|  |  |  | | | | | TERMSC | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Ultrastructural morphology of equine adipose-derived mesenchymal stem cells. Histol Histopathol. 2010 Oct;25(10):1277-85 Authors: Pascucci L, Mercati F, Marini C, Ceccarelli P, Dall'aglio C, Pedini V, Gargiulo AM Mesenchymal stem cells are a virtually ubiquitous population of adult stem cells, able to differentiate into various tissue lineages. As they are multipotent and easy to grow in culture, they are at present considered very attractive candidates for tissue repair and gene therapy. With the exception of a few reports, mesenchymal stem cell morphology has been widely disregarded in the past years. In this paper we discuss the establishment of mesenchymal stem cell cultures from equine adipose tissue and describe their fine structure by transmission electron microscopy. The cultured cells revealed a fibroblastoid appearance and were characterized by an eccentric nucleus with multiple nucleoli, dense cytoplasm rich in ribosomes, a rough endoplasmic reticulum with dilated cisternae, elongated mitochondria and heterogeneous vacuolar inclusions. In addition, they were often interconnected by adhesion structures located on the cell body and on cytoplasmic processes contacting other cells. The features observed are evocative of an undifferentiated cellular phenotype and of an intense synthetic and metabolic activity. PMID: 20712012 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Hematopoietic stem cell activity in the aorta-gonad-mesonephros region enhances after mid-day 11 of mouse development. Int J Dev Biol. 2010;54(6-7):1055-60 Authors: Taylor E, Taoudi S, Medvinsky A The E11.5 aorta-gonad-mesonephros (AGM) region is a site of hematopoietic stem cell (HSC) development prior to colonisation of the embryonic liver. The generation of HSCs in the embryo starting from E11 is very rapid. Here, we have assessed hematopoietic development in the AGM region during E11 at precise somitic ages. Although the numbers of committed hematopoietic precursors fluctuate throughout the day, the repopulation activity in the AGM region noticeably increases from mid (44 s.p.) to end (48 s.p.) day 11 of gestation. While prior to mid day 11 two thirds of AGM regions contain no definitive HSCs, shortly prior to liver colonisation, all older day 11 embryos contain definitive HSC. Nevertheless, all E11 AGM regions even at early somitic stages have the capacity to expand numbers of definitive HSCs ex vivo. Quantitative anatomical analysis confirmed preferential localization of intra-aortic clusters (IACs) to the ventral domain of the dorsal aorta during entire day 11 of development. No clear correlation was established between IAC numbers and the presence of definitive HSCs. PMID: 20711982 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | The origin and fate of yolk sac hematopoiesis: application of chimera analyses to developmental studies. Int J Dev Biol. 2010;54(6-7):1019-31 Authors: Ueno H, Weissman IL During mammalian development, as exemplified by mice, hematopoietic cells first appear in the yolk sac blood islands, then in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region and the placenta, eventually seeding into liver, spleen and then bone marrow. The formation of hematopoietic stem cells from mesodermal precursors has finished by mid-fetal life. Once established, the hematopoietic system must supply blood cells to host circulation and tissues for the entire life of the animal. Easy access to hematopoietic cells has enabled a vast number of studies over the last several decades, and much is now understood about the different hematopoietic lineages, how they differentiate, and their derivation from immature progenitors. Yet to be elucidated are the following two intriguing questions: do yolk sac and AGM hematopoietic cells arise from a common precursor or from distinct precursor cells?; and what is the lineage relationship between blood and endothelial cells. In this review, we will survey the state of our current knowledge in these areas, and discuss the potential use of multicolor chimera analyses to elucidate unresolved questions. PMID: 20711980 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Dissecting hematopoietic differentiation using the embryonic stem cell differentiation model. Int J Dev Biol. 2010;54(6-7):991-1002 Authors: Huber TL Embryonic stem cells (ESCs) have been successfully used to study the generation of the hematopoietic lineage. The ESC differentiation model provides access to distinct developmental stages during hematopoietic differentiation enabling us to study developmental transitions in a manner that is difficult to do with embryos. The identification of the bipotential hemangioblast/blast-colony forming cell (BL-CFC) which represents the earliest stage of hematopoietic commitment in ESC cultures has enabled the study of signalling pathways, transcription factors and enzymes at the level of this developmental stage. Reporter ESC lines, flow cytometry and serum-free culture reagents are helping the field to transition from serum-containing protocols to step-wise serum-free differentiation strategies that attempt to mimic the developmental processes in the embryo. This serves as a framework with which to approach directed differentiation of human ESCs for the purposes of regenerative medicine. This review is focused on the contributions that the ESC differentiation system has made to understanding hematopoiesis and will highlight the strengths of this model of development and the challenges it still faces. PMID: 20711977 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Developmental hematopoiesis - preface. Int J Dev Biol. 2010;54(6-7):947-9 Authors: Durand C, Jaffredo T, Medvinsky A Stem cell biology is one of the most rapidly developing and exciting fields in the Life Sciences. The biology of embryonic and adult stem cells is critically important for understanding development, tissue homeostasis, reprogramming and cancer. Stem cells are characterised by their capacity to self-renew and differentiate into one or several cell types and hold great promise for regenerative medicine. The purification and tissue localization of stem cells, as well as identification of intrinsic and extrinsic factors regulating their functions, are central topics in stem cell biology. Hematopoietic stem cells (HSCs) are one of the best studied models and represent a key paradigm for analysis of various stem cell types. The availability of antibodies recognizing surface markers has been instrumental for enrichment and localization of HSCs. In vivo and in vitro assays offer researchers the possibility to study hematopoietic stem and progenitor cells at the functional level. For several decades bone marrow and more recently cord blood HSCs have been used for treatment of patients with hematopoietic disorders such as leukemia. PMID: 20711975 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | ROCK Inhibitor Y-27632 Increases Thaw-Survival Rates and Preserves Stemness and Differentiation Potential of Human Wharton's Jelly Stem Cells After Cryopreservation. Stem Cell Rev. 2010 Aug 14; Authors: Gauthaman K, Fong CY, Subramanian A, Biswas A, Bongso A The ROCK inhibitor Y-27632 inhibits apoptosis and increases proliferation of frozen-thawed cells. We examined the role of Y-27632 on human umbilical cord Wharton's jelly stem cells (hWJSCs) for (1) thaw-survival (2) proliferation and (3) preservation of stemness and differentiation potential after cryopreservation. hWJSCs were allotted to 4 groups [Gp I: Untreated hWJSC controls; Gp II: Pretreatment with Y-27632 (10 muM) for 24 h before freezing; Gp III: Y-27632 (10 muM) in freezing medium and Gp IV: Pretreatment with Y-27632 (10 muM) for 24 h and inclusion in freezing medium]. All groups were frozen using a rapid freezing method and stored at -196 degrees C in liquid nitrogen for 90 days before evaluation for apoptosis, cell proliferation, stemness and differentiation. After thawing, Groups II, III and IV showed improved cell attachment, increased thaw-survival (live/dead cell counts) and increased cell proliferation (Trypan blue and MTT assay) compared to controls. CD marker stemness profiles, morphology and normal karyotypes were maintained in the treatment groups after thawing and there was no obvious evidence of apoptosis (Annexin V-FITC and TUNEL assays). After thawing, qRT-PCR demonstrated up-regulation of the anti-apoptotic BCL2 gene and down-regulation of the pro-apoptotic BAX gene and cell cycle regulators (P53 and P21) in the treatment groups. Treated frozen-thawed hWJSCs from all groups differentiated into a neuronal phenotype (neuronal morphology and expression of GFAP, beta-3 tubulin and SOX2). Increased thaw-survival and retention of stemness and differentiation potential in hWJSCs following cryopreservation is useful for their storage in cord blood banks for future regenerative medicine purposes. PMID: 20711690 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | [Mesenchymal stem cells for bone tissue engineering.] Pathologe. 2010 Aug 15; Authors: Schneider RK, Neuss S, Knüchel R, Perez-Bouza A Human mesenchymal stem cells (MSC) represent an attractive option for cell replacement strategies (tissue engineering, TE). TE applications require stability of a stem cell/biomaterial-hybrid via cell migration, matrix-remodelling and differentiation. We focus on these mechanisms in organotypic culture systems for bone TE using MSC from the umbilical cord (UC-MSC) and from bone marrow (BM-MSC). For the organotypic differentiation of MSC into functional osteoblasts, MSC were embedded in a collagenous matrix and subjected to osteogenic differentiation. Under these culture conditions, UC-MSC exceeded BM-MSC in the expression and synthesis of extracellular matrix (ECM) proteins, while BM-MSC show enhanced osteogenic gene upregulation. In both cell types the biosynthetic activity was accompanied by the ultrastructural appearance of hydroxyapatite/calcium crystals. Following secretion of matrix metalloproteinases, both MSC types migrated into and colonised the collagenous matrix causing matrix strengthening and contraction. In conclusion, MSC promise a broad therapeutical application for a variety of connective tissues requiring ECM synthesis and remodelling. PMID: 20711587 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Interactions between Spider Silk and Cells - NIH/3T3 Fibroblasts Seeded on Miniature Weaving Frames. PLoS One. 2010;5(8): Authors: Kuhbier JW, Allmeling C, Reimers K, Hillmer A, Kasper C, Menger B, Brandes G, Guggenheim M, Vogt PM BACKGROUND: Several materials have been used for tissue engineering purposes, since the ideal matrix depends on the desired tissue. Silk biomaterials have come to focus due to their great mechanical properties. As untreated silkworm silk has been found to be quite immunogenic, an alternative could be spider silk. Not only does it own unique mechanical properties, its biocompatibility has been shown already in vivo. In our study, we used native spider dragline silk which is known as the strongest fibre in nature. METHODOLOGY/PRINCIPAL FINDINGS: Steel frames were originally designed and manufactured and woven with spider silk, harvesting dragline silk directly out of the animal. After sterilization, scaffolds were seeded with fibroblasts to analyse cell proliferation and adhesion. Analysis of cell morphology and actin filament alignment clearly revealed adherence. Proliferation was measured by cell count as well as determination of relative fluorescence each after 1, 2, 3, and 5 days. Cell counts for native spider silk were also compared with those for trypsin-digested spider silk. Spider silk specimens displayed less proliferation than collagen- and fibronectin-coated cover slips, enzymatic treatment reduced adhesion and proliferation rates tendentially though not significantly. Nevertheless, proliferation could be proven with high significance (p<0.01). CONCLUSION/SIGNIFICANCE: Native spider silk does not require any modification to its application as a biomaterial that can rival any artificial material in terms of cell growth promoting properties. We could show adhesion mechanics on intracellular level. Additionally, proliferation kinetics were higher than in enzymatically digested controls, indicating that spider silk does not require modification. Recent findings concerning reduction of cell proliferation after exposure could not be met. As biotechnological production of the hierarchical composition of native spider silk fibres is still a challenge, our study has a pioneer role in researching cellular mechanics on native spider silk fibres. PMID: 20711495 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Targeting of embryonic stem cells by Peptide-conjugated quantum dots. PLoS One. 2010;5(8): Authors: Lu S, Xu X, Zhao W, Wu W, Yuan H, Shen H, Zhou C, Li LS, Ma L BACKGROUND: Targeting stem cells holds great potential for studying the embryonic stem cell and development of stem cell-based regenerative medicine. Previous studies demonstrated that nanoparticles can serve as a robust platform for gene delivery, non-invasive cell imaging, and manipulation of stem cell differentiation. However specific targeting of embryonic stem cells by peptide-linked nanoparticles has not been reported. METHODOLOGY/PRINCIPAL FINDINGS: Here, we developed a method for screening peptides that specifically recognize rhesus macaque embryonic stem cells by phage display and used the peptides to facilitate quantum dot targeting of embryonic stem cells. Through a phage display screen, we found phages that displayed an APWHLSSQYSRT peptide showed high affinity and specificity to undifferentiated primate embryonic stem cells in an enzyme-linked immunoabsorbent assay. These results were subsequently confirmed by immunofluoresence microscopy. Additionally, this binding could be completed by the chemically synthesized APWHLSSQYSRT peptide, indicating that the binding capability was specific and conferred by the peptide sequence. Through the ligation of the peptide to CdSe-ZnS core-shell nanocrystals, we were able to, for the first time, target embryonic stem cells through peptide-conjugated quantum dots. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that our established method of screening for embryonic stem cell specific binding peptides by phage display is feasible. Moreover, the peptide-conjugated quantum dots may be applicable for embryonic stem cell study and utilization. PMID: 20711469 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Adipose Stem Cell Treatment in Mice Attenuates Lung and Systemic Injury Induced by Cigarette Smoking. Am J Respir Crit Care Med. 2010 Aug 13; Authors: Schweitzer K, Johnstone BH, Garrison J, Rush N, Cooper S, Traktuev DO, Feng D, Adamowicz JJ, Van Demark M, Fisher AJ, Kamocki K, Brown MB, Presson Jr RG, Broxmeyer HE, March KL, Petrache I RATIONALE: Adipose-derived stem cells express multiple growth factors that inhibit endothelial cell apoptosis, and demonstrate substantial pulmonary trapping following intravascular delivery. OBJECTIVES: We hypothesized that adipose stem cells would ameliorate chronic lung injury associated with endothelial cell apoptosis, such as that occurring in emphysema. METHODS: Therapeutic effects of systemically-delivered human or mouse adult adipose stem cells were evaluated in murine models of emphysema induced by chronic exposure to cigarette smoke or by inhibition of vascular endothelial growth factor receptors. MEASUREMENTS AND MAIN RESULTS: Adipose stem cells were detectable in the parenchyma and large airways of lungs up to 21 days following injection. Adipose stem cell treatment was associated with reduced inflammatory infiltration in response to cigarette smoke exposure, as well as markedly decreased lung cell death and airspace enlargement in both models of emphysema. Remarkably, therapeutic results of adipose stem cells extended beyond lung protection by rescuing the suppressive effects of cigarette smoke on bone marrow hematopoietic progenitor cell function, and by restoring weight loss sustained by mice during cigarette smoke exposure. Pulmonary vascular protective effects of adipose stem cells were recapitulated by application of cell-free conditioned medium, which improved lung endothelial cell repair and recovery in a wound injury repair model and antagonized effects of cigarette smoke in vitro. CONCLUSIONS: These results suggest a useful therapeutic effect of adipose stem cells on both lung and systemic injury induced by cigarette smoke, and implicate a lung vascular protective function of adipose stem cell-derived paracrine factors. PMID: 20709815 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | CAV1 inhibits metastatic potential in melanomas through suppression of the Integrin/Src/FAK signaling pathway. Cancer Res. 2010 Aug 13; Authors: Trimmer C, Whitaker-Menezes D, Bonuccelli G, Milliman JN, Daumer KM, Aplin AE, Pestell RG, Sotgia F, Lisanti MP, Capozza F Caveolin-1 (CAV1) is the main structural component of Caveolae which are plasma membrane invaginations that participate in vesicular trafficking and signal transduction events. Although, evidence has recently accumulated describing the function of CAV1 in several cancer types, its role in melanoma tumor formation and progression remains poorly explored. Here, by employing B16F10 melanoma cells as an experimental system, we directly explore the function of CAV1 in melanoma tumor growth and metastasis. We first show that CAV1 expression promotes proliferation while it suppresses migration and invasion of B16F10 cells in vitro. When orthotopically implanted in the skin of mice, B16F10 cells expressing CAV1 form tumors that are similar in size to their control counterpart. An experimental metastasis assay demonstrates that CAV1 expression suppresses the ability of B16F10 cells to form lung metastases in C57Bl/6 syngeneic mice. Additionally, CAV1 protein and mRNA levels are found to be significantly reduced in human metastatic melanoma cell lines and human tissue from metastatic lesions. Finally, we demonstrate that following integrin activation, B16F10 cells expressing CAV1 display reduced expression levels and activity of FAK and Src proteins. CAV1 expression also markedly reduces the expression levels of beta3 Integrin in B16F10 melanoma cells. In summary, our findings provide experimental evidence that CAV1 may function as an antimetastatic gene in malignant melanoma. PMID: 20709760 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Macrophage-Mediated Degradation of Crosslinked Collagen Scaffolds. Acta Biomater. 2010 Aug 12; Authors: Yahyouche A, Zhidao X, Czernuszka JT, Clover AJ Biological scaffolds used in tissue engineering are incorporated in vivo by a process of cellular in growth, followed by host mediated degradation and replacement of these scaffolds, in which phagocytic cells from the monocyte/ macrophage cell lineage play a key role. The chemical degradation of scaffolds with collagenases is well established, but to date this has not been correlated with an in vitro model of cell mediated scaffold degradation. RAW264.7, a murine monocyte/macrophage cell line was cultured on collagen scaffolds crosslinked either by dehydrothermal treatment (DHT) or by carbodiimide (EDC). These cells attached to collagen scaffolds, proliferated, and exhibited macrophage aggregation to form giant cells. Crosslinking the scaffolds with either DHT or EDC increased the resistance of the scaffold to degradation by macrophages. Increasing the amount of crosslinking in the scaffold made them more resistant to degradation by collagenase. However, while EDC increased the scaffolds' thermal and mechanical properties and decreased swelling ratio, DHT increased the mechanical properties but decreased the denaturation temperature and swelling ratio. Altering the scaffold properties by crosslinking affects the rate of degradation by macrophages and this is correlated with chemical degradation(r = 0.658, p<0.01). This will help to design scaffolds with task specific profile for use in tissue engineering. PMID: 20709200 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Mechano-topographic modulation of stem cell nuclear shape on nanofibrous scaffolds. Acta Biomater. 2010 Aug 12; Authors: Nathan AS, Baker BM, Nerurkar NL, Mauck RL Stem cells transit along a variety of lineage-specific routes towards differentiated phenotypes. These fate decisions are dependent not just on the soluble chemical cues that are encountered or enforced in vivo and in vitro, but also on physical cues from the cellular microenvironment. These physical cues can consist of both nano- and micro-scale topographical features, as well as mechanical inputs provided passively (from the base properties of the materials to which they are adhered) or actively (from extrinsic applied mechanical deformations). A suitable tool for investigating the coordination of these cues lies in nanofibrous scaffolds, which can both dictate cellular and cytoskeletal orientation, and facilitate mechanical perturbations to seeded cells. Here, we demonstrate a coordinated influence of scaffold architecture (aligned versus randomly organized fibers) and tensile deformation on nuclear shape and orientation. Sensitivity of nuclear morphology to scaffold architecture was more pronounced in stem cell populations than in terminally differentiated fibrochondrocytes. Tension applied to the scaffold elicited further alterations in nuclear morphology, greatest in stem cells, that were mediated by the filamentous actin cytoskeleton, but not the microtubule or intermediate filament network. With loading, nuclear perturbations were time- and direction-dependent, suggesting that the modality and direction of loading influence nuclear architecture. The present work may provide additional insight into the mechanisms by which the physical microenvironment influences cell fate decisions, and has specific application to the design of new materials for regenerative medicine applications with adult stem cells. PMID: 20709198 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Chemokine profile of synovial fluid from normal, osteoarthritis and rheumatoid arthritis patients: CCL25, CXCL10 and XCL1 recruit human subchondral mesenchymal progenitor cells. Osteoarthritis Cartilage. 2010 Aug 12; Authors: Endres M, Andreas K, Kalwitz G, Freymann U, Neumann K, Ringe J, Sittinger M, Häupl T, Kaps C OBJECTIVE: The microfracture technique activates mesenchymal progenitors that enter the cartilage defect and form cartilage repair tissue. Synovial fluid (SF) has been shown to stimulate the migration of subchondral progenitors. The aim of our study was to determine the chemokine profile of SF from normal, rheumatoid arthritis (RA) and osteoarthritis (OA) donors and evaluate the chemotactic effect of selected chemokines on human subchondral progenitor cells. METHOD: Chemokine levels of SF were analyzed using human chemokine antibody membrane arrays. The chemotactic potential of selected chemokines on human mesenchymal progenitors derived from subchondral cortico-spongious bone was tested using 96-well chemotaxis assays. Chemokine receptor expression of subchondral progenitors was assessed by real-time gene expression analysis and immuno-histochemistry. RESULTS: Chemokine antibody array analysis showed that SF contains a broad range of chemokines. Ten chemokines that showed significantly reduced levels in RA or OA compared to normal SF or robustly high levels in all SF tested were used for further chemotactic analysis. Chemotaxis assays showed that the chemokines MDC/CCL22, CTACK/CCL27, ENA78/CXCL5 and SDF1alpha/CXCL12 significantly inhibited migration of progenitors, while TECK/CCL25, IP10/CXCL10 and Lymphotactin/XCL1 effectively stimulated cell migration. MCP1/CCL2, Eotaxin2/CCL24 and NAP2/CXCL7 showed no chemotactic effect on subchondral progenitors. Gene expression and immuno-histochemical analysis of corresponding chemokine receptors document presence of low levels of chemokine receptors in subchondral progenitors, with the CXCL10 receptor CXCR3 showing the highest expression level. CONCLUSION: These results suggest that synovial fluid contains chemokines that may contribute to the recruitment of human mesenchymal progenitors from the subchondral bone in microfracture. PMID: 20709179 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Adult Stem Cells for Cardiac Tissue Engineering. J Mol Cell Cardiol. 2010 Aug 12; Authors: Martinez EC, Kofidis T Cell therapy and tissue engineering attract increasing attention as a potential approach for cardiac repair. Adult stem cells from autologous origin are a practical safe and appealing source for cell-based regenerative therapies that may hold realistic clinical potential. A plethora of interesting concepts have been introduced aiming at regenerating ischemic myocardium through adult stem cell based- bioartificial cardiac tissue supplements. Yet, current pre-clinical concepts have not reached translational applicability, and successes are only episodic. This review will provide a brief overview of the latest concepts and breakthroughs in the emerging field of cardiac adult stem cell-based tissue engineering, and discuses the challenges that this field needs to overcome to achieve realistic therapeutic translation into the clinical arena. PMID: 20709074 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Transcriptional Regulation of a Brown Adipocyte-specific Gene, UCP1, by KLF11 and KLF15. Biochem Biophys Res Commun. 2010 Aug 12; Authors: Yamamoto KI, Sakaguchi M, Medina RJ, Niida A, Sakaguchi Y, Miyazaki M, Kataoka K, Huh NH Several growth factors and transcription factors have been reported to play important roles in brown adoipocyte differentiation and modulation of thermogenic gene expression, especially the expression of UCP1. In this study, we focused on KLF11 and KLF15, which were expressed highly in brown adipose tissue. Our data demonstrated that KLF11 and KLF15 interacted directly with the UCP1 promoter using GC-box and GT-boxes, respectively. Co-transfection of KLF11 and KLF15 in the mesenchymal stem cell line muBM3.1 during brown adipocyte differentiation enhanced the expression level of UCP1. KLF11, but not KLF15, was essential for UCP1 expression during brown adipocyte differentiation of muBM3.1. PMID: 20709022 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Angiogenesis induced by controlled release of neuropeptide substance P. Biomaterials. 2010 Aug 13; Authors: Kohara H, Tajima S, Yamamoto M, Tabata Y The in vivo recruitment of circulating host cells to the site to be regenerated is one of the promising strategies for therapeutic angiogenesis. Substance P (SP), a member of neuropeptides, mediates pain perception and regulates wound healing, inflammation, tumor cell proliferation, and angiogenesis. This SP enhanced the migration, adhesion, and angiogenic gene expression of granulocytes in vitro. A biodegradable hydrogel was prepared from an anionic derivative of gelatin to achieve the controlled release of SP in vivo. When the anionic gelatin hydrogels incorporating SP were subcutaneously implanted into the mouse back, significant angiogenesis was induced around the site implanted, in contrast to the injection of SP solution. In vivo accumulation of granulocytes around the implanted sites was observed. It is concluded that the controlled release of SP efficiently induced the recruitment and the subsequent activation of granulocytes, one of the circulating cells with angiogenic activities, from the blood circulation into the site implanted, resulting in enhanced angiogenesis. PMID: 20708795 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Tissue engineering the mechanosensory circuit of the stretch reflex arc: Sensory neuron innervation of intrafusal muscle fibers. Biomaterials. 2010 Aug 12; Authors: Rumsey JW, Das M, Bhalkikar A, Stancescu M, Hickman JJ The sensory circuit of the stretch reflex arc, composed of specialized intrafusal muscle fibers and type Ia proprioceptive sensory neurons, converts mechanical information regarding muscle length and stretch to electrical action potentials and relays them to the central nervous system. Utilizing a non-biological substrate, surface patterning photolithography and a serum-free medium formulation a co-culture system was developed that facilitated functional interactions between intrafusal muscle fibers and sensory neurons. The presence of annulospiral wrappings (ASWs) and flower-spray endings (FSEs), both physiologically relevant morphologies in sensory neuron-intrafusal fiber interactions, were demonstrated and quantified using immunocytochemistry. Furthermore, two proposed components of the mammalian mechanosensory transduction system, BNaC1 and PICK1, were both identified at the ASWs and FSEs. To verify functionality of the mechanoreceptor elements the system was integrated with a MEMS cantilever device, and Ca(2+) currents were imaged along the length of an axon innervating an intrafusal fiber when stretched by cantilever deflection. This system provides a platform for examining the role of this mechanosensory complex in the pathology of myotonic and muscular dystrophies, peripheral neuropathy, and spasticity inducing diseases like Parkinson's. These studies will also assist in engineering fine motor control for prosthetic devices by improving our understanding of mechanosensitive feedback. PMID: 20708792 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Non-destructive label-free monitoring of collagen gel remodeling using optical coherence tomography. Biomaterials. 2010 Aug 12; Authors: Levitz D, Hinds MT, Ardeshiri A, Hanson SR, Jacques SL Matrix remodeling plays a fundamental role in physiological and pathological processes, as well as in tissue engineering applications. In this paper, optical coherence tomography (OCT), a non-destructive optical imaging technology, was used to image collagen gel remodeling by smooth muscle cells (SMCs). The optical scattering properties of collagen-SMC gels were characterized quantitatively by fitting OCT data to a theoretical model. Matrix remodeling over 5 days produced a 10-fold increase in the reflectivity of the collagen gels, corresponding to a decrease in scattering anisotropy from 0.91 to 0.46. The increase in reflectivity was corroborated in confocal mosaic images. Blocking matrix degradation in collagen-SMC gels with doxycycline, a non-specific matrix metalloproteinases (MMPs) inhibitor, impeded the decrease in scattering anisotropy and resulted in few macroscopic signs of remodeling. Causing matrix degradation in acellular gels with a 3 h treatment of MMP-8 (collagenase 2) partially mimicked the decrease in anisotropy measured in collagen-SMC gels after 5 days. These results suggest that the decrease in scattering anisotropy in the collagen-SMC gels was due to MMP activity that degrades collagen fibrils into smaller fragments. PMID: 20708790 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Efficiently Differentiating Vascular Endothelial Cells from Adipose Tissue Derived Mesenchymal Stem Cells in Serum-Free Culture. Biochem Biophys Res Commun. 