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| Spatial and temporal expression pattern of germ layer markers during human embryonic stem cell differentiation in embryoid bodies.
April 7, 2010 at 8:22 AM
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| | Spatial and temporal expression pattern of germ layer markers during human embryonic stem cell differentiation in embryoid bodies. Histochem Cell Biol. 2010 Apr 6; Authors: Pekkanen-Mattila M, Pelto-Huikko M, Kujala V, Suuronen R, Skottman H, Aalto-Setälä K, Kerkelä E Human embryonic stem cell (hESC) differentiation in embryoid bodies (EBs) provides a valuable tool to study the interplay of different germ layers and their influence on cell differentiation. The gene expression of the developing EBs has been shown in many studies, but the protein expression and the spatial composition of different germ layers in human EBs have not been systematically studied. The aim of the present work was to study the temporal and spatial organisation of germ layers based on the expression of mesoderm (Brachyury T), endoderm (AFP) and ectoderm (SOX1) markers during the early stages of differentiation in eight hESC lines. Tissue multi-array technology was applied to study the protein expression of a large number of EBs. According to our results, EB formation and the organisation of germ layers occurred in a similar manner in all the lines. During 12 days of differentiation, all the germ layer markers were present, but no obvious distinct traject! ories were formed. However, older EBs were highly organised in structure. Pluripotency marker OCT3/4 expression persisted unexpectedly long in the differentiating EBs. Cavity formation was observed in the immunocytological sections, and caspase-3 expression was high, suggesting a role of apoptosis in hESC differentiation and/or EB formation. The expression of Brachyury T was notably low in all the lines, also those with the best cardiac differentiation capacity, while the expression of SOX1 was higher in some lines, suggesting that the neural differentiation propensity may be detectable already in the early stages of EB differentiation. PMID: 20369364 [PubMed - as supplied by publisher] | | |
| Implantation of ferumoxides labeled human mesenchymal stem cells in cartilage defects.
April 7, 2010 at 8:22 AM
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| | Implantation of ferumoxides labeled human mesenchymal stem cells in cartilage defects. J Vis Exp. 2010;(38): Authors: Nedopil AJ, Mandrussow LG, Daldrup-Link HE The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo(1, 2). In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular m! atrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses(3). Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques(4). This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging. PMID: 20368696 [PubMed - in process] | | |
| Role of Glycans and Glycosyltransferases in the Regulation of Notch Signaling.
April 7, 2010 at 8:22 AM
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| | Role of Glycans and Glycosyltransferases in the Regulation of Notch Signaling. Glycobiology. 2010 Apr 5; Authors: Jafar-Nejad H, Leonardi J, Fernandez-Valdivia R The evolutionarily conserved Notch signaling pathway plays broad and important roles during embryonic development and in adult tissue homeostasis. Unlike most other pathways used during animal development, Notch signaling does not rely on second messengers and intracellular signaling cascades. Instead, pathway activation results in the cleavage of the Notch intracellular domain and its translocation into the nucleus, where it functions as a transcriptional co-activator of the Notch target genes. To ensure tight spatial and temporal regulation of a pathway with such an unusually direct signaling transduction, animal cells have devised a variety of specialized modulatory mechanisms. One such mechanism takes advantage of decorating the Notch extracellular domain with rare types of O-linked glycans. In this review, we will discuss the genetic and biochemical data supporting the notion that carbohydrate modification is essential for Notch signaling, and attempt to prov! ide a brief historical overview of how we have learned what we know about the glycobiology of Notch. We will also summarize what is known about the contribution of specific nucleotide sugar transporters to Notch biology and the roles-enzymatic and nonenzymatic-played by specific glycosyltransferases in the regulation of this pathway. Mutations in the Notch pathway components cause a variety of human diseases, and manipulation of Notch signaling is emerging as a powerful tool in regenerative medicine. Therefore, studying how sugar modification modulates Notch signaling provides a framework for better understanding the role of glycosylation in animal development and might offer new tools to manipulate Notch signaling for therapeutic purposes. PMID: 20368670 [PubMed - as supplied by publisher] | | |
| Human Adult Vena Saphena Contains Perivascular Progenitor Cells Endowed With Clonogenic and Proangiogenic Potential.
April 7, 2010 at 8:22 AM
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| | Human Adult Vena Saphena Contains Perivascular Progenitor Cells Endowed With Clonogenic and Proangiogenic Potential. Circulation. 2010 Apr 5; Authors: Campagnolo P, Cesselli D, Al Haj Zen A, Beltrami AP, Kränkel N, Katare R, Angelini G, Emanueli C, Madeddu P BACKGROUND: -Clinical trials in ischemic patients showed the safety and benefit of autologous bone marrow progenitor cell transplantation. Non-bone marrow progenitor cells with proangiogenic capacities have been described, yet they remain clinically unexploited owing to their scarcity, difficulty of access, and low ex vivo expansibility. We investigated the presence, antigenic profile, expansion capacity, and proangiogenic potential of progenitor cells from the saphenous vein of patients undergoing coronary artery bypass surgery. Methods and Results-CD34-positive cells, negative for the endothelial marker von Willebrand factor, were localized around adventitial vasa vasorum. After dissection of the vein from surrounding tissues and enzymatic digestion, CD34-positive/CD31-negative cells were isolated by selective culture, immunomagnetic beads, or fluorescence-assisted cell sorting. In the presence of serum, CD34-positive/CD31-negative cells gave rise to a highly pr! oliferative population that expressed pericyte/mesenchymal antigens together with the stem cell marker Sox2 and showed clonogenic and multilineage differentiation capacities. We called this population "saphenous vein-derived progenitor cells" (SVPs). In culture, SVPs integrated into networks formed by endothelial cells and supported angiogenesis through paracrine mechanisms. Reciprocally, endothelial cell-released factors facilitated SVP migration. These interactive responses were inhibited by Tie-2 or platelet-derived growth factor-BB blockade. Intramuscular injection of SVPs in ischemic limbs of immunodeficient mice improved neovascularization and blood flow recovery. At 14 days after transplantation, proliferating SVPs were still detectable in the recipient muscles, where they established N-cadherin-mediated physical contact with the capillary endothelium. Conclusions-SVPs generated from human vein CD34-positive/CD31-negative progenitor cells might represent a new therap! eutic tool for angiogenic therapy in ischemic patients. PMID: 20368523 [PubMed - as supplied by publisher] | | |
| Functional repair of human donor lungs by IL-10 gene therapy.
April 7, 2010 at 8:22 AM
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| | Functional repair of human donor lungs by IL-10 gene therapy. Sci Transl Med. 2009 Oct 28;1(4):4ra9 Authors: Cypel M, Liu M, Rubacha M, Yeung JC, Hirayama S, Anraku M, Sato M, Medin J, Davidson BL, de Perrot M, Waddell TK, Slutsky AS, Keshavjee S More than 80% of potential donor lungs are injured during brain death of the donor and from complications experienced in the intensive care unit, and therefore cannot be used for transplantation. These lungs show inflammation and disruption of the alveolar-capillary barrier, leading to poor gas exchange. Although the number of patients in need of lung transplantation is increasing, the number of donors is static. We investigated the potential to use gene therapy with an adenoviral vector encoding human interleukin-10 (AdhIL-10) to repair injured donor lungs ex vivo before transplantation. IL-10 is an anti-inflammatory cytokine that mainly exerts its suppressive functions by the inactivation of antigen-presenting cells with consequent inhibition of proinflammatory cytokine secretion. In pigs, AdhIL-10-treated lungs exhibited attenuated inflammation and improved function after transplantation. Lungs from 10 human multiorgan donors that had suffered brain death were ! determined to be clinically unsuitable for transplantation. They were then maintained for 12 hours at body temperature in an ex vivo lung perfusion system with or without intra-airway delivery of AdhIL-10 gene therapy. AdhIL-10-treated lungs showed significant improvement in function (arterial oxygen pressure and pulmonary vascular resistance) when compared to controls, a favorable shift from proinflammatory to anti-inflammatory cytokine expression, and recovery of alveolar-blood barrier integrity. Thus, treatment of injured human donor lungs with the cytokine IL-10 can improve lung function, potentially rendering injured lungs suitable for transplantation into patients. PMID: 20368171 [PubMed - in process] | | |
| Optimization of Fibrinogen Isolation for Manufacturing Autologous Fibrin Glue for Use as Scaffold in Tissue Engineering.
April 7, 2010 at 8:22 AM
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| | Optimization of Fibrinogen Isolation for Manufacturing Autologous Fibrin Glue for Use as Scaffold in Tissue Engineering. Artif Cells Blood Substit Immobil Biotechnol. 2010 Apr 6; Authors: Froelich K, Pueschel RC, Birner M, Kindermann J, Hackenberg S, Kleinsasser NH, Hagen R, Staudenmaier R Abstract: Numerous manufacturing techniques for autogenous fibrin glue used as scaffold material have been described. As there is no consensus regarding the influence of chemical additives on cell biology, it was the aim of this study to establish a method for manufacturing autologous fibrin glue without any additives. The serum part was separated from whole blood. After fibrinogen precipitation, centrifugation was performed to obtain the fibrinogen pellet. Various experimental series were run to examine influences of various temperatures or substituting centrifugation for sedimentation. The method as described here is effective, simple, and performed without any additives, which could potentially influence cell biology. PMID: 20367549 [PubMed - as supplied by publisher] | | |
| Prospective isolation of mesenchymal stem cells from multiple mammalian species using cross-reacting anti human monoclonal antibodies.
April 7, 2010 at 8:22 AM
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| | Prospective isolation of mesenchymal stem cells from multiple mammalian species using cross-reacting anti human monoclonal antibodies. Stem Cells Dev. 2010 Apr 5; Authors: Rozemuller H, Prins HJ, Naaijkens BA, Staal J, Bühring HJ, Martens AC Mesenchymal stem cells (MSC) of human and non-human mammalian species are often studied for various applications in regenerative medicine research. These MSC can be derived from human bone marrow (BM) and identified by their ability to form fibroblast-like colony forming units (CFU-F) that develop into stromal like cells when expanded in culture. These cells are characterized by their spindle-shaped morphology, their characteristic phenotype (CD73+, CD90+, CD105+, CD45- and CD34-) and their ability to differentiate into cells of the osteogenic, adipogenic, and chondrogenic lineages. However, the identification and purification of MSC from non-human mammalian species is hampered by the lack of suitable monoclonal antibodies (mAb). In this report, primary BM as well as cultured BM-derived MSC of human and monkey, goat, sheep, dog and pig were screened for cross-reactivity using a panel of 43 mAb, of which 22 react with either human BM mononuclear cells or cultured h! uman MSC. We found seven mAb with specificity for CD271, MSCA-1 (W8B2 antigen), W4A5, CD56, W3C4 (CD349), W5C4 and 58B1, which showed interspecies cross-reactivity. These mAb proved to be useful for prospective sorting of MSC from the BM of the six mammalian species studied as well as for the characterization of their cultured offspring. Flow sorting with the cross-reacting mAb resulted in an up to 2400-fold enrichment of the clonogenic cell fraction (CFU-F). This study provides an important contribution for the comparative prospective isolation of primary BM-MSC and the characterization of cultured MSC from multiple mammalian species for preclinical research. PMID: 20367498 [PubMed - as supplied by publisher] | | |
| Cancellous Bone Osseointegration is Enhanced by In Vivo Loading.
