cells showed different expression levels of these stemness genes at each passage, but their expression was maintained throughout the passages. Taken together, our data suggest that a primary culture of HERS/ERM cells contains a population of primitive stem cells that express epithelial stem cell markers and embryonic stem cell markers. Furthermore, these cell populations were maintained during the sub-culturing process in our culture conditions. Therefore, our findings suggest that there is a strong possibility of accomplishing cementum tissue engineering with HERS/ERM cells. PMID: 21359676 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Mesenchymal stem cells as a therapeutic tool in tissue and organ regeneration. Postepy Hig Med Dosw (Online). 2011;65:124-32 Authors: Bajek A, Olkowska J, Drewa T Tissue engineering is an interdisciplinary field that offers new opportunities for regeneration of diseased and damaged tissue with the use of many different cell types,including adult stem cells. In tissue engineering and regenerative medicine the most popular are mesenchymal stem cells (MSCs) isolated from bone marrow. Bone marrow mesenchymal stem cells are a potential source of progenitor cells for osteoblasts, chondroblasts, adipocytes, skeletal muscles and cardiomyocytes. It has also been shown that these cells can differentiate into ecto- and endodermal cells, e.g. neuronal cells, glial cells, keratinocytes and hepatocytes. The availability of autologous MSCs, their proliferative potential and multilineage differentiation capacity make them an excellent tool for tissue engineering and regenerative medicine. The aim of this publication is to present characteristic and biological properties of mesenchymal stem cells isolated from bone marrow.<br /> PMID: 21358000 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Evaluation of Traumatic Brain Injury: Brain Potentials in Diagnosis, Function, Prognosis. Int J Psychophysiol. 2011 Feb 25; Authors: Duncan CC, Summers AC, Perla EJ, Coburn KL, Mirsky AF The focus of this review is an analysis of the use of event-related brain potential (ERP) abnormalities as indices of functional pathophysiology in survivors of traumatic brain injury (TBI). TBI may be the most prevalent but least understood neurological disorder in both civilian and military populations. In the military, thousands of new brain injuries occur yearly; this lends considerable urgency to the use of highly sensitive ERP tools to illuminate brain changes and to address remediation issues. We review the processes thought to be indexed by the cognitive components of the ERP and outline the rationale for applying ERPs to evaluate deficits after TBI. Studies in which ERPs were used to clarify the nature of cognitive complaints of TBI survivors are reviewed, emphasizing impairment in attention, information processing, and cognitive control. Also highlighted is research on the application of ERPs to predict emergence from coma and eventual outcome. We describe primary blast injury, the leading cause of TBI for active duty military personnel in present day warfare. The review concludes with a description of an ongoing investigation of mild TBI, aimed at using indices of brain structure and function to predict the course of posttraumatic stress disorder. An additional goal of this ongoing investigation is to characterize the structural and functional sequelae of blast injury. PMID: 21356253 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Adipose-derived stem cells: current findings and future perspectives. Discov Med. 2011 Feb;11(57):160-70 Authors: Tobita M, Orbay H, Mizuno H Adipose tissue is an abundant source of mesenchymal stem cells, which have shown promise in the field of regenerative medicine. Furthermore, these cells can be readily harvested in large numbers with low donor-site morbidity. During the past decade, numerous studies have provided preclinical data on the safety and efficacy of adipose-derived stem cells, supporting the use of these cells in future clinical applications. Various clinical trials have shown the regenerative capability of adipose-derived stem cells in subspecialties of medical fields such as plastic surgery, orthopedic surgery, oral and maxillofacial surgery, and cardiac surgery. In addition, a great deal of knowledge concerning the harvesting, characterization, and culture of adipose-derived stem cells has been reported. This review will summarize data from in vitro studies, pre-clinical animal models, and recent clinical trials concerning the use of adipose-derived stem cells in regenerative medicine. PMID: 21356171 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | IL-17 and VEGF Are Necessary for Efficient Corneal Nerve Regeneration. Am J Pathol. 