2010 Aug 11; Authors: Konno M, Hamazaki TS, Fukuda S, Tokuhara M, Uchiyama H, Okazawa H, Okochi H, Asashima M Adipose tissue-derived mesenchymal stem cells (ASCs) have been reported to be multipotent and to differentiate into various cell types, including osteocytes, adipocytes, chondrocytes, and neural cells. Recently, many authors have reported that ASCs are also able to differentiate into vascular endothelial cells (VECs) in vitro. However, these reports included the use of medium containing fetal bovine serum for endothelial differentiation. In the present study, we have developed a novel method for differentiating mouse ASCs into VECs under serum-free conditions. After the differentiation culture, over 80% of the cells expressed vascular endothelial-specific marker proteins and could take up low-density lipoprotein in vitro. This protocol should be helpful in clarifying the mechanisms of ASC differentiation into the VSC lineage. PMID: 20708604 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | ADAM13 Induces Cranial Neural Crest by Cleaving Class B Ephrins and Regulating Wnt Signaling. Dev Cell. 2010 Aug 17;19(2):345-352 Authors: Wei S, Xu G, Bridges LC, Williams P, White JM, Desimone DW The cranial neural crest (CNC) consists of multipotent embryonic cells that contribute to craniofacial structures and other cells and tissues of the vertebrate head. During embryogenesis, CNC is induced at the neural plate boundary through the interplay of several major signaling pathways. Here, we report that the metalloproteinase activity of ADAM13 is required for early induction of CNC in Xenopus. In both cultured cells and X. tropicalis embryos, membrane-bound Ephrins (Efns) B1 and B2 were identified as substrates for ADAM13. ADAM13 upregulates canonical Wnt signaling and early expression of the transcription factor snail2, whereas EfnB1 inhibits the canonical Wnt pathway and snail2 expression. We propose that by cleaving class B Efns, ADAM13 promotes canonical Wnt signaling and early CNC induction. PMID: 20708595 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Immune responses to coiled coil supramolecular biomaterials. Biomaterials. 2010 Aug 11; Authors: Rudra JS, Tripathi PK, Hildeman DA, Jung JP, Collier JH Self-assembly has been increasingly utilized in recent years to create peptide-based biomaterials for 3D cell culture, tissue engineering, and regenerative medicine, but the molecular determinants of these materials' immunogenicity have remained largely unexplored. In this study, a set of molecules that self-assembled through coiled coil oligomerization was designed and synthesized, and immune responses against them were investigated in mice. Experimental groups spanned a range of oligomerization behaviors and included a peptide from the coiled coil region of mouse fibrin that did not form supramolecular structures, an engineered version of this peptide that formed coiled coil bundles, and a peptide-PEG-peptide triblock bioconjugate that formed coiled coil multimers and supramolecular aggregates. In mice, the native peptide and engineered peptide did not produce any detectable antibody response, and none of the materials elicited detectable peptide-specific T cell responses, as evidenced by the absence of IL-2 and interferon-gamma in cultures of peptide-challenged splenocytes or draining lymph node cells. However, specific antibody responses were elevated in mice injected with the multimerizing peptide-PEG-peptide. Minimal changes in secondary structure were observed between the engineered peptide and the triblock peptide-PEG-peptide, making it possible that the triblock's multimerization was responsible for this antibody response. PMID: 20708258 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Nuclear receptors in stem cells and their therapeutic potential. Adv Drug Deliv Rev. 2010 Aug 10; Authors: Sun G, Shi Y The core transcriptional regulatory circuitries are important for controlling stem cell self-renewal and differentiation. Nuclear receptors provide an ideal model to regulate gene expression in both ligand-dependent and ligand-independent manners. Recent studies of regulatory events by nuclear receptors in neural stem cells, embryonic stem cells, and induced pluripotent stem cells (iPSCs), provided unique insights into mechanisms of stem cell regulation and provided invaluable resources for regenerative medicine. Nuclear receptors have been shown to be key players in stem cell self-renewal, pluripotency, and reprogramming. We summarize recent progress of studies on nuclear receptors in stem cell field as well as the potential therapeutic implications of these nuclear receptors and their cognate ligands. These studies not only uncover molecular mechanisms of stem cell regulation, but also provide unique opportunities for drug discovery. PMID: 20708051 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Induced pluripotent stem cells - alchemist's tale or clinical reality? Expert Rev Mol Med. 2010;12:25 Authors: Rashid ST, Vallier L Following Shinya Yamanaka's first report describing the reprogramming of fibroblasts into stem cells over three years ago, some sceptics initially drew analogies between this new field of research and the quasi-mystical practice of 'alchemy'. Unlike the alchemist, however, stem cell researchers have rigorously tested and repeated experiments, proving their very own brand of cellular 'alchemy' to be a reality, with potentially massive implications for the study of human biology and clinical medicine. These investigations have resulted in an explosion of related publications and initiated the field of stem cell research known as 'induced pluripotency'. In this review, we give an account of the historical development, current technologies and potential clinical applications of induced pluripotency and conclude with a perspective on the possible future directions for this dynamic field. PMID: 20707936 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Cardiac progenitor cells: potency and control. J Cell Physiol. 2010 Sep;224(3):590-600 Authors: Di Nardo P, Forte G, Ahluwalia A, Minieri M Stem cell-based regeneration of the heart has focused much scientific and public attention being cardiac diseases the major cause of disability and death in industrialized countries. Innumerable efforts have been taken to unveil the mechanisms undergoing stem cell proliferation and fate, but much remains to be endeavoured for their application in clinical practice. Nevertheless, the discovery of progenitor cells resident within the cardiac tissue has sparked off enthusiasm about the possibility of efficiently and safely engineering them to repair the injured myocardium. Indeed, the early applications of the cardiac progenitor cells, mostly based on simplistic concepts and techniques, have failed highlighting the prerequisite of expanding the knowledge about progenitor cell features and microenvironmental conditioning. In this review, recent information on resident cardiac progenitor cells has been systematically gathered in order to create a valuable instrument to support investigators in their efforts to establish an efficient cardiac cell therapy. PMID: 20578234 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Direct and progressive differentiation of human embryonic stem cells into the chondrogenic lineage. J Cell Physiol. 