April 7, 2010 at 8:22 AM
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| | Cancellous Bone Osseointegration is Enhanced by In Vivo Loading. Tissue Eng Part C Methods. 2010 Apr 2; Authors: Willie BM, Yang X, Kelly NH, Han J, Nair T, Wright TM, van der Meulen M, Bostrom M Biophysical stimuli may be an effective therapy to counteract age-related changes in bone structure that affect the primary stability of implants used in joint replacement or fracture fixation. The influence of controlled mechanical loading on osseointegration was investigated using an in vivo device implanted in the distal lateral femur of twelve male rabbits. Compressive loads (1 MPa, 1 Hz, 50 cycles/day, 4 weeks) were applied to a porous titanium foam implant and the underlying cancellous bone. The contralateral limbs served as nonloaded controls. Backscattered electron imaging indicated that the amount of bone ingrowth was significantly greater in the loaded limb compared to the nonloaded control limb, while the amount of underlying cancellous periprosthetic bone was similar. No significant difference in the mineral apposition rate of the bone ingrowth or periprosthetic bone was measured in the loaded compared to the control limb. Histological analysis demonst! rated newly formed woven bone in direct apposition to the implant coating, with a lack of fibrous tissue at the implant-periprosthetic bone interface in both loaded and nonloaded implants. The lack of fibrous tissue demonstrates that mechanical stimulation using this model significantly enhanced cancellous bone ingrowth without the detrimental effects of micromotion. These results suggest that biophysical therapy should be further investigated to augment current treatments to enhance long-term fixation of orthopaedic devices. Additionally, this novel in vivo loading model can be used to further investigate the influence of biophysical stimulation on other tissue engineering approaches requiring bone ingrowth into both metallic and nonmetallic cell seeded scaffolds. PMID: 20367497 [PubMed - as supplied by publisher] | | |
| KRas and BRaf mutational status analysis from formalin-fixed, paraffin-embedded tissues using multiplex polymerase chain reaction-based assay.
April 7, 2010 at 8:22 AM
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| | KRas and BRaf mutational status analysis from formalin-fixed, paraffin-embedded tissues using multiplex polymerase chain reaction-based assay. Arch Pathol Lab Med. 2010 Apr;134(4):620-4 Authors: Jakubauskas A, Griskevicius L CONTEXT: Monoclonal anti-epidermal growth factor receptor antibodies bind to the epidermal growth factor receptor and inhibit receptor kinase activity. Clinical trials have indicated that evaluation of the mutational status of KRas and BRaf is necessary to exclude patients who are resistant to the clinical benefit of anti-epidermal growth factor receptor therapy. OBJECTIVE: To develop a multiplex polymerase chain reaction-based assay for the evaluation of KRas and BRaf mutational status. DESIGN: A sample-saving and cost-effective, multiplex polymerase chain reaction-based assay to detect somatic mutations in KRAS exon 2 and exon 3 as well as in BRAF exon 15 was developed. The same primer pairs could be successfully used in amplification of a single DNA fragment under the same conditions. RESULTS: DNA isolated from 20 retrospective formalin-fixed, paraffin-embedded samples of colorectal cancer was screened for mutations using the multiplex polymerase chain reaction! assay followed by dideoxy-termination sequencing. Five samples bearing mutations-p.G12D (identified twice), p.G12V, p.G12S, and p.G13D, all encoded in KRAS exon 2-were identified. Three samples were found bearing amino acid substitution p.V600E of BRaf. The detected KRas and BRaf mutations were found to be mutually exclusive. CONCLUSIONS: A multiplex polymerase chain reaction-based amplification followed by dideoxy-termination sequencing may be used advantageously for the evaluation of KRas and BRaf mutational status from formalin-fixed, paraffin-embedded samples. PMID: 20367313 [PubMed - in process] | | |
| Electric field stimulation integrated into perfusion bioreactor for cardiac tissue engineering.
April 7, 2010 at 8:22 AM
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| | Electric field stimulation integrated into perfusion bioreactor for cardiac tissue engineering. Tissue Eng Part C Methods. 2010 Apr 2; Authors: Barash Y, Dvir T, Tandeitnik P, Ruvinov E, Guterman H, Cohen S We describe herein the features of a novel cultivation system, combining electrical stimulation with medium perfusion for producing thick, functional cardiac patches. A custom-made electrical stimulator was integrated via inserting two carbon rod electrodes into a perfusion bioreactor, housing multiple neonatal SD rat cardiac cell constructs between two 96% open-pore-area fixing nets. The stimulator produced adjustable stimulation waveform (i.e. duty cycle, number of stimulating channels, maximum stimulation amplitude, etc.), specially-designed for cardiac cell stimulation. The cell constructs were subjected to a homogenous fluid flow regime and electrical stimulation under conditions optimal for cell excitation. The stimulation threshold in the bioreactor was set by first determining its value in a Petri dish under a microscope, and then matching the current density in the two cultivation systems by constructing electric field models. The models were built by Com! sol Multiphysics software using the exact three-dimensional geometry of the two cultivation systems. These models illustrate, for the first time, the local electric conditions required for cardiomyocyte field excitation and they confirmed the uniformity of the electrical field around the cell constructs. Bioreactor cultivation for only 4 d under perfusion and continuous electrical stimulus (74.4 mA/cm;2 , 2 ms, bipolar, 1 Hz) promoted cell elongation and striation in the cell constructs and enhanced the expression level of connexin-43, the gap junction protein responsible for cell-cell coupling. These results thus confirm the validity of the electrical field model in predicting the optimal electrical stimulation in a rather complex cultivation system, a perfusion bioreactor. PMID: 20367291 [PubMed - as supplied by publisher] | | |
| Enhanced transfection efficiency of human embryonic stem cells by the incorporation of DNA liposomes in extracellular matrix.
April 7, 2010 at 8:22 AM
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| | Enhanced transfection efficiency of human embryonic stem cells by the incorporation of DNA liposomes in extracellular matrix. Stem Cells Dev. 2010 Apr 1; Authors: Villa-Diaz LG, Garcia-Perez JL, Krebsbach PH Because they can be differentiated to virtually any cell type in the human body, human embryonic stem (hES) cells hold promise for regenerative medicine. The genetic manipulation of hES cells will enhance our understanding of genes involved in early development and will accelerate their potential use and applications for regenerative medicine. The objective of this study was to increase the transfection efficiency of plasmid DNA into hES cells by modifying a standard reverse transfection protocol of lipofection. We hypothesized that immobilization of plasmid DNA in extracellular matrix (ECM) would be a more efficient method for plasmid transfer, due to the affinity of hES cells to substrates such as Matrigel and to the prolonged exposure of cells to plasmid DNA. Our results demonstrate that this modification doubled the transient transfection efficiency of hES cells and the generation of clonal cell lines containing a piece of foreign DNA stably inserted in their ! genomes compared to results obtained with standard forward transfection. In addition, treatment with DMSO further increased the transfection efficiency of hES cells. In conclusion, modifications to the reverse transfection protocol of lipofection result in a significant and robust increase in the transfection efficiency of hES cells. PMID: 20367256 [PubMed - as supplied by publisher] | | |
| New Dimensions in Vascular Engineering: Opportunities for Cancer Biology.
April 7, 2010 at 8:22 AM
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| | New Dimensions in Vascular Engineering: Opportunities for Cancer Biology. Tissue Eng Part A. 2010 Apr 1; Authors: Rabbany SY, James D, Rafii S Abstract. Angiogenesis is a fundamental pre-requisite for tissue growth, and thus an attractive target for cancer therapeutics. However, current efforts to halt tumor growth using anti-angiogenic agents have been met with limited success. A reason for this may be that studies aimed at understanding tissue and organ formation have to this point utilized two-dimensional cell culture techniques, which fail to faithfully mimic the pathological architecture of disease in an in vivo context. In this issue of Tissue Engineering, the work of Fischbach-Teschl's group manipulate such variables as oxygen concentration, culture three-dimensionality, and cell-extracellular matrix interactions to more closely approximate the biophysical and biochemical microenvironment of tumor angiogenesis. In this manuscript, we discuss how novel tissue engineering platforms provide a framework for the study of tumorigenesis under pathophysiologically relevant in-vitro culture conditions. PMID: 20367255 [PubMed - as supplied by publisher] | | |
| EVALUATION OF THE COMPLEX TRANSCRIPTIONAL TOPOGRAPHY OF MESENCHYMAL STEM CELL CHONDROGENESIS FOR CARTILAGE TISSUE ENGINEERING.
April 7, 2010 at 8:22 AM
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| | EVALUATION OF THE COMPLEX TRANSCRIPTIONAL TOPOGRAPHY OF MESENCHYMAL STEM CELL CHONDROGENESIS FOR CARTILAGE TISSUE ENGINEERING. Tissue Eng Part A. 2010 Apr 1; Authors: Huang AH, Stein A, Mauck RL Mesenchymal stem cells (MSCs) are a promising cell source for cartilage tissue engineering given their chondrogenic potential. This potential has yet to be fully realized, as the mechanical properties of MSC-based constructs are lower than those of chondrocyte-based constructs cultured identically. Therefore the aim of the current study was to better understand the transcriptional underpinnings of this functional limitation. Matched chondrocytes and MSCs from three donors were cultured in agarose in a defined media containing TGF-beta3. We evaluated the compressive mechanical properties and matrix deposition of maturing constructs over 56 days. Transcriptional differences between the two cell types were assessed on day 0 and day 28 constructs via microarray analysis and real-time PCR; differential deposition of matrix molecules was assessed by immunohistochemistry. Although the mechanical and biochemical properties of cell-seeded constructs improved with culture d! uration, MSC values plateaued at day 28, and remained lower than chondrocyte values. Using microarray analysis, 324 genes were identified as mis-expressed during chondrogenesis. Differential expression of 18 genes was validated, and differential deposition of proteoglycan 4 (PRG4) and transforming-growth-factor-beta induced 68kDa protein (TGFBI) was confirmed. Temporal expression profiles of these 18 genes showed that some genes were never expressed (chondromodulin), some were expressed at lower levels (PRG4), and some were expressed only at later time points (TGFBI) in MSCs compared to chondrocytes. These findings further define the complex transcriptional topography and mechanobiology of MSC chondrogenesis, and provide new benchmarks for optimizing the growth of MSC-based engineered cartilage. PMID: 20367254 [PubMed - as supplied by publisher] | | |
| Wnt/beta-catenin signaling regulates postnatal development and regeneration of the salivary gland.
April 7, 2010 at 8:22 AM
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| | Wnt/beta-catenin signaling regulates postnatal development and regeneration of the salivary gland. Stem Cells Dev. 2010 Mar 30; Authors: Hai B, Yang Z, Millar SE, Choi YS, Taketo MM, Nagy A, Liu F Regenerative therapy of the salivary gland is a promising therapeutic approach for irreversible hyposalivation in Head and Neck Cancer patients treated with radiotherapy. However, little is known about the molecular regulators of stem/progenitor cell activity and regenerative processes in the salivary gland. Wnt/beta-catenin signaling regulates the proliferation of several adult stem cell populations, but its role in salivary gland development and regeneration is unknown. Using BAT-gal Wnt reporter transgenic mice, we demonstrate that in the submandibular glands (SMG) of newborn mice Wnt/beta-catenin signaling is active in a few cells at the basal layer of intercalated ducts, the putative location of salivary gland stem/progenitor cells. Wnt activity decreases as mice age, but is markedly enhanced in salivary gland ducts during regeneration of adult SMG after ligation of the main secretory duct. The Hedgehog pathway is also activated after duct ligation. Inhibitio! n of epithelial beta-catenin signaling in young Keratin5-rtTA/tetO-Dkk1 mice impairs the postnatal development of SMG, particularly affecting maturation of Granular Convoluted Tubules. Conversely, forced activation of epithelial beta-catenin signaling in adult Keratin5-rtTA/tetO-Cre/Ctnnb1(Ex3)fl mice promotes proliferation of ductal cells, expansion of the salivary gland stem/progenitor cell compartment, and ectopic activation of Hedgehog signaling. Taken together, these results indicate that Wnt/beta-catenin signaling regulates the activity of salivary gland stem/progenitor cells during postnatal develepment and regeneration upstream of the Hedgehog pathway, and suggest the potential of modulating Wnt/beta-catenin and/or Hedgehog pathways for functional restoration of salivary glands after irradiation. PMID: 20367250 [PubMed - as supplied by publisher] | | |
| Sonic Hedgehog influences the balance of osteogenesis and adipogenesis in mouse adipose-derived stromal cells.
April 7, 2010 at 8:22 AM
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| | Sonic Hedgehog influences the balance of osteogenesis and adipogenesis in mouse adipose-derived stromal cells. Tissue Eng Part A. 2010 Mar 30; Authors: James AW, Leucht P, Levi B, Carre AL, Xu Y, Helms J, Longaker M Adipose-derived stromal cells (ASCs) present a great potential for tissue engineering, as they are capable of differentiating into osteogenic and adipogenic cell types, among others. In this study, we examined the role of Hedgehog signaling in the balance of osteogenic and adipogenic differentiation in mouse ASCs. Results showed Hedgehog signaling increased during early osteogenic differentiation (Shh, Ptc1, Gli1), but decreased during adipogenic differentiation. N-terminal Sonic Hedgehog (Shh-N) significantly increased in vitro osteogenic differentiation in mASCs, by all markers examined (*P < 0.01). Concomitantly, Shh-N abrogated adipogenic differentiation, by all markers examined (*P < 0.01). Conversely, blockade of endogenous Hedgehog signaling, with the Hedgehog antagonist cyclopamine, enhanced adipogenesis at the expense of osteogenesis. We next translated these results to a mouse model of appendicular skeletal regeneration. Using qRT-PCR and in situ h! ybridization, we found that skeletal injury (a monocortical 1mm defect in the tibia) results in a localized increase in Hedgehog signaling. Moreover, grafting of ASCs treated with Shh-N resulted in significantly increased bone regeneration within the defect site. In conclusion, Hedgehog signaling enhances the osteogenic differentiation of mASCs, at the expense of adipogenesis. These data suggest that Hedgehog signaling directs the lineage differentiation of mesodermal stem cells and represents a promising strategy for skeletal tissue regeneration. PMID: 20367246 [PubMed - as supplied by publisher] | | |
| Osteoblastic Cell Proliferation with Uniform Distribution in a Large Scaffold using Radial-Flow Bioreactor.
April 7, 2010 at 8:22 AM
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| | Osteoblastic Cell Proliferation with Uniform Distribution in a Large Scaffold using Radial-Flow Bioreactor. Tissue Eng Part C Methods. 2010 Mar 30; Authors: Arano T, Sato T, Matsuzaka K, Ikada Y, Yoshinari MY Bioreactors employing different types of in vitro physiological cell stimulation have been developed to obtain three-dimensional cultivation for tissue engineering. The purpose of this study was to determine whether osteoblastic cells proliferated uniformly over a large scaffold with a diameter of 18 mm and height of 10 mm under dynamic cultivation with the radial-flow bioreactor (RFB), and thereby ascertain the potential of this system in the regeneration of jaw bone. Mouse osteoblastic cells (MC3T3-E1) were seeded onto type-1 collagen sheets. Cells were then incubated outside the reactor for 6 hours to produce pre-cultured sheets. The 6 pre-cultured sheets were then placed in the RFB to fabricate the scaffolds. Cells were dynamically cultured for one week at 37 C, pH 7.4, DO 6.86 ppm, and with the culture medium circulating at 3 mL/min. As a control, static cultivation cultured in the same manner without circulating culture medium and single cultivation in a cul! ture dish were performed. The resulting cell proliferation and cell distribution were analyzed. After one week of dynamic cultivation, cells showed a 5-fold and 4-fold increase with uniform distribution throughout the three-dimensional scaffolds than those under the static and single cultivation as a control, respectively. These results indicate that the RFB is a promising system for three-dimensional cultivation of osteoblastic cells for treating large bone defects by tissue engineering. PMID: 20367244 [PubMed - as supplied by publisher] | | |
| Mesenchymal stem cell-encapsulated collagen microspheres for bone tissue engineering.
April 7, 2010 at 8:22 AM
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| | Mesenchymal stem cell-encapsulated collagen microspheres for bone tissue engineering. Tissue Eng Part C Methods. 2010 Apr;16(2):225-35 Authors: Chan BP, Hui TY, Wong MY, Yip KH, Chan GC There is a demonstrated clinical need for alternatives of autologous fresh bone graft with excellent biological performance in osteoconductivity, osteoinductivity, and osteogenicity. We previously developed a collagen microencapsulation technology entrapping bone marrow-derived mesenchymal stem cells (MSCs) in a biomimetic collagen fiber meshwork and produced injectable collagen-MSC microspheres. In this study, we hypothesize that injectable microspheres with osteoconductivity, osteogenicity, and osteoinductivity can be fabricated by differentiating the encapsulated MSCs, from either human or mouse sources, toward osteogenic lineages in these three-dimensional microspheres. The osteogenicity, osteoconductivity, and osteoinductivity of the microspheres were evaluated in vitro. Osteogenic markers of the differentiating MSCs including alkaline phosphatase and calcium deposition showed positive staining. Osteoconductivity of the collagen meshwork in the microsphere wa! s demonstrated by the presence of calcium phosphate deposits among the collagen fibers and by the significantly increased calcium content extracted from the microspheres. Moreover, osteoinductivity of the MSC-encapsulated microspheres was demonstrated by the ability to induce osteogenic differentiation of undifferentiated MSCs in both contact and noncontact coculture. This study contributes toward the future development of injectable alternatives for fresh bone grafts using autologous MSCs. PMID: 20367213 [PubMed - in process] | | |
| Stem cells for tendon tissue engineering and regeneration.
April 7, 2010 at 8:22 AM
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| | Stem cells for tendon tissue engineering and regeneration. Expert Opin Biol Ther. 2010 Apr 2; Authors: Yin Z, Chen X, Chen JL, Ouyang HW Importance of the field: Tendon injuries are common especially in sports activities, but tendon is a unique connective tissue with poor self-repair capability. With advances in stem cell biology, tissue engineering is becoming increasingly powerful for tissue regeneration. Stem cells with capacity of multipotency and self-renewal are an ideal cell source for tissue engineering. Areas covered in this review: This review focus on discussing the potential strategies including inductive growth factors, bio-scaffolds, mechanical stimulation, genetic modification and co-culture techniques to direct tendon-lineage differentiation of stem cells for complete tendon regeneration. Attempting to use embryonic stem cells as seed cells for tendon tissue engineering have achieved encouraging results. The combination of chemical and physical signals in stem cell microenvironment could be regulated to induce differentiation of the embryonic stem cells into tendon. What the reader ! will gain: We summarize fundamental questions, as well as future directions in tendon biology and tissue engineering. Take home message: Multifaceted technologies are increasingly required to control stem cell differentiation, to develop novel stem cell-based therapy, and, ultimately, to achieve more effective repair or regeneration of injured tendons. PMID: 20367125 [PubMed - as supplied by publisher] | | |
| Acellular dermal matrix seeded with autologous gingival fibroblasts for the treatment of gingival recession: a proof-of-concept study.
April 7, 2010 at 8:22 AM
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| | Acellular dermal matrix seeded with autologous gingival fibroblasts for the treatment of gingival recession: a proof-of-concept study. J Periodontol. 2010 Apr;81(4):616-25 Authors: Jhaveri HM, Chavan MS, Tomar GB, Deshmukh VL, Wani MR, Miller PD BACKGROUND: One of the most common esthetic concerns associated with periodontal tissues is gingival recession. There are multiple periodontal plastic surgery approaches documented in the literature for the treatment of such defects. With the tremendous advances being made in periodontal science and technology, tissue engineering could be considered among the latest exciting techniques for recession management. METHODS: In this split-mouth, controlled, double-masked clinical case series, 20 sites from 10 patients with Miller Class I or II recessions affecting canines or premolars in the maxillary arch were selected. One tooth in each patient was randomized to receive either a subepithelial connective tissue graft (SCTG) (control group) or an acellular dermal matrix allograft (ADMA) seeded with autologous gingival fibroblasts (test group) under a coronally positioned flap. Clinical parameters, including recession depth, probing depth, clinical attachment level, wid! th of keratinized tissue, attached gingiva, and plaque scores, were recorded by a calibrated examiner at baseline and 3 and 6 months. The inflammation of grafted sites was scored, and the healing time was calculated. The final esthetic outcome of treated sites was assessed by the root coverage esthetic score at the end of 6 months. RESULTS: There were no significant differences between test and control sites for all measured clinical parameters. However, the test sites demonstrated less inflammation in the early postoperative period. CONCLUSION: Within the limits of this case series, the results indicate that an ADMA seeded with autologous gingival fibroblasts by tissue-engineering technology may be explored as a substitute to an SCTG for the treatment of Miller Class I and II recession defects. PMID: 20367104 [PubMed - in process] | | |
| Introducing the scanning air puff tonometer for biological studies.
April 7, 2010 at 8:22 AM
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| | Introducing the scanning air puff tonometer for biological studies. Phys Rev E Stat Nonlin Soft Matter Phys. 2010 Feb;81(2 Pt 1):021920 Authors: Fleury V, Al-Kilani A, Boryskina OP, Cornelissen AJ, Nguyen TH, Unbekandt M, Leroy L, Baffet G, le Noble F, Sire O, Lahaye E, Burgaud V It is getting increasingly evident that physical properties such as elastoviscoplastic properties of living materials are quite important for the process of tissue development, including regulation of genetic pathways. Measuring such properties in vivo is a complicated and challenging task. In this paper, we present an instrument, a scanning air puff tonometer, which is able to map point by point the viscoelastic properties of flat or gently curved soft materials. This instrument is an improved version of the air puff tonometer used by optometrists, with important modifications. The instrument allows one to obtain a direct insight into gradients of material properties in vivo. The instrument capabilities are demonstrated on substances with known elastoviscoplastic properties and several biological objects. On the basis of the results obtained, the role of the gradients of elastoviscoplastic properties is outlined for the process of angiogenesis, limb development, ! bacterial colonies expansion, etc. which is important for bridging the gaps in the theory of the tissue development and highlighting new possibilities for tissue engineering, based on a clarification of the role of physical features in developing biological material. PMID: 20365608 [PubMed - in process] | | |
| Stochastic model of self-assembly of cell-laden hydrogels.
April 7, 2010 at 8:22 AM
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| | Stochastic model of self-assembly of cell-laden hydrogels. Phys Rev E Stat Nonlin Soft Matter Phys. 2009 Dec;80(6 Pt 1):061901 Authors: Shi Z, Chen N, Du Y, Khademhosseini A, Alber M Recent progress in bottom-up tissue engineering has demonstrated that three-dimensional tissue constructs with predefined architectures may be obtained by assembling shape-controlled hydrogels in multiphase reactor systems. Driven by the hydrophobic force between gel unit and liquid media, highly ordered hydrogel clusters can be formed. Many complex factors occurring at microscale (i.e., gel unit collisions, hydrophobic forces, and gel unit movement) are involved in the self-assembly process. In this paper a two-dimensional off-lattice Monte Carlo model with Lennard-Jones-type potential describing unit-unit interactions is introduced for studying this process. Simulations are shown to agree well with the experimental results for hydrogel assembly in mineral oil. The simulation method is demonstrated for rectangular hydrogel units of different aspect ratios as well as extended to the case of more complex hydrogel unit geometries. PMID: 20365184 [PubMed - in process] | | |
| Intraepidermal injection of dissociated epidermal cell suspension improves vitiligo.
April 7, 2010 at 8:22 AM
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| | Intraepidermal injection of dissociated epidermal cell suspension improves vitiligo. Arch Dermatol Res. 2010 Apr 4; Authors: Khodadadi L, Shafieyan S, Sotoudeh M, Dizaj AV, Shahverdi A, Aghdami N, Baharvand H This study was initiated to evaluate the safety and effectiveness of intraepidermal injection of dissociated epidermal cells into the lesions of stable vitiligo patients. Autologous dissociated epidermal cell suspensions were injected intraepidermally into 10 stable vitiligo patients. None of the patients received adjuvant therapy. The response was evaluated as: marked (76-100%), moderate (51-75%), mild (26-50%) and minimal repigmentation (0-25%). Transmission electron microscopy was used to evaluate the transplanted cells and immunohistochemical staining with HMB-45 was performed to assess the repigmentation in vivo. In all cases, repigmentation started during the 4-week period after transplantation. Six months after transplantation, a marked repigmentation in four (40%), moderate repigmentation in two (20%) and mild repigmentation in two (20%) patients were observed. Two (20%) patients with white patches on their lids showed minimal repigmentation. No side effec! ts were observed in any patients. Interestingly, repigmentation of gray hair in one patient, 4 months post transplantation was observed. Analysis of the ultrastructure of transplanted cells showed 1.5% of the cells had melanocyte morphology. HMB-45 positive cells were observed after cell transplantation. This method is an effective, simple and safe therapeutic option for stable vitiligo lesions. PMID: 20364383 [PubMed - as supplied by publisher] | | |
| Generation of induced pluripotent stem cells from newborn marmoset skin fibroblasts.
April 7, 2010 at 8:22 AM
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| | Generation of induced pluripotent stem cells from newborn marmoset skin fibroblasts. Stem Cell Res. 2010 Mar 6; Authors: Wu Y, Zhang Y, Mishra A, Tardif SD, Hornsby PJ Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine. For the application of iPSCs to forms of autologous cell therapy, suitable animal models are required. Among species that could potentially be used for this purpose, nonhuman primates are particularly important, and among these the marmoset offers significant advantages. In order to demonstrate the feasibility of the application of iPSC technology to this species, here we derived lines of marmoset iPSCs. Using retroviral transduction with human Oct4, Sox2, Klf4 and c-Myc, we derived clones that fulfil critical criteria for successful reprogramming: they exhibit typical iPSC morphology; they are alkaline phosphatase positive; they express high levels of NANOG, OCT4 and SOX2 mRNAs, while the corresponding vector genes are silenced; they are immunoreactive for Oct4, TRA-1-81 and SSEA-4; and when implanted into immunodeficient mice they produce teratomas that have derivatives of all t! hree germ layers (endoderm, alpha-fetoprotein; ectoderm, betaIII-tubulin; mesoderm, smooth muscle actin). Starting with a population of 4x10(5) newborn marmoset skin fibroblasts, we obtained approximately 100 colonies with iPSC-like morphology. Of these, 30 were expanded sufficiently to be cryopreserved, and, of those, 8 were characterized in more detail. These experiments provide proof of principle that iPSC technology can be adapted for use in the marmoset, as a future model of autologous cell therapy. PMID: 20363201 [PubMed - as supplied by publisher] | | |
| How far are induced pluripotent stem cells from the clinic?
April 7, 2010 at 8:22 AM
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| | How far are induced pluripotent stem cells from the clinic? Ageing Res Rev. 2010 Mar 31; Authors: Li M, Chen M, Han W, Fu X Induced pluripotent stem cells (iPSCs) can be derived from diverse different somatic cells and share many of the characteristics of embryonic stem cells (ESCs). Because iPSCs avoid many of the ethical concerns associated with the use of embryonic or fetal material, iPSCs have great potential in cell-based regenerative medicine. However, several hurdles will need to be surmounted before their potential can be realized in therapeutic applications. For example, the use of viral vectors, some of which are oncogenes raises the risk of tumor formation in patients, the differentiation of iPSCs into required functional cells in vivo remains to be established, the obtaining of pure populations of target cells from iPSCs is still difficult. Of these, some are shared by both iPSCs and ESCs, others are unique to iPSCs. We will describe these stumbling blocks in detail and discuss possible ways to overcome them. Despite many significant advances, there is as yet no technologic! al framework that would allow the exploitation of iPSCs in a clinical setting in the immediate future. Further research will be required before directed reprogramming can provide a source of cells suitable for application in regenerative medicine. PMID: 20362696 [PubMed - as supplied by publisher] | | |
| Cell contraction forces in scaffolds with varying pore size and cell density.
April 7, 2010 at 8:22 AM
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| | Cell contraction forces in scaffolds with varying pore size and cell density. Biomaterials. 2010 Mar 31; Authors: Corin KA, Gibson LJ The contractile behavior of cells is relevant in understanding wound healing and scar formation. In tissue engineering, inhibition of the cell contractile response is critical for the regeneration of physiologically normal tissue rather than scar tissue. Previous studies have measured the contractile response of cells in a variety of conditions (e.g. on two-dimensional solid substrates, on free-floating tissue engineering scaffolds and on scaffolds under some constraint in a cell force monitor). Tissue engineering scaffolds behave mechanically like open-cell elastomeric foams: between strains of about 10 and 90%, cells progressively buckle struts in the scaffold. The contractile force required for an individual cell to buckle a strut within a scaffold has been estimated based on the strut dimensions (radius, r, and length, l) and the strut modulus, E(s). Since the buckling force varies, according to Euler's law, with r(4)/l(2), and the relative density of the scaf! fold varies as (r/l)(2), the cell contractile force associated with strut buckling is expected to vary with the square of the pore size for scaffolds of constant relative density. As the cell density increases, the force per cell to achieve a given strain in the scaffold is expected to decrease. Here we model the contractile response of fibroblasts by analyzing the response of a single tetrakaidecahedron to forces applied to individual struts (simulating cell contractile forces) using finite element analysis. We model tetrakaidecahedra of different strut lengths, corresponding to different scaffold pore sizes, and of varying numbers of loaded struts, corresponding to varying cell densities. We compare our numerical model with the results of free-floating contraction experiments of normal human dermal fibroblasts (NHDF) in collagen-GAG scaffolds of varying pore size and with varying cell densities. PMID: 20362329 [PubMed - as supplied by publisher] | | |
| 5-Aza-2'-deoxycytidine treatment induces skeletal myogenic differentiation of mouse dental pulp stem cells.
April 7, 2010 at 8:22 AM
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| | 5-Aza-2'-deoxycytidine treatment induces skeletal myogenic differentiation of mouse dental pulp stem cells. Arch Oral Biol. 2010 Mar 31; Authors: Nakatsuka R, Nozaki T, Uemura Y, Matsuoka Y, Sasaki Y, Shinohara M, Ohura K, Sonoda Y OBJECTIVE: Tissue stem cells in dental pulp are assumed to possess differentiation potentials similar to mesenchymal stem cells (MSCs). The aim of this in vitro study is to examine the differentiation potentials of mouse dental pulp stem cells (DPSCs) and develop the appropriate differentiation assay systems for skeletal myogenic differentiation of these cells. METHODS: Dental pulps were extracted from mandible sections of C57/BL6 mice, and adherent dental pulp cells were isolated in culture. These cells were cultured in osteogenic or adipogenic induction medium to induce osteogenic and adipogenic differentiation. On the other hand, the skeletal myogenic differentiation potential of these cells was investigated using different conditions, such as serum-free medium, Myod1 overexpression, or 5-Aza-2'-deoxycytidine (5-Aza) treatment for DNA demethylation. Muscle-specific transcriptional factor expression was evaluated by RT-PCR, and myotube formation and myosin heavy! chain expression were evaluated by phase-contrast microscopy and immunofluorescence staining, respectively. RESULTS: The adherent dental pulp cells exhibited a proliferative capacity and they showed osteogenic and adipogenic differentiation as seen in previous studies. Although the expression of Myod1 mRNA and myotube formation was not detected in serum-free conditions, the forced expression of Myod1 up-regulated the expression of Myogenin and Pax7 mRNA. However, myotube formation was not confirmed. Interestingly, myosin heavy chain expression and myotube formation were observed following 5-Aza treatment of these cells. CONCLUSIONS: These results demonstrated that mouse DPSCs possess MSC-like differentiation potential. DNA demethylation induced by 5-Aza treatment resulted in the skeletal muscle differentiation in mouse DPSCs, suggesting that DNA demethylation might trigger this differential induction of mouse DPSCs. PMID: 20362276 [PubMed - as supplied by publisher] | | |
| Biocomposites containing Natural Polymers and Hydroxyapatite for Bone Tissue Engineering.
April 7, 2010 at 8:22 AM
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| | Biocomposites containing Natural Polymers and Hydroxyapatite for Bone Tissue Engineering. Int J Biol Macromol. 2010 Mar 30; Authors: Swetha M, Sahithi K, Moorthi A, Srinivasan N, Ramasamy K, Selvamurugan N Bone tissue engineering is an alternative strategy to generate bone utilizing a combination of biomaterials and cells. Biomaterials that mimic the structure and composition of bone tissues at nano scale are important for the development of bone tissue engineering applications. Natural or biopolymer-based composites containing chitin, chitosan, or collagen have advantages such as biocompatibility, biodegradability that are essential for bone tissue engineering. The inclusion of nanoparticles of hydroxyapatite (one of the most widely used bioceramic materials) into the biopolymer matrix improves the mechanical properties and incorporates the nanotopographic features that mimic the nanostructure of bone. This review summarizes the recent work on the development of biocomposites containing natural polymers with hydroxyapatite particles suitable for use in bone defects/bone regeneration. PMID: 20361991 [PubMed - as supplied by publisher] | | |
| Human stem cell delivery for treatment of large segmental bone defects.
April 7, 2010 at 8:22 AM
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| | Human stem cell delivery for treatment of large segmental bone defects. Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3305-10 Authors: Dupont KM, Sharma K, Stevens HY, Boerckel JD, García AJ, Guldberg RE Local or systemic stem cell delivery has the potential to promote repair of a variety of damaged or degenerated tissues. Although various stem cell sources have been investigated for bone repair, few comparative reports exist, and cellular distribution and viability postimplantation remain key issues. In this study, we quantified the ability of tissue-engineered constructs containing either human fetal or adult stem cells to enhance functional repair of nude rat critically sized femoral defects. After 12 weeks, defects treated with cell-seeded polymer scaffolds had significantly higher bone ingrowth and torsional strength compared to those receiving acellular scaffolds, although there were no significant differences between the cell sources. Next, stem cells were labeled with fluorescent quantum dots (QDs) in an attempt to noninvasively track their distribution after delivery on scaffolds. Clear fluorescence was observed at implantation sites throughout the study;! however, beginning 7-10 days after surgery, signals were also observed at contralateral sites treated with acellular QD-free scaffolds. Although immunostaining for human nuclei revealed retention of some cells at the implantation site, no human cells were detected in the control limb defects. Additional histological analysis of implantation and control defect tissues revealed macrophages containing endocytosed QDs. Furthermore, QD-labeling appeared to diminish transplanted cell function resulting in reduced healing responses. In summary, augmentation of polymeric scaffolds with stem cells derived from fetal and adult tissues significantly enhanced healing of large segmental bone defects; however, QD labeling of stem cells eliminated the observed therapeutic effect and failed to conclusively track stem cell location long-term in vivo. PMID: 20133731 [PubMed - indexed for MEDLINE] | | |
| Autologous engineering of cartilage.
April 7, 2010 at 8:22 AM
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| | Autologous engineering of cartilage. Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3418-23 Authors: Emans PJ, van Rhijn LW, Welting TJ, Cremers A, Wijnands N, Spaapen F, Voncken JW, Shastri VP Treatment of full-thickness damage to hyaline cartilage is hampered by the limited availability of autologous healthy cartilage and the lengthy, cost-prohibitive cell isolation and expansion steps associated with autologous cartilage implantation (ACI). Here we report a strategy for de novo engineering of ectopic autologous cartilage (EAC) within the subperiosteal space (in vivo bioreactor), through the mere introduction of a biocompatible gel that might promote hypoxia-mediated chondrogenesis, thereby effectively overcoming the aforementioned limitations. The EAC is obtained within 3 wk post injection of the gel, and can be press-fit into an osteochondral defect where it undergoes remodeling with good lateral and subchondral integration. The implanted EAC showed no calcification even after 9 mo and attained an average O'Driscoll score of 11 (versus 4 for controls). An "on demand" autologous source of autologous cartilage with remodeling capacity is expected to si! gnificantly impact the clinical options in repair of trauma to articular cartilage. PMID: 20133690 [PubMed - indexed for MEDLINE] | | |
| Engineered vascularized bone grafts.
April 7, 2010 at 8:22 AM
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| | Engineered vascularized bone grafts. Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3311-6 Authors: Tsigkou O, Pomerantseva I, Spencer JA, Redondo PA, Hart AR, O'Doherty E, Lin Y, Friedrich CC, Daheron L, Lin CP, Sundback CA, Vacanti JP, Neville C Clinical protocols utilize bone marrow to seed synthetic and decellularized allogeneic bone grafts for enhancement of scaffold remodeling and fusion. Marrow-derived cytokines induce host neovascularization at the graft surface, but hypoxic conditions cause cell death at the core. Addition of cellular components that generate an extensive primitive plexus-like vascular network that would perfuse the entire scaffold upon anastomosis could potentially yield significantly higher-quality grafts. We used a mouse model to develop a two-stage protocol for generating vascularized bone grafts using mesenchymal stem cells (hMSCs) from human bone marrow and umbilical cord-derived endothelial cells. The endothelial cells formed tube-like structures and subsequently networks throughout the bone scaffold 4-7 days after implantation. hMSCs were essential for stable vasculature both in vitro and in vivo; however, contrary to expectations, vasculature derived from hMSCs briefly cul! tured in medium designed to maintain a proliferative, nondifferentiated state was more extensive and stable than that with hMSCs with a TGF-beta-induced smooth muscle cell phenotype. Anastomosis occurred by day 11, with most hMSCs associating closely with the network. Although initially immature and highly permeable, at 4 weeks the network was mature. Initiation of scaffold mineralization had also occurred by this period. Some human-derived vessels were still present at 5 months, but the majority of the graft vasculature had been functionally remodeled with host cells. In conclusion, clinically relevant progenitor sources for pericytes and endothelial cells can serve to generate highly functional microvascular networks for tissue engineered bone grafts. PMID: 20133604 [PubMed - indexed for MEDLINE] | | |
| Tissue regeneration of the vocal fold using bone marrow mesenchymal stem cells and synthetic extracellular matrix injections in rats.
April 7, 2010 at 8:22 AM
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| | Tissue regeneration of the vocal fold using bone marrow mesenchymal stem cells and synthetic extracellular matrix injections in rats. Laryngoscope. 2010 Mar;120(3):537-45 Authors: Johnson BQ, Fox R, Chen X, Thibeault S OBJECTIVES/HYPOTHESIS: To determine the effectiveness of bone marrow mesenchymal stem cell (BM-MSC) transplantation in isolation or within a synthetic extracellular matrix (sECM) for tissue regeneration of the scarred vocal fold lamina propria. METHODS: In vitro stability and compatibility of mouse BM-MSC embedded in sECM was assessed by flow cytometry detection of BM-MSC marker expression and proliferation. Eighteen rats were subjected to vocal fold injury bilaterally, followed by 1 month post-treatment with unilateral injections of saline or sECM hydrogel (Extracel; Glycosan BioSystems, Inc., Salt Lake City, UT), green fluorescence protein (GFP)-mouse BM-MSC, or BM-MSC suspended in sECM. Outcomes measured 1 month after treatment included procollagen-III, fibronectin, hyaluronan synthase-III (HAS3), hyaluronidase (HYAL3), smooth muscle actin (SMA), and transforming growth factor-beta 1(TGF-beta1) mRNA expression. The persistence of GFP BM-MSC, proliferation, apop! tosis, and myofibroblast differentiation was assessed by immunofluorescence. RESULTS: BM-MSC grown in vitro within sECM express Sca-1, are positive for hyaluronan receptor CD44, and continue to proliferate. In the in vivo study, groups injected with BM-MSC had detectable GFP-labeled BM-MSC remaining and showed proliferation and low apoptotic or myofibroblast markers compared to the contralateral side. Embedded BM-MSC in the sECM group exhibited increased levels of procollagen III, fibronectin, and TGF-beta1. BM-MSC within sECM downregulated the expression of SMA compared to BM-MSC alone and exhibited upregulation of HYAL3 and no change in HAS3 compared to saline. CONCLUSIONS: Treatment of vocal fold scarring with BM-MSC injected in a sECM displayed the most favorable outcomes in ECM production, hyaluronan metabolism, myofibroblast differentiation, and production of TGF-beta1. Furthermore, the combined treatment had no detectable cytotoxicity and preserved local cell prolife! ration. PMID: 20131370 [PubMed - indexed for MEDLINE] | | |
| HPLC purification of adenoviral vectors.
April 7, 2010 at 8:22 AM
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| | HPLC purification of adenoviral vectors. Methods Mol Biol. 2010;594:395-408 Authors: Eglon M, McGrath B, O'Brien T Adenoviruses are attractive vectors for gene therapy where short-term transgene expression is required. In order to meet the clinical requirements of adenovirus for use beyond the laboratory, advanced methods are required for the purification and quantitation of recombinant adenoviral vectors (rAd). Chromatographic systems offer the advantages of linear scalability and reproducibility, and this method describes a laboratory-scale process based on liquid chromatography, which can be technically transferred and readily scaled-up according to the demands of the laboratory or clinic in which it will be used. PMID: 20072933 [PubMed - indexed for MEDLINE] | | |
| Generation of antioxidant adenovirus gene transfer vectors encoding CuZnSOD, MnSOD, and catalase.
April 7, 2010 at 8:22 AM
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| | Generation of antioxidant adenovirus gene transfer vectors encoding CuZnSOD, MnSOD, and catalase. Methods Mol Biol. 2010;594:381-93 Authors: Duffy AM, O'Brien T, McMahon JM Replication-deficient adenovirus gene transfer vectors are very useful for the experimental delivery of genes into cells and are widely used both in vitro and in vivo to determine the effects of transgene expression. Having a broad cell tropism, these vectors allow efficient transduction of many cell types and permit transfer of large amounts of DNA with resulting high expression levels within the target cell. Manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (CuZnSOD) and catalase are all known antioxidants whose over-expression can result in amelioration of pathology brought about by an excess of reactive oxygen species within a cell. Their use has been suggested as therapies for many conditions, including cardiovascular disease, arthritis, diabetes, cancer, and damage to central nervous system cells. This chapter describes the methodology commonly used for production of replication-deficient adenovirus vectors encoding MnSOD, CuZnSOD, and! catalase. PMID: 20072932 [PubMed - indexed for MEDLINE] | | |
| Scaffold-free tissue-engineered cartilage implants for laryngotracheal reconstruction.