2011 Mar;178(3):1106-16 Authors: Li Z, Burns AR, Han L, Rumbaut RE, Smith CW The contribution of acute inflammation to sensory nerve regeneration was investigated in the murine cornea using a model of corneal abrasion that removes the stratified epithelium and subbasal nerve plexus. Abrasion induced accumulation of IL-17(+) CCR6(+) γδ T cells, neutrophils, and platelets in the cornea followed by full restoration of the epithelium and ∼19% regeneration of sensory nerves within 96 hours. Mice deficient in γδ T cells (TCRδ(-/-)) or wild-type mice treated systemically with anti-IL-17 had >50% reduction in leukocyte and platelet infiltration and >50% reduction in nerve regeneration. Strategies used to prevent neutrophil and platelet accumulation (eg, wild-type mice treated with anti-Ly6G or anti-GP1bα antibody to deplete neutrophils or platelets) also resulted in >50% reductions in corneal nerve density. Infiltrating neutrophils and platelets stained positively for VEGF-A, tissue levels of VEGF-A peaked coincidentally with peak tissue levels of neutrophils and platelets, depletion of neutrophils before injury reduced tissue VEGF-A levels by >70%, and wild-type mice treated systemically with anti-VEGF-A antibody exhibited >80% reduction in corneal nerve regeneration. Given the known trophic effects of VEGF-A for neurite growth, the results in this report demonstrate a previously unrecognized beneficial role for the γδ T cell-dependent inflammatory cascade involving IL-17, neutrophils, platelets, and VEGF-A in corneal nerve regeneration. PMID: 21356362 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Harnessing the pancreatic stem cell. Endocrinol Metab Clin North Am. 2010 Dec;39(4):763-76 Authors: Taylor-Fishwick DA, Pittenger GL Building on the elaborate research studies that have helped map out key decision points in the process of pancreas development, reprogramming of pluripotent embryonic stem cells or induced pluripotent stem cells offers the possibility of overcoming restrictions on tissue supply associated with transplantation of donor islets. In a healthy pancreas, the beta-cell mass can exhibit significant plasticity, as reflected in the normal adaptive response in beta-cell mass to offset the metabolic challenge associated with pregnancy and obesity. In this article, alternative strategies and potential sources of pancreatic stem cells are considered. PMID: 21095543 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Translation of myelin basic protein mRNA in oligodendrocytes is regulated by integrin activation and hnRNP-K. J Cell Biol. 2011 Feb 28; Authors: Laursen LS, Chan CW, Ffrench-Constant C Myelination in the central nervous system provides a unique example of how cells establish asymmetry. The myelinating cell, the oligodendrocyte, extends processes to and wraps multiple axons of different diameter, keeping the number of wraps proportional to the axon diameter. Local regulation of protein synthesis represents one mechanism used to control the different requirements for myelin sheath at each axo-glia interaction. Prior work has established that β1-integrins are involved in the axoglial interactions that initiate myelination. Here, we show that integrin activation regulates translation of a key sheath protein, myelin basic protein (MBP), by reversing the inhibitory effect of the mRNA 3'UTR. During oligodendrocyte differentiation and myelination α6β1-integrin interacts with hnRNP-K, an mRNA-binding protein, which binds to MBP mRNA and translocates from the nucleus to the myelin sheath. Furthermore, knockdown of hnRNP-K inhibits MBP protein synthesis during myelination. Together, these results identify a novel pathway by which axoglial adhesion molecules coordinate MBP synthesis with myelin sheath formation. PMID: 21357748 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Translating the lessons from gene therapy to the development of regenerative medicine. Mol Ther. 2011 Mar;19(3):439-41 Authors: Porteus M PMID: 21358706 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Spinal cord injury: A regenerative medicine. Nat Rev Drug Discov. 2011 Mar;10(3):178 Authors: Kingwell K PMID: 21358736 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Automated mitosis detection of stem cell populations in phase-contrast microscopy images. IEEE Trans Med Imaging. 2011 Mar;30(3):586-96 Authors: Huh S, Ker DF, Bise R, Chen M, Kanade T Due to the enormous potential and impact that stem cells may have on regenerative medicine, there has been a rapidly growing interest for tools to analyze and characterize the behaviors of these cells in vitro in an automated and high throughput fashion. Among these behaviors, mitosis, or cell division, is important since stem cells proliferate and renew themselves through mitosis. However, current automated systems for measuring cell proliferation often require destructive or sacrificial methods of cell manipulation such as cell lysis or in vitro staining. In this paper, we propose an effective approach for automated mitosis detection using phase-contrast time-lapse microscopy, which is a nondestructive imaging modality, thereby allowing continuous monitoring of cells in culture. In our approach, we present a probabilistic model for event detection, which can simultaneously 1) identify spatio-temporal patch sequences that contain a mitotic event and 2) localize a birth event, defined as the time and location at which cell division is completed and two daughter cells are born. Our approach significantly outperforms previous approaches in terms of both detection accuracy and computational efficiency, when applied to multipotent C3H10T1/2 mesenchymal and C2C12 myoblastic stem cell populations. PMID: 21356609 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | An enzyme-modulated oxygen-producing micro-system for regenerative therapeutics. Int J Pharm. 