2010 Sep;224(3):664-71 Authors: Gong G, Ferrari D, Dealy CN, Kosher RA Treatment of common and debilitating degenerative cartilage diseases particularly osteoarthritis is a clinical challenge because of the limited capacity of the tissue for self-repair. Because of their unlimited capacity for self-renewal and ability to differentiate into multiple lineages, human embryonic stem cells (hESCs) are a potentially powerful tool for repair of cartilage defects. The primary objective of the present study was to develop culture systems and conditions that enable hESCs to directly and uniformly differentiate into the chondrogenic lineage without prior embryoid body (EB) formation, since the inherent cellular heterogeneity of EBs hinders obtaining homogeneous populations of chondrogenic cells that can be used for cartilage repair. To this end, we have subjected undifferentiated pluripotent hESCs to the high density micromass culture conditions we have extensively used to direct the differentiation of embryonic limb bud mesenchymal cells into chondrocytes. We report that micromass cultures of pluripotent hESCs undergo direct, rapid, progressive, and substantially uniform chondrogenic differentiation in the presence of BMP2 or a combination of BMP2 and TGF-beta1, signaling molecules that act in concert to regulate chondrogenesis in the developing limb. The gene expression profiles of hESC-derived cultures harvested at various times during the progression of their differentiation has enabled us to identify cultures comprising cells in different phases of the chondrogenic lineage ranging from cultures just entering the lineage to well differentiated chondrocytes. Thus, we are poised to compare the abilities of hESC-derived progenitors in different phases of the chondrogenic lineage for cartilage repair. PMID: 20432462 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Cyclically stretching developing tissue in vivo enhances mechanical strength and organization of vascular grafts. Acta Biomater. 2010 Jul;6(7):2448-56 Authors: Stickler P, De Visscher G, Mesure L, Famaey N, Martin D, Campbell JH, Van Oosterwyck H, Meuris B, Flameng W Tissue-engineered vascular grafts must have qualities that rival native vasculature, specifically the ability to remodel, the expression of functional endothelial components and a dynamic and functional extracellular matrix (ECM) that resists the forces of the arterial circulation. We have developed a device that when inserted into the peritoneal cavity, attracts cells around a tubular scaffold to generate autologous arterial grafts. The device is capable of cyclically stretching (by means of a pulsatile pump) developing tissue to increase the mechanical strength of the graft. Pulsed (n=8) and unpulsed (n=8) devices were implanted for 10 days in Lovenaar sheep (n=8). Pulsation occurred for a period of 5-8 days before harvest. Thick unadhered autologous tissue with cells residing in a collagen ECM was produced in all devices. Collagen organization was greater in the circumferential direction of pulsed tissue. Immunohistochemical labelling revealed the hematopoietic origin of >90% cells and a significantly higher coexpression with vimentin in pulsed tissue. F-actin expression, mechanical failure strength and strain were also significantly increased by pulsation. Moreover, tissue could be grafted as carotid artery patches. This paper shows that unadhered tissue tubes with increased mechanical strength and differentiation in response to pulsation can be produced with every implant after a period of 10 days. However, these tissue tubes require a more fine-tuned exposure to pulsation to be suitable for use as vascular grafts. PMID: 20123137 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | On the biomechanical function of scaffolds for engineering load-bearing soft tissues. Acta Biomater. 2010 Jul;6(7):2365-81 Authors: Stella JA, D'Amore A, Wagner WR, Sacks MS Replacement or regeneration of load-bearing soft tissues has long been the impetus for the development of bioactive materials. While maturing, current efforts continue to be confounded by our lack of understanding of the intricate multi-scale hierarchical arrangements and interactions typically found in native tissues. The current state of the art in biomaterial processing enables a degree of controllable microstructure that can be used for the development of model systems to deduce fundamental biological implications of matrix morphologies on cell function. Furthermore, the development of computational frameworks which allow for the simulation of experimentally derived observations represents a positive departure from what has mostly been an empirically driven field, enabling a deeper understanding of the highly complex biological mechanisms we wish to ultimately emulate. Ongoing research is actively pursuing new materials and processing methods to control material structure down to the micro-scale to sustain or improve cell viability, guide tissue growth, and provide mechanical integrity, all while exhibiting the capacity to degrade in a controlled manner. The purpose of this review is not to focus solely on material processing but to assess the ability of these techniques to produce mechanically sound tissue surrogates, highlight the unique structural characteristics produced in these materials, and discuss how this translates to distinct macroscopic biomechanical behaviors. PMID: 20060509 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Progenitor-enriched adipose tissue transplantation as rescue for breast implant complications. Breast J. 2010 Mar;16(2):169-75 Authors: Yoshimura K, Asano Y, Aoi N, Kurita M, Oshima Y, Sato K, Inoue K, Suga H, Eto H, Kato H, Harii K Breast enhancement with artificial implants is one of the most frequently performed cosmetic surgeries but is associated with various complications, such as capsular contracture, that lead to implant removal or replacement at a relatively high rate. For replacement, we used transplantation of progenitor-supplemented adipose tissue (cell-assisted lipotransfer; CAL) in 15 patients. The stromal vascular fraction containing adipose tissue progenitor cells obtained from liposuction aspirates was used to enrich for progenitor cells in the graft. Overall, clinical results were very satisfactory, and no major abnormalities were seen on magnetic resonance imaging or mammogram after 12 months. Postoperative atrophy of injected fat was minimal and did not change substantially after 2 months. Surviving fat volume at 12 months was 155 +/- 50 mL (Right; mean +/- SD) and 143 +/- 80 mL (Left) following lipoinjection from an initial mean of 264 mL. These preliminary results suggest that CAL is a suitable methodology for the replacement of breast implants. PMID: 19912236 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Candidate bone-tissue-engineered product based on human-bone-derived cells and polyurethane scaffold. Acta Biomater. 2010 Jul;6(7):2484-93 Authors: Woźniak P, Bil M, Ryszkowska J, Wychowański P, Wróbel E, Ratajska A, Hoser G, Przybylski J, Kurzydłowski KJ, Lewandowska-Szumieł M Biodegradable polyurethanes (PURs) have recently been investigated as candidate materials for bone regenerative medicine. There are promising reports documenting the biocompatibility of selected PURs in vivo and the tolerance of certain cells toward PURs in vitro - potentially to be used as scaffolds for tissue-engineered products (TEPs). The aim of the present study was to take a step forward and create a TEP using human osteogenic cells and a polyurethane scaffold, and to evaluate the quality of the obtained TEP in vivo. Human-bone-derived cells (HBDCs) were seeded and cultured on polyurethane scaffolds in a bioreactor for 14 days. The TEP examination in vitro was based on the evaluation of cell number, cell phenotype and cell distribution within the scaffold. TEPs and control samples (scaffolds without cells) were implanted subcutaneously into SCID mice for 4 and 13 weeks. Explants harvested from the animals were examined using histological and immunohistochemical methods. They were also tested in mechanical trials. It was found that dynamic conditions for cell seeding and culture enable homogeneous distribution, maintaining the proliferative potential and osteogenic phenotype of the HBDCs cultured on polyurethane scaffolds. It was also found that HBDCs implanted as a component of TEP survived and kept their ability to produce the specific human bone extracellular matrix, which resulted in higher mechanical properties of the harvested explants when preseeded with HBDCs. The whole system, including the investigated PUR scaffold and the method of human cell seeding and culture, is recommended as a candidate bone TEP. PMID: 19837193 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Follow-up of collagen meniscus implant patients: clinical, radiological, and magnetic resonance imaging results at 5 years. Knee. 2010 Jun;17(3):224-9 Authors: Bulgheroni P, Murena L, Ratti C, Bulgheroni E, Ronga M, Cherubino P This study investigated at medium term follow-up the clinical outcomes and any progression of knee osteoarthritis in a population of patients that underwent arthroscopic placement of a collagen meniscus implant. Thirty-four patients underwent arthroscopic placement of a collagen meniscus implant for a symptomatic deficiency of medial meniscal tissue. Follow-up evaluation included Lysholm II score and Tegner activity scores and MR arthrography of the knee at 2 and 5 years after surgery. Plain radiographs were also obtained at 5 years. Six patients were excluded. In eight cases arthroscopic second look evaluation was performed. Lysholm and Tegner activity scores at 2 and 5 years after surgery improved significantly compared to the preoperative score. These patients showed good to excellent clinical results after 5 years from a CMI placement. The chondral surfaces of the medial compartment had not degenerated further since placement of the CMI. MR signal had continued to mature between 2 and 5 years after implant, progressively decreasing signal intensity but in any case comparable to the low signal of a normal meniscus. In most of cases the CMI-new tissue complex had a slight reduction in size, compared to a normal medial meniscus, but the new tissue had no apparent negative effects. PMID: 19800801 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Bone formation in a rat calvarial defect model after transplanting autogenous bone marrow with beta-tricalcium phosphate. Acta Histochem. 2010 May;112(3):270-7 Authors: Shirasu N, Ueno T, Hirata Y, Hirata A, Kagawa T, Kanou M, Sawaki M, Wakimoto M, Ota A, Imura H, Matsumura T, Yamada T, Yamachika E, Sano K In the present study, we evaluated the osteogenic potential of an autogenous bone marrow graft combined with beta-tricalcium phosphate (beta-TCP) in a rat calvarial bone defect model. The bone marrow harvested from the tibia of 7-week-old rats was grafted autogenously in a calvarial defect together with beta-TCP (=BTG group, n=16) or without beta-TCP (=BG group, n=16). Groups of animals were also treated with beta-TCP alone (=TG group, n=16) and control animals (n=8) received no graft implanted into the defect. We then observed the process of bone formation by histology, enzyme histochemistry and immunohistochemistry. Five days after grafting, in the BTG and BG groups, cell proliferation and osteogenic differentiation were observed. From 5 to 10 days after surgery, active Runx2, osteopontin (OPN), and TRAP- positive cells appeared in the BTG and BG groups. New bone formation started in the defect in both the BTG and BG groups. At 30 days after grafting, the BTG group showed new bone development and replacement of beta-TCP to fill the bone defect. New bone formation in the BTG group was significantly greater than in the BG group (P<0.01). The TG group showed no marked bone formation in the defect. The combination graft of bone marrow with beta-TCP showed marked bone formation in rat calvarial defects. Our results indicate that the combination grafts of bone marrow with beta-TCP may be an effective technique for repairing bone defects Beta-TCPgraft (TG) group. PMID: 19403161 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | A prefabricated, tissue-engineered integra free flap. Plast Reconstr Surg. 2008 Aug;122(2):682; author reply 682-3 Authors: Agaoglu G, Erol OO PMID: 18626411 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Discussion: Sources of processed lipoaspirate cells: influence of donor site on cell concentration. Plast Reconstr Surg. 2008 Aug;122(2):619-20 Authors: Pu LL PMID: 18626382 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Sources of processed lipoaspirate cells: influence of donor site on cell concentration. Plast Reconstr Surg. 2008 Aug;122(2):614-8 Authors: Padoin AV, Braga-Silva J, Martins P, Rezende K, Rezende AR, Grechi B, Gehlen D, Machado DC BACKGROUND: Recently, adipose tissue harvested by liposuction has been identified as a source of processed lipoaspirate cells. The aim of this study was to determine the concentration of processed lipoaspirate cells in adipose tissue obtained by liposuction from different harvest areas in women. METHODS: A prospective cross-sectional study was conducted in 25 women in whom liposuction in four or more different zones in the same procedure was indicated. After selective liposuction, the material was sent to the laboratory, where it was processed for extraction of processed lipoaspirate cells, which were separated from the adipose tissue, quantified, and characterized through determination of c-kit expression. The following harvest regions were evaluated: upper abdomen, lower abdomen, trochanteric region, inner thigh, knee, and flank. The cell concentration obtained at each site was compared by analysis of variance for mixed models. RESULTS: A significant difference was found for cell concentration obtained at the different harvest sites. The cell concentration in the lower abdomen was greater than in other areas, but no significant difference was found in relation to the inner thigh. CONCLUSIONS: The lower abdomen and the inner thigh may have higher processed lipoaspirate cell concentrations. These sites may turn out to be better sources of adult mesenchymal stem cells. PMID: 18626381 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Differential effects of three preparations of human serum on expansion of various types of human cells. Plast Reconstr Surg. 2008 Aug;122(2):438-48 Authors: Kurita M, Aiba-Kojima E, Shigeura T, Matsumoto D, Suga H, Inoue K, Eto H, Kato H, Aoi N, Yoshimura K BACKGROUND: To avoid potential risks of animal-derived products such as viral transmission and immunologic reactions, usefulness of human-derived products in manipulation of cells for cell-based therapies has been investigated but has not yet been completely clarified. METHODS: Three types of human sera-serum from whole blood, serum from platelet-rich plasma, and serum from platelet-poor plasma-were prepared from blood samples obtained from the same four volunteers. The authors investigated the biochemical profiles of the three serum preparations as well as their potential as culture additives using three types of human cells: dermal fibroblasts, adipose-derived stem/stromal cells, and umbilical vein endothelial cells. RESULTS: Platelet counts differed among serum from whole blood (100 percent), platelet-rich plasma (75.1 percent), and platelet-poor plasma (12.6 percent), resulting in differential concentrations of platelet-derived growth factor and epidermal growth factor, although other biochemical values such as total protein and albumin were similar. Serum from whole blood and platelet-rich plasma highly enhanced proliferation of dermal fibroblasts compared with the effects of serum from platelet-poor plasma, but no differences in proliferative efficacy were observed in cultures of adipose-derived stem/stromal cells and vascular endothelial cells. CONCLUSIONS: Serum from platelet-rich plasma, which is less invasive to prepare than serum from whole blood, was superior to serum from platelet-poor plasma as a substitute for animal-derived serum in culture expansion of dermal fibroblasts. Although autologous or human-derived serum preparations may be of great use in cell-based therapies, this usefulness strongly depends on the target cell species and the purpose of