April 7, 2010 at 8:22 AM
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| | Scaffold-free tissue-engineered cartilage implants for laryngotracheal reconstruction. Laryngoscope. 2010 Mar;120(3):612-7 Authors: Gilpin DA, Weidenbecher MS, Dennis JE OBJECTIVES/HYPOTHESIS: Donor site morbidity, including pneumothorax, can be a considerable problem when harvesting cartilage grafts for laryngotracheal reconstruction (LTR). Tissue engineered cartilage may offer a solution to this problem. This study investigated the feasibility of using autologous chondrocytes to tissue-engineer scaffold-free cartilage grafts for LTR in rabbits to avoid degradation that often arises from an inflammatory reaction to scaffold carrier matrix. STUDY DESIGN: Animal study. METHODS: Auricular cartilage was harvested from seven New Zealand white rabbits, the chondrocytes expanded and loaded onto a custom-made bioreactor for 7 to 8 weeks to fabricate autologous scaffold-free cartilage sheets. The sheets were cut to size and used for LTR, and the rabbits were sacrificed 4, 8, and 12 weeks after the LTR and prepared for histology. RESULTS: None of the seven rabbits showed signs of respiratory distress. A smooth, noninflammatory scar was vis! ible intraluminally; the remainder of the tracheal lumen was unremarkable. Histologically, the grafts showed no signs of degradation or inflammatory reaction, were covered with mucosal epithelium, but did show signs of mechanical failure at the implantation site. CONCLUSIONS: These results show that autologous chondrocytes can be used to fabricate an implantable sheet of cartilage that retains a cartilage phenotype, becomes integrated, and does not produce a significant inflammatory reaction. These findings suggest that with the design of stronger implants, these implants can be successfully used as a graft for LTR. PMID: 20058322 [PubMed - indexed for MEDLINE] | | |
| [Development of injectable bone repair biomaterials]
April 7, 2010 at 8:22 AM
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| | [Development of injectable bone repair biomaterials] Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009 Nov;23(11):1382-7 Authors: Li Z, Huang S, Guan L, Wu L, Zhang X OBJECTIVE: To introduce the basic research and clinical application of the injectable bone repair biomaterials. METHODS: The recent original articles about the injectable bone repair biomaterials were extensively reviewed. RESULTS: The injectable bone repair biomaterials could fill irregularly shaped defects and might allow bone augmentation, both with minimal surgical intervention, and the injectable bone repair material had a good prospect by the medical profession and attach great importance to the academic material, but there were some deficiencies and shortcomings. CONCLUSION: The injectable bone repair biomaterials may be a future approach to repair bone defect. PMID: 19968185 [PubMed - indexed for MEDLINE] | | |
| [A new composite matrix bridging both stumps of spinal cord transection in rats to promote recovery of motor function]
April 7, 2010 at 8:22 AM
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| | [A new composite matrix bridging both stumps of spinal cord transection in rats to promote recovery of motor function] Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009 Nov;23(11):1376-81 Authors: Liang H, Liang P, Gao A, Qi Q, Liu E, Wu J, Xu X OBJECTIVE; To investigate a new composite matrix (BMSCs seeded on the denuded human amniotic membrane, BMSCs-DHAM) bridging the both stumps of spinal cord injury in rats to promote axon regeneration and improve motor function of hind limbs. METHODS: The human amniotic membrane (HAM) was voluntarily donated by the healthy pregnant women after a caesarean section. The cells on the HAM were completely removed with a tryptic and mechanical approach to prepare DHAM. The BMSCs were separated and cultured from 4-week-old female rats (n = 4), then the forth passage of BMSCs were labeled by PKH26 and seeded on DHAM (BMSCs-DHAM). The growing state of BMSCs was observed under the microscopy. Moreover, 40 female rats (8-week-old, weighting 200-220 g) were made spinal cord injury models by transecting at T9 level, and were randomly divided into 4 groups (each group, n = 10). The both stumps were respectively wrapped by BMSCs-DHAM or simple DHAM in groups A and C, and the same ! dose of BMSCs or physiological saline were also respectively injected the central lesion in groups B and D. At 12 weeks after surgery, the functional recovery of the hindlimbs was evaluated by the BBB locomotor rating score, and other indexes were tested including cortical motion evoked potential (MEP), anterograde biotinylated dextran amine (BDA) tracing, and immunofluorescence of neurofilament protein 200 (NF-200). RESULTS: HE staining proved that the DHAM was devoid of cellular components by this way, and BMSCs grew well on the substrate under the microscopy. At 12 weeks after operation, the BBB score (12.50 +/- 1.26) in group A was significantly higher than those of other groups (P < 0.05), and the recovery in latency (3.52 +/- 2.45) ms and amplitude (480.68 +/- 18.41) microV of MEP was also obviously improved in group A (P < 0.05) when compared with other groups. In addition, anterograde BDA tracing revealed that the rate of the positive BDA axons 54.12% +/- 3.30! % under the lesion level in group A was higher than those of o! ther gro ups (P < 0.05), and lots of the regeneration axons (positive NF-200) were found to grow into the spinal cord under the composite matrix in group A. CONCLUSION: The BMSCs-DHAM composite matrix can improve hindlimb motor function to some extent after spinal cord injury. It will be widely applied as the matrix material in the future. PMID: 19968184 [PubMed - indexed for MEDLINE] | | |
| [Biocompatibility of silk fibroin nanofibers scaffold with olfactory ensheathing cells]
April 7, 2010 at 8:22 AM
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| | [Biocompatibility of silk fibroin nanofibers scaffold with olfactory ensheathing cells] Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009 Nov;23(11):1365-70 Authors: Qian Y, Shen Y, Lu Z, Fan Z, Liu T, Zhang J, Zhang F OBJECTIVE: To investigate the biocompatibility of silk fibroin nanofibers scaffold with olfactory ensheathing cells (OECs) and to provide an ideal tissue engineered scaffold for the repair of spinal cord injury (SCI). METHODS: Silk fibroin nanofibers were prepared using electrospinning techniques and were observed by scanning electron microscope (SEM). Freshly isolated OECs from SD rats purified by the modified differential adherent velocity method were cultured. The cells at passage 1 (1 x 10(4) cells/cm2) were seeded on the poly-l-lysine (control group) and the silk fibroin nanofibers (experimental group) coated coverslips in Petri dish. At desired time points, the morphological features, growth, and adhesion of the cells were observed using phase contrast inverted microscopy. The OECs were identified by the nerve growth factor receptor p75 (NGFR p75) immunofluorescence staining. The viability of OECs was examined by live/dead assay. The proliferation of OECs wa! s examined by MTT assay. The cytotoxicity of the nanofibers was evaluated. RESULTS: The SEM micrographs showed that the nanofibers had a smooth surface with solid voids among the fibers, interconnecting a porous network, constituted a fibriform three dimensional structure and the average diameter of the fibers was about (260 +/- 84) nm. The morphology of OECs on the experimental group was similar to the cell morphology on the control group, the cells distributed along the fibers, and the directions of the cell protrusions were in the same as that of the fibers. Fluorescence microscopy showed that the purity of OECs was 74.21% +/- 2.48% in the experimental group and 79.05% +/- 2.52% in the control group 5 days after culture. There was no significant difference on cell purity between two groups (P > 0.05). The OECs in the experimental group stained positive for NGFR p75 compared to the control group, indicating that the cells in the experimental group still maintained the ! OECs characteristic phenotype. Live/dead staining showed that ! high via bility was observed in both groups 3 days after culture. There was no significant difference on cell viability between two groups. The proliferation activity at 1, 3, 5, 7, and 10 days was examined by MTT assay. The absorbency values of the control group and the experimental group had significant differences 3 and 5 days after culture (P < 0.05). The relative growth rates were 95.11%, 90.35%, 92.63%, 94.12%, and 94.81%. The cytotoxicity of the material was grade 1 and nonvenomous according to GB/T 16886 standard. CONCLUSION: Silk fibroin nanofibers scaffold has good compatibility with OECs and is a promising tissue engineered scaffold for the repair of SCI. PMID: 19968182 [PubMed - indexed for MEDLINE] | | |
| [Effects of NGF on proliferation, mitotic cycle, collagen synthesis and migration of human dermal fibroblasts in vitro]
April 7, 2010 at 8:22 AM
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| | [Effects of NGF on proliferation, mitotic cycle, collagen synthesis and migration of human dermal fibroblasts in vitro] Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009 Nov;23(11):1350-4 Authors: Gan H, Xie H, Chen X, Luo J OBJECTIVE: To investigate the effects of NGF on the proliferation, mitotic cycle, collagen synthesis and migration of human dermal fibroblasts (HDFs), and to explore the function of NGF on the wound healing. METHODS: The 3rd generation of HDFs were incubated with various concentrations of NGF (0, 25, 50, 100, 200 and 400 ng/mL), the cell proliferation was measured with MTT assay. After treated with NGF at 0, 100 ng/mL, the cell cycle of HDFs was determined by flow cytometry (FCM). Hydroxyproline and real-time fluorescence quantitative PCR (FQ-PCR) were used to measure collagen synthesis at protein level and mRNA level respectively. The in vitro cell scratch wound model was set up to observe the effect of NGF (0, 50, 100 and 200 ng/mL) on the migration of HDFs after 24 hours of culture. RESULTS: Absorbance value of HDFs for different concentrations of NGF (0, 25, 50, 100, 200, and 400 ng/mL) showed that NGF did not influence the proliferation of HDFs (P > 0.05).! When HDFs were treated with NGF at 0 and 100 ng/mL, the result of FCM analysis showed that percentage of HDFs in G0/G1, S, G2/M phases were not changed (P > 0.05). Compared with control group, the expression of Col I and Col III were not significantly different, measured by both hydroxyproline and FQ-PCR (P > 0.05). The rates of HDFs' migration at various concentrations of NGF (0, 50, 100, 200 ng/mL) were 52.12% +/- 6.50%, 80.67% +/- 8.51%, 66.33% +/- 3.58%, and 61.19% +/- 0.97%, respectively, indicating that NGF could significantly enhanced the migration of HDFs at 50 and 100 ng/mL (P < 0.05). CONCLUSION: NGF does not influence proliferation, mitotic cycle and collagen synthesis of HDFs, but significantly enhanced migration in an in vitro model of wounded fibroblasts. PMID: 19968179 [PubMed - indexed for MEDLINE] | | |
| Enabling tools for engineering collagenous tissues integrating bioreactors, intravital imaging, and biomechanical modeling.
April 7, 2010 at 8:22 AM
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| | Enabling tools for engineering collagenous tissues integrating bioreactors, intravital imaging, and biomechanical modeling. Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3335-9 Authors: Niklason LE, Yeh AT, Calle EA, Bai Y, Valentín A, Humphrey JD Many investigators have engineered diverse connective tissues having good mechanical properties, yet few tools enable a global understanding of the associated formation of collagen fibers, the primary determinant of connective tissue stiffness. Toward this end, we developed a biomechanical model for collagenous tissues grown on polymer scaffolds that accounts for the kinetics of polymer degradation as well as the synthesis and degradation of multiple families of collagen fibers in response to cyclic strains imparted in a bioreactor. The model predicted well both overall thickness and stress-stretch relationships for tubular engineered vessels cultured for 8 weeks, and suggested that a steady state had not yet been reached. To facilitate future refinements of the model, we also developed bioreactors that enable intravital nonlinear optical microscopic imaging. Using these tools, we found that collagen fiber alignment was driven strongly by nondegraded polymer fiber! s at early times during culture, with subsequent mechano-stimulated dispersal of fiber orientations as polymer fibers degraded. In summary, mathematical models of growth and remodeling of engineered tissues cultured on polymeric scaffolds can predict evolving tissue morphology and mechanics after long periods of culture, and related empirical observations promise to further our understanding of collagen matrix development in vitro. PMID: 19955446 [PubMed - indexed for MEDLINE] | | |
| Bioengineered corporal tissue for structural and functional restoration of the penis.
April 7, 2010 at 8:22 AM
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| | Bioengineered corporal tissue for structural and functional restoration of the penis. Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3346-50 Authors: Chen KL, Eberli D, Yoo JJ, Atala A Various reconstructive procedures have been attempted to restore a cosmetically acceptable phallus that would allow normal reproductive, sexual, and urinary function in patients requiring penile reconstruction. However, these procedures are limited by a shortage of native penile tissue. We previously demonstrated that a short segment of the penile corporal body can be replaced using naturally derived collagen matrices with autologous cells. In the current study, we examined the feasibility of engineering the entire pendular penile corporal bodies in a rabbit model. Neocorpora were engineered from cavernosal collagen matrices seeded with autologous cells using a multistep static/dynamic procedure, and these were implanted to replace the excised corpora. The bioengineered corpora demonstrated structural and functional parameters similar to native tissue and male rabbits receiving the bilateral implants were able to successfully impregnate females. This study demonst! rates that neocorpora can be engineered for total pendular penile corporal body replacement. This technology has considerable potential for patients requiring penile reconstruction. PMID: 19915140 [PubMed - indexed for MEDLINE] | | |
| The cyclooxygenase-2 selective inhibitor, etodolac, but not aspirin reduces neovascularization in a murine ischemic hind limb model.