2011 Feb 25; Authors: Abdi SI, Ng SM, Lim JO This study suggests the idea of treating oxygen as a drug in a biological environment and demonstrates that it will exhibit a dosage-dependent trend. To accomplish this, a micro-system was fabricated, having hydrogen peroxide as the oxygen-generating source, which was decomposed using catalase, a common enzyme found in nearly all living organisms. The relevance of the proposed micro-system was justified using cell viability assays under well-controlled and fixed conditions. This study was performed under two controlled conditions, normoxia and hypoxia, and tests were carried out using three different configurations of samples under each condition: direct addition of H(2)O(2), H(2)O(2) encapsulated with single layer, and H(2)O(2) encapsulated with double layers. This study demonstrates that the elegantly designed micro-system managed to control the decomposition of H(2)O(2) and avoided direct contact with cells, while also maintaining cell viability under a low oxygen environment. PMID: 21356297 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Mesenchymal stem cells as a therapeutic tool in tissue and organ regeneration. Postepy Hig Med Dosw (Online). 2011;65:124-32 Authors: Bajek A, Olkowska J, Drewa T Tissue engineering is an interdisciplinary field that offers new opportunities for regeneration of diseased and damaged tissue with the use of many different cell types,including adult stem cells. In tissue engineering and regenerative medicine the most popular are mesenchymal stem cells (MSCs) isolated from bone marrow. Bone marrow mesenchymal stem cells are a potential source of progenitor cells for osteoblasts, chondroblasts, adipocytes, skeletal muscles and cardiomyocytes. It has also been shown that these cells can differentiate into ecto- and endodermal cells, e.g. neuronal cells, glial cells, keratinocytes and hepatocytes. The availability of autologous MSCs, their proliferative potential and multilineage differentiation capacity make them an excellent tool for tissue engineering and regenerative medicine. The aim of this publication is to present characteristic and biological properties of mesenchymal stem cells isolated from bone marrow.<br /> PMID: 21358000 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Amino acid sequence preferences to control cell-specific organization of endothelial cells, smooth muscle cells, and fibroblasts. J Pept Sci. 2011 Feb 24; Authors: Kanie K, Kato R, Zhao Y, Narita Y, Okochi M, Honda H Effective surface modification with biocompatible molecules is known to be effective in reducing the life-threatening risks related to artificial cardiovascular implants. In recent strategies in regenerative medicine, the enhancement and support of natural repair systems at the site of injury by designed biocompatible molecules have succeeded in rapid and effective injury repair. Therefore, such a strategy could also be effective for rapid endothelialization of cardiovascular implants to lower the risk of thrombosis and stenosis. To achieve this enhancement of the natural repair system, a biomimetic molecule that mimics proper cellular organization at the implant location is required. In spite of the fact that many reported peptides have cell-attracting properties on material surfaces, there have been few peptides that could control cell-specific adhesion. For the advanced cardiovascular implants, peptides that can mimic the natural mechanism that controls cell-specific organization have been strongly anticipated. To obtain such peptides, we hypothesized the cellular bias toward certain varieties of amino acids and examined the cell preference (in terms of adhesion, proliferation, and protein attraction) of varieties and of repeat length on SPOT peptide arrays. To investigate the role of specific peptides in controlling the organization of various cardiovascular-related cells, we compared endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts (FBs). A clear, cell-specific preference was found for amino acids (longer than 5-mer) using three types of cells, and the combinational effect of the physicochemical properties of the residues was analyzed to interpret the mechanism. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. PMID: 21360630 [PubMed - as supplied by publisher] | | | | | | | | | |  | |  | |  |
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