the cell culture. PMID: 18626359 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Fluid shear stress magnitude, duration, and total applied load regulate gene expression and nitric oxide production in primary calvarial osteoblast cultures. Plast Reconstr Surg. 2008 Aug;122(2):419-28 Authors: González O, Fong KD, Trindade MC, Warren SM, Longaker MT, Smith RL BACKGROUND: Successful bone engineering requires an understanding of the effects of mechanical stress on osteoblast differentiation. Therefore, we examined the effects of varying magnitude and duration of fluid shear stress on factors associated with osteoblastic differentiation. METHODS: Using a cone viscometer, primary neonatal rat calvarial osteoblasts were exposed to continuous fluid shear stress at varying doses: 0.21, 0.43, and 0.85 Pa for varying time periods. Gene expression was analyzed using Northern blots and nitric oxide production was quantified with the colorimetric Griess reaction. RESULTS: Fluid shear stress stimulated comparable transient increases in TGF-beta1 and TGF-beta3 expression by 3 hours. TGF-beta1 expression returned to baseline by 12 hours at all shear doses. In contrast, TGF-beta3 expression decreased by 22 percent and 47 percent at 12 hours in response to 0.43 Pa and 0.85 Pa, respectively. Osteopontin and Msx-2 expression patterns were consistent with a more differentiated phenotype at all shear levels. The maximum level of shear stress increased nitric oxide production 2.5-fold at 12 hours and 6.0-fold at 24 hours. CONCLUSIONS: These data demonstrate differential regulation of TGF-beta1 and TGF-beta3 isoforms with fluid shear stress. Furthermore, because osteopontin and Msx-2 changes were consistent with progressive differentiation at all levels of shear stress, dosage appears to be less important than the presence of an effective physical stimulus. Lastly, nitric oxide does not appear to be the primary regulator of early transcriptional changes found in this study. PMID: 18626357 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Repairing critical-sized rat calvarial defects with a periosteal cell-seeded small intestinal submucosal layer. Plast Reconstr Surg. 2008 Aug;122(2):400-9 Authors: Keskin M, Kelly CP, Moreira-Gonzalez A, Lobocki C, Yarim M, Kaplan S, Jackson IT BACKGROUND: Small intestinal submucosa was evaluated as a bioscaffold candidate for periosteum-derived osteoblasts, and its suitability as a bone replacement material for cranial defects was investigated. METHODS: In the in vitro phase, osteoblasts were expanded in osteogenic medium and were then seeded onto small intestinal submucosa. To confirm osteoblast phenotype, they were tested for alkaline phosphatase, collagen type 1, and calcium expression. In the in vivo phase, calvarial critical-sized defects were created in 35 rats. The defects were either left untreated for surgical control (group 1), treated with small intestinal submucosa alone (group 2), treated with an osteoblast-embedded construct (group 3), or treated with an autogenous bone graft (group 4). The results were evaluated 12 weeks after surgery with radiopacity measurements and with stereologic analysis. RESULTS: Periosteal cells grew successfully in vitro. The percentage radiopaque area at the defect was measured to be 42, 74, 76, and 89 percent for groups 1, 2, 3, and 4, respectively. The pixel intensity of the same site was 36.4, 48.1, 47.5, and 54.5 for the same groups, respectively. Tissue-engineered constructs did not achieve enough bone formation and calcification to be effective as autogenous bone grafts and were not superior to the small intestinal submucosa alone. However, both small intestinal submucosa and cell-seeded small intestinal submucosa showed significantly more bone formation compared with the untreated group. CONCLUSIONS: Although it was demonstrated that the small intestinal submucosa itself has osteogenic properties, it was not significantly increased by adding periosteum-derived osteoblasts to it. The osteogenic properties of small intestinal submucosa are promising, and its role as a scaffold should be investigated further. PMID: 18626355 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Microarray analysis of the role of regional dura mater in cranial suture fate. Plast Reconstr Surg. 2008 Aug;122(2):389-99 Authors: Kwan MD, Wan DC, Wang Z, Gupta DM, Slater BJ, Longaker MT BACKGROUND: Craniosynostosis, the premature fusion of cranial sutures, results in serious neurologic and morphologic abnormalities when left untreated. Surgical excision of the fused sutures and remodeling of the skull remains the standard therapy. Development of novel, minimally invasive therapies for craniosynostosis will undoubtedly be dependent on a more thorough understanding of the molecular mechanisms underlying this abnormality. Significant evidence suggests the influence of regional dura mater on the behavior of the overlying suture complex. The mouse model has been instrumental in investigating this observation because of the natural juxtaposition of the posterior frontal suture, which fuses early in life, with the other cranial sutures, which remain patent. METHODS: The authors used microarray analysis to compare genomic changes in the dura mater underlying the posterior frontal and sagittal sutures of mice. Suture-associated dura mater was harvested from mice before (postnatal day 5), during (postnatal day 10), and after (postnatal day 20) posterior frontal suture fusion (n = 20 mice for each of the three time points). RESULTS: Microarray results confirmed differential regulation of genes involved in paracrine signaling, extracellular matrix, and bone remodeling between the dura mater underlying the fusing posterior frontal suture and the patent sagittal suture. CONCLUSIONS: These data confirm global differences in gene expression between regional dura mater underlying fusing and patent sutures. These results provide further insight into potential molecular mechanisms that may play a role in cranial suture biology. PMID: 18626354 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | A hydrophobic perfluoropolyether elastomer as a patternable biomaterial for cell culture and tissue engineering. Biomaterials. 2010 Aug 12; Authors: Schulte VA, Hu Y, Diez M, Bünger D, Möller M, Lensen MC We present a systematic study of a perfluoropolyether (PFPE)-based elastomer as a new biomaterial. Besides its excellent long-term stability and inertness, PFPE can be decorated with topographical surface structures by replica molding. Micrometer-sized pillar structures led to considerably different cell morphology of fibroblasts. Although PFPE is a very hydrophobic material we could show that PFPE substrates allow cell adhesion and spreading of primary human fibroblasts (HDF) very similar to that observed on standard cell culture substrates. Less advanced cell spreading was observed for L929 (murine fibroblast cell line) cells during the first 5 h in culture which was accompanied by retarded recruitment of alpha(v)beta(3)-integrin into focal adhesions (FAs). After 24 h distinct FAs were evident also in L929 cells on PFPE. Furthermore, organization of soluble FN into a fibrillar ECM network was shown for hdF and L929 cells. Based on these results PFPE is believed to be a suitable substrate for several biological applications. On the one hand it is an ideal cell culture substrate for fundamental research of substrate-independent adhesion signaling due to its different characteristics (e.g. wettability, elasticity) compared to glass or TCPS. On the other hand it could be a promising implant material, especially due to its straightforward patternability, which is a tool to direct cell growth and differentiation. PMID: 20708794 [PubMed - as supplied by publisher] | | | | | | | | | |  | |  | |  |
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