April 7, 2010 at 8:22 AM
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| | The cyclooxygenase-2 selective inhibitor, etodolac, but not aspirin reduces neovascularization in a murine ischemic hind limb model. Eur J Pharmacol. 2010 Feb 10;627(1-3):223-8 Authors: Tanaka K, Yamamoto Y, Tsujimoto S, Uozumi N, Kita Y, Yoshida A, Shimizu T, Hisatome I Cyclooxygenase inhibitors are often prescribed to relieve severe ischemic leg pain in critical ischemic limb patients. Prescription of high doses of aspirin and selective cyclooxygenase-2 inhibitors is reported to increase cardiovascular events through suppression of the vasodilative prostanoid prostaglandin I(2) in endothelium. Here, we evaluated the influence of aspirin and etodolac, a selective cyclooxygenase-2 inhibitor, on neovascularization using a murine ischemia hind limb model. C57BL/6J mice were treated with aspirin or etodolac for twenty-eight days after induction of ischemia. We exploited a concentration of the agents that suppressed cyclooxygenase activity efficiently, especially in prostaglandin I(2) production. Recovery of limb blood perfusion and capillary density in ischemic limbs was significantly suppressed by etodolac treatment when compared to the aspirin treated group and untreated group. Production of 6-keto prostaglandin F(1alpha) and prost! aglandin E(2) was lower in the aspirin treated group when compared with the etodolac-treated group. Also, these concentrations were lower in both treatment groups compared with the untreated group. Immunohistochemical analysis suggested cyclooxygenase-2 was expressed in endothelium but not in inflammatory cells in ischemic tissue from the acute to chronic phase. Cyclooxygenase-1 was expressed strongly in inflammatory cells in the acute phase. Furthermore, bone marrow-derived mononuclear cell transplantation improved neovascularization, whereas aspirin and etodolac did not inhibit these effects. Production of arachidonic acid metabolites by transplanted cells was independent of the improvement of neovascularization. In conclusion, cyclooxygenase-2 inhibition reduces ischemia-induced neovascularization. PMID: 19879866 [PubMed - indexed for MEDLINE] | | |
| Interrogating functional integration between injected pluripotent stem cell-derived cells and surrogate cardiac tissue.
April 7, 2010 at 8:22 AM
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| | Interrogating functional integration between injected pluripotent stem cell-derived cells and surrogate cardiac tissue. Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3329-34 Authors: Song H, Yoon C, Kattman SJ, Dengler J, Massé S, Thavaratnam T, Gewarges M, Nanthakumar K, Rubart M, Keller GM, Radisic M, Zandstra PW Myocardial infarction resulting in irreversible loss of cardiomyocytes (CMs) remains a leading cause of heart failure. Although cell transplantation has modestly improved cardiac function, major challenges including increasing cell survival, engraftment, and functional integration with host tissue, remain. Embryonic stem cells (ESCs), which can be differentiated into cardiac progenitors (CPs) and CMs, represent a candidate cell source for cardiac cell therapy. However, it is not known what specific cell type or condition is the most appropriate for transplantation. This problem is exasperated by the lack of efficient and predictive strategies to screen the large numbers of parameters that may impact cell transplantation. We used a cardiac tissue model, engineered heart tissue (EHT), and quantitative molecular and electrophysiological analyses, to test transplantation conditions and specific cell populations for their potential to functionally integrate with the ho! st tissue. In this study, we validated our analytical platform using contractile mouse neonatal CMs (nCMs) and noncontractile cardiac fibroblasts (cFBs), and screened for the integration potential of ESC-derived CMs and CPs (ESC-CMs and -CPs). Consistent with previous in vivo studies, cFB injection interfered with electrical signal propagation, whereas injected nCMs improved tissue function. Purified bioreactor-generated ESC-CMs exhibited a diminished capacity for electrophysiological integration; a result correlated with lower (compared with nCMs) connexin 43 expression. ESC-CPs, however, appeared able to appropriately mature and integrate into EHT, enhancing the amplitude of tissue contraction. Our results support the use of EHT as a model system to accelerate development of cardiac cell therapy strategies. PMID: 19846783 [PubMed - indexed for MEDLINE] | | |
| Engineering anatomically shaped human bone grafts.
April 7, 2010 at 8:22 AM
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| | Engineering anatomically shaped human bone grafts. Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3299-304 Authors: Grayson WL, Fröhlich M, Yeager K, Bhumiratana S, Chan ME, Cannizzaro C, Wan LQ, Liu XS, Guo XE, Vunjak-Novakovic G The ability to engineer anatomically correct pieces of viable and functional human bone would have tremendous potential for bone reconstructions after congenital defects, cancer resections, and trauma. We report that clinically sized, anatomically shaped, viable human bone grafts can be engineered by using human mesenchymal stem cells (hMSCs) and a "biomimetic" scaffold-bioreactor system. We selected the temporomandibular joint (TMJ) condylar bone as our tissue model, because of its clinical importance and the challenges associated with its complex shape. Anatomically shaped scaffolds were generated from fully decellularized trabecular bone by using digitized clinical images, seeded with hMSCs, and cultured with interstitial flow of culture medium. A bioreactor with a chamber in the exact shape of a human TMJ was designed for controllable perfusion throughout the engineered construct. By 5 weeks of cultivation, tissue growth was evidenced by the formation of confl! uent layers of lamellar bone (by scanning electron microscopy), markedly increased volume of mineralized matrix (by quantitative microcomputer tomography), and the formation of osteoids (histologically). Within bone grafts of this size and complexity cells were fully viable at a physiologic density, likely an important factor of graft function. Moreover, the density and architecture of bone matrix correlated with the intensity and pattern of the interstitial flow, as determined in experimental and modeling studies. This approach has potential to overcome a critical hurdle-in vitro cultivation of viable bone grafts of complex geometries-to provide patient-specific bone grafts for craniofacial and orthopedic reconstructions. PMID: 19820164 [PubMed - indexed for MEDLINE] | | |
| [Experimental study on adhesiveness of osteoblasts and vascular endothelial cells from rat BMSCs co-cultured on allogeneic freeze-dried partially bone in vitro]
April 7, 2010 at 8:22 AM
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| | [Experimental study on adhesiveness of osteoblasts and vascular endothelial cells from rat BMSCs co-cultured on allogeneic freeze-dried partially bone in vitro] Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009 Sep;23(9):1129-33 Authors: Chen C, Li Q, Sun R, Bai J, Wang Z OBJECTIVE: To investigate the adhesiveness of osteoblasts and vascular endothelial cells from rat BMSCs. METHODS: The BMSCs were isolated from 4-week-old SD co-cultured on allogeneic freeze-dried partially bone in vitro. rats (weighing 100-110 g) and cultured in vitro. The third generation of BMSCs were induced into osteoblasts and vascular endothelial cells. The osteoblasts and vascular endothelial cells after being induced for 7 days in a ratio of 1 to 1 were directly co-cultured (experimental group), while the second generation of uninduced BMSCs was used as a control (control group). The growth and proliferation ability were analyzed by MTT examination and the growth curve was drawn at 1-8 days. The osteoblasts and vascular endothelial cells after being induced for 14 days were implanted in the allogeneic freeze-dried partially bone coated by 20% Col I or not at different densities (0.25 x 10(6)/mL, 0.50 x 10(6)/mL, 1.00 x 10(6)/mL, 2.00 x 10(6)/mL, 4.00 x 10(! 6)/mL), as modified group and unmodified group, the cell adherence rate was calculated after 24 hours. These two kinds of cells were implanted in the pre-disposal treated allogeneic freeze-dried partially bone and observed by scanning electron microscope. RESULTS: ALP staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days. CD31 and CD34 immunocytochemical staining of vascular endothelial cell showed that there were positive signals in the cytoplasm at 14 days. The MTT test showed that the proliferation level of the experimental group was lower than those of the control group. There were significant differences in absorbance value between two group from 3 days to 8 days (P < 0.05). The cell adherence rate increased with increasing seeding density when the seeding density was (0.25-1.00) x 10(6)/mL. The cell adherence rate reached the peak when the seeding density was 1.00 x 10(6)/mL. The cell adherence rate decreased when the seeding density was! more than 2.00 x 106/mL. There were significant differences i! n cell a dherence rate between modified group and unmodified group at different seeding densities (P < 0.05). The proliferation of the osteoblasts and endothelial cells presented better growth and histocompatibility under scanning electron microscope. CONCLUSION: The growing behavior of two kinds of cells is good in the allogeneic freeze-dried partially bone coated by 20% Col I, which can be used in reconstruction of vascularized tissue engineered bone. PMID: 19817304 [PubMed - indexed for MEDLINE] | | |
| [Mechanisms and effects of biosynthesis and apoptosis in repair of full-thickness skin defect with collagen-chitosan dermal stent]
April 7, 2010 at 8:22 AM
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| | [Mechanisms and effects of biosynthesis and apoptosis in repair of full-thickness skin defect with collagen-chitosan dermal stent] Zhonghua Zheng Xing Wai Ke Za Zhi. 2009 May;25(3):208-12 Authors: Xu SJ, Huang AB, Ma L, Teng JY, Gao CY, Zhang ZL, Ni YD, Ye S, Wang YG OBJECTIVE: To investigate biosynthetic and apoptotic mechanisms in repair of full thickness skin defect with collagen-chitosan porous scaffold transplantation, and to determinate differences between wound repair with the scaffold transplantation and scar healing without the scaffold transplantation. METHODS: The full thickness skin defects were made on 10 Bama miniature pigs and the bilayer dermal equivalent (BDE) composed of collagen-chitosan porous scaffold and silicone membrane was transplanted on wounds. Surfaces of wounds were observed at 1, 2, and 3 weeks after the BDE transplantation, and so were done the wound repairs after epidermis had been grafted for 2 weeks on surface of the scaffold which had been transplanted on skin defect wounds for 2 weeks. At the same time, TGF-beta1 expressions, apoptosis and self collagen replacement of scaffolds in wounds were detected in situ by immunohistochemical staining, terminal deoxynucleotidyl transferase-mediated deo! xyuridine triphosphate-biotin nick end labeling (TUNEL) and picrosirius red polarized light. Wounds without scaffold transplantation were studied as control. RESULTS: 1) Wounds with the scaffold transplantation were different from granulation tissue. 2) The peak of TGF-beta1 expression in the scaffold wounds was from 1 to 2 weeks after BDE transplantation, and TGF-beta1 expressions decreased continuously from 3 to 4 weeks. TGF-beta1 expressions increased continuously in the control wounds from 1 to 3 weeks and decreased on 4 weeks. TGF-beta1 expressions in the scaffold wounds on 1st and 2nd week were significantly higher than those in the corresponding control wounds, whereas, TGF-beta1 expressions in the scaffold wounds on 3rd and 4th week were significantly lower than those in the corresponding control wounds. 3) Apoptosis increased continuously in the scaffold wounds from 2 to 4 weeks after BDE transplantation, and so did in the control wounds from 3 to 4 weeks. However,! apoptosis signals in the scaffold wounds on 2nd, 3rd, and 4th! week af ter BDE transplantation were significantly more than those in the corresponding control wounds, and there was no difference between apoptosis signals in the scaffold wounds on 1st week after BDE transplantation and those in the corresponding control wounds. 4) Observation by picrosirius red polarized light method: self collagen began to synthesize in the scaffold wounds on 1st week after BDE transplantation, and scaffolds had been replaced by self collagen from 2 to 3 weeks after BDE transplantation. CONCLUSIONS: Collagen-chitosan porous scaffold plays a very important role in wound healing of full thickness skin defect. The mechanisms of wound repair by dermal scaffold are different from those by granulation and scar healing. It has a good future in repairing skin defect. PMID: 19803205 [PubMed - indexed for MEDLINE] | | |
| [Experimental study of tissue-engineered skin loaded with keratinocyte growth factor nanocapsules for skin defect]
April 7, 2010 at 8:22 AM
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| | [Experimental study of tissue-engineered skin loaded with keratinocyte growth factor nanocapsules for skin defect] Zhonghua Zheng Xing Wai Ke Za Zhi. 2009 May;25(3):200-4 Authors: Yang B, Qiu RS, Hong QQ, Ji CY OBJECTIVE: To study the effect of tissue-engineered skin loaded with keratinocyte growth factor (KGF) nanocapsules for skin defect on athymic mice. METHODS: The acellular dermal matrix (ADM) loaded with KGF-ADM was constructed by means of phacoemulsification solvent evaporation and low temperature drying. The human epidermal stem cells and fibroblasts were captured and identified, then cultivated on the surface of the KGF-ADM. The cell growth was observed. The tissue-engineered skin without KGF was used as sham group. The autogenous skin graft was used as control group. 2 and 6 weeks after the skin was transplanted to the back of athymic mice, the contraction and histological healing of the transplanted skins were observed respectively. Then the immunofluorescence examination with anti-human K10-FITC and beta1-integrin-Cy3 were applied to detect the origin, growth and differentiation of epidermal and dermal cells in tissue-engineered skin. RESULTS: The epidermal s! tem cells grew well and attached tightly on KGF-ADM. There were small round stem cells and polygonal terminally-differentiated cells, which appeared a partly cloning growth and a tendency of merging. The tissue-engineered skin with KGF nanocapsules gained better result in repairing the skin defects as compared with the blank group and the control group 2 and 6 weeks after transplantation. The regenerative skin cells could connect and mix closely with the athymic mouse skin cells on the border of skin defect. Meanwhile, the regenerative skin existed some contraction. The histological observation with HE staining showed that the regenerative skin possessed intact epidermis with several cell layers and normal keratose stratum, among which there were still some beta1-integrin (+) cells which represented epidermal stem cells or transient amplifying cells when they were tested by immunofluorescence after 6 weeks of transplantation. CONCLUSIONS: The tissue-engineered skin loaded w! ith KGF nanocapsules had a better result in repairing athymic ! mice ski n defects than common tissue-engineered skin without KGF nanocapsules or skin auto-graft. PMID: 19803203 [PubMed - indexed for MEDLINE] | | |
| Dynamic loading stimulates chondrocyte biosynthesis when encapsulated in charged hydrogels prepared from poly(ethylene glycol) and chondroitin sulfate.
April 7, 2010 at 8:22 AM
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| | Dynamic loading stimulates chondrocyte biosynthesis when encapsulated in charged hydrogels prepared from poly(ethylene glycol) and chondroitin sulfate. Matrix Biol. 2010 Jan;29(1):51-62 Authors: Villanueva I, Gladem SK, Kessler J, Bryant SJ This study aimed to elucidate the role of charge in mediating chondrocyte response to loading by employing synthetic 3D hydrogels. Specifically, neutral poly(ethylene glycol) (PEG) hydrogels were employed where negatively charged chondroitin sulfate (ChS), one of the main extracellular matrix components of cartilage, was systematically incorporated into the PEG network at 0%, 20% or 40% to control the fixed charge density. PEG hydrogels were employed as a control environment for extracellular events which occur as a result of loading, but which are not associated with a charged matrix (e.g., cell deformation and fluid flow). Freshly isolated bovine articular chondrocytes were embedded in the hydrogels and subject to dynamic mechanical stimulation (0.3Hz, 15% amplitude strains, 6h) and assayed for nitric oxide production, cell proliferation, proteoglycan synthesis, and collagen deposition. In the absence of loading, incorporation of charge inhibited cell proliferat! ion by approximately 75%, proteoglycan synthesis by approximately 22-50% depending on ChS content, but had no affect on collagen deposition. Dynamic loading had no effect on cellular responses in PEG hydrogels. However, dynamically loading 20% ChS gels inhibited nitrite production by 50%, cell proliferation by 40%, but stimulated proteoglycan and collagen deposition by 162% and 565%, respectively. Dynamic loading of 40% ChS hydrogels stimulated nitrite production by 62% and proteoglycan synthesis by 123%, but inhibited cell proliferation by 54% and collagen deposition by 52%. Upon removing the load and culturing under free-swelling conditions for 36h, the enhanced matrix synthesis observed in the 20% ChS gels was not maintained suggesting that loading is necessary to stimulate matrix production. In conclusion, extracellular events associated with a charged matrix have a dramatic affect on how chondrocytes respond to mechanical stimulation within these artificial 3D matrices! suggesting that streaming potentials and/or dynamic changes i! n osmola rity may be important regulators of chondrocytes while cell deformation and fluid flow appear to have less of an effect. PMID: 19720146 [PubMed - indexed for MEDLINE] | | |
| [Constructing tissue engineered trachea-like cartilage graft in vitro by using bone marrow stromal cells sheet and PLGA internal support: experimental study in bioreactor]
April 7, 2010 at 8:22 AM
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| | [Constructing tissue engineered trachea-like cartilage graft in vitro by using bone marrow stromal cells sheet and PLGA internal support: experimental study in bioreactor] Zhonghua Zheng Xing Wai Ke Za Zhi. 2009 Mar;25(2):124-8 Authors: Zhang JR, Chen FL, Wu W, Wei JH, Feng XH, Mao TQ OBJECTIVE: To explore the feasibility of constructing tissue engineered trachea-like cartilage graft in vitro by using bone marrow stromal cells (BMSCs) sheet and PLGA internal support. METHODS: Rabbit BMSCs were expanded and induced by transforming growth factor-1 to improve chondrocyte phenotype of BMSCs. BMSCs sheets were obtained by continuous culture and wrapped the PGLA scaffold in the shape of cylinder. The constructs were incubated in spinner flask for 8 weeks and cartilage formation was investigated by gross inspection, histology, glycosaminoglycan and mechanical strength content. RESULTS: After in vitro culture, cartilage like tissue in cylindrical shape had been regenerated successfully. Stiff, shiny, pearly opalescence tissues were observed. Histological analysis showed engineered trachea cartilage consisted of evenly spaced lacunae embedded in matrix, cells stationed in the lacunae could be noticed clearly. Safranin-O staining on the sections showed h! omogenous and positive red staining, which demonstrated that the engineered tissue was rich in proteoglycans. CONCLUSIONS: Based on the cell sheet and internal support strategy, trachea-like cartilage in cylindrical shape could be successfully fabricated which provided a highly effective cartilage graft substitute and could be useful in many situations of trachea-cartilage loss encountered in clinical practice. PMID: 19558168 [PubMed - indexed for MEDLINE] | | |
| [Application of adipose-derived cells in reconstruction of tissue engineered cartilage in vitro]
April 7, 2010 at 8:22 AM
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| | [Application of adipose-derived cells in reconstruction of tissue engineered cartilage in vitro] Zhonghua Zheng Xing Wai Ke Za Zhi. 2008 Nov;24(6):464-7 Authors: Zhang Y, Zhou GD, Gui L, Qi ZL, Liu W, Cao YL OBJECTIVE: To explore the feasibility of application of adipose-derived cells (ADCs) in reconstruction of tissue engineered cartilage in vitro. METHODS: Adipose tissue were obtained from human liposuction aspirate (19 cases, 31.5 +/- 5.8 years old). ADCs were isolated by collagenase digestion from liposuction aspirates. 3rd passage cells were seeded into PLGA scaffolds. The copolymer constructs were cultured in conditioned or non-conditioned medium in vitro for 4 weeks. The constructs were evaluated though gross morphology, histology, and immunohistochemistry. RESULTS: The cell-polymer constructs kept its original shape in the induced group, but lost its original shape in the non-induced group. The scaffold group were collapsed. Histologically, the induced groups showed dense cellularity and lacunae-containing cells embedded in a basophilic matrix, while non-induced groups showed connective tissue-like morphology. Collagen and proteoglycan deposition was revealed ! by Massons's trichome and Safranin' O staining, and minor collagen II expression in the matrix was detected by immunohistochemistry staining in the induced group. They were all negative in the non-induced groups. CONCLUSIONS: Although ADCs included many kinds of cells, it is feasible to use ADCs as seeds cells for reconstruction of tissue engineered cartilage. PMID: 19241711 [PubMed - indexed for MEDLINE] | | |
| [An experimental study on rabbit's radial bone defect healed by application of mimetic periosteum with tissue-engineered bone]
April 7, 2010 at 8:22 AM
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| | [An experimental study on rabbit's radial bone defect healed by application of mimetic periosteum with tissue-engineered bone] Zhonghua Zheng Xing Wai Ke Za Zhi. 2008 Jan;24(1):63-7 Authors: Guo HG, Yao FL, Ma XL, Yao KD OBJECTIVE: The purpose of this study is to investigate the osteogenic potential and possibility of combination application of mimetic osteoinductive periosteum with tissue-engineered bone. METHODS: The three-dimensional construction of tissue-engineered bone was made by implantation of adipose derived stromal cells (ADSCs) into rhBMP-2 mediated bio-derived carrier, and mimetic periosteum was constructed by loading ADSCs into Cs-Col-beta3-TCP with rhBMP-2. 10 mm defects of right radiuses were established in adult New Zealand rabbits, group A was transplanted by tissue-engineered bone with mimetic periosteum, group B was implanted by tissue-engineered bone, and group C was implanted by mimetic periosteum, group D was transplanted by bio-derived compound bone as blank scaffold. X-ray, histology, immunohistochemistry stain, dural energy X-ray absorptiometry (DEXA) and transmission electron microscopy (TEM) examinations were performed at different periods. RESULTS: Gro! up A played a predominant role in process of new tissue regeneration and mature bone reconstitution, defect completely healed at 12 weeks. Group B showed primary repair, group C also existed in modeling stage. While, group D displayed retard regeneration with poor osteogenic capacity. DEXA result showed that group A had statistical significance over control group according to data of BMC and BMD ( P < 0.05). CONCLUSIONS: Enhanced osteogenic potential can be obtained by using tissue-engineered bone with mimetic osteoinductive periosteum. Defect can be healed with concord pattern of osteoinductive and osteopromotive and osteoconductive effects. PMID: 18437989 [PubMed - indexed for MEDLINE] | | |
| [The long-term contrastive result of neo-urethra reconstructed with different kinds of tissue]
April 7, 2010 at 8:22 AM
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| | [The long-term contrastive result of neo-urethra reconstructed with different kinds of tissue] Zhonghua Zheng Xing Wai Ke Za Zhi. 2008 Jan;24(1):36-8 Authors: Fan JF, Li SK, Li YQ, Xu JJ OBJECTIVE: To observe the long-term apparent and structural change of the neo-urethra inner wall reconstructed with different kinds of tissue. To compare the difference between normal and reconstructed urethra for urethra reconstruction with more appropriate materials. METHODS: The neo-urethra inner wall was observed during operation, through urography and urethroscope. The tissue section of neo-urethra inner wall was observed through light microscope and electron microscope. RESULTS: With unchanged structure, the appearance of neo-urethra reconstructed with skin or mucosa could be close to normal urethra after a long time. Neo-urethra made of buccal mucosa possesses some of the most important basic structures for normal urethral microenvironment to form. The structure of neo-urethra made of bladder could be close to normal urethra after a long time. CONCLUSIONS: Buccal and bladder mucosa may be a better material than skin for urethra reconstruction. PMID: 18437982 [PubMed - indexed for MEDLINE] | | | | 
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