5,871 research outputs found
Tissue engineering of a tracheal substitute
No full textLectin histochemistry and scanning electron microscopy (SEM) was used to assess the growth and characterise the differentiation of human respiratory epithelial cells (REC) cultured on two biomaterial scaffolds. The first scaffold, based on a hyaluronic acid derivative, was observed to be non-adhesive for REC. This lack of adhesion was found to be unrelated to the presence of the hyaluronic acid binding domain on the surface of isolated REC. The other scaffold, consisting of equine collagen, was observed to encourage REC spreading and adhesion. Positive Ulex Europaeus agglutinin (UEA) lectin staining of this preparation indicated the presence of ciliated REC on the scaffold surface. However, the marked decrease in peanut agglutinin (PNA) positive staining, relative to that of control cultures and native tissue, indicates a dedifferentiation of the secretory cells in monolayer. SEM analysis of REC cultured on the collagen scaffold confirmed the presence of ciliated cells thereby validating the UEA positive staining. The presence of both established and developing cilia was also verified. This indicates that collagen biomaterials are appropriate for the tissue engineering of REC. Furthermore, that UEA and PNA staining is a useful tool in the characterisation of cells cultured on biomaterials, therefore helpful in identifying biomaterials that are suitable for specific tissue engineering purposes.
The culture of REC at an air liquid interface (ALI) was investigated. Both conventional ALI inserts and the Biofleece scaffold were used. The cells grown the on conventional inserts became multilayered and showed some degree of ciliation after the period of ten days. The cells grown on the Biofleece scaffold became necrotic and died due to nutrient deprivation. The use of ALI culture techniques on scaffold materials needs to be adjusted to allow for sufficient nutrient supply to the cells.
The Biofleece scaffold was found to be suitable for the tissue engineering of cartilage in vitro. Constructs with a cartilage-like morphology were generated with the scaffold after two weeks in culture. The tissue-engineered cartilage was found to contain a higher number of cells and less extracellular matrix (ECM) than the native tissue controls. Suction seeding techniques were used to improve the distribution of cells within the scaffold and thereby increase the overall efficiency of cartilage tissue engineering within the scaffold. Alcian blue (AB) and Papanicolau (PN) stains of the tissue engineered cartilage described two distinct regions within the constructs, namely the developed cartilage-like region and the developing region. The latter is thought to be areas in which the cartilage cells are yet to fully remodel the scaffold material and deposit their own “native” ECM. However, the Biofleece scaffold material was observed to loose 40-50% of its initial volume during the tissue engineering process over a period of two weeks. Thus the degradation of the Biofleece scaffold exceeds the rate of maturation of the cartilage tissue within the scaffold. This rapid biodegradation is most likely a result of matrixmetalloproteinase (MMP), in particular collagenase, production by the maturing chondrocytes. This reduction in size means that the Biofleece scaffold is not an appropriate material for the tissue engineering of a trachea. The optimal biomaterial for the tissue engineering of a trachea would degrade at a rate equal too, or slower than, the time taken for the cells within the scaffold to mature into functional tissue.
The co-culture of REC and chondrocytes was achieved through the use of matrigel as a basement membrane replacement (note that direct growth of REC on cartilage tissue has been observed to be difficult). The co-cultured constructs were not stable because the Biofleece scaffold degrades at a high rate in the presence of both cell types. The constructs were observed to shrink to approximately 35-30% of the original dimensions in a period of 3-7 days. The reason for this accelerated degradation is not known but is most likely the result of severe MMP production by the two cell types when in combination.
It was concluded that the characterisation procedures used in this study (histochemical staining, fluorescent staining and scanning electron microscopy) for both REC and chondrocyte tissue engineered constructs are appropriate for this and further studies. The chondrocyte seeding methodologies in particular are a useful tool for tissue engineering. This study succeeds in many ways to investigate the tissue engineering of a tracheal substitute by detailing how REC and chondrocytes can be cultured on biomaterials and assessed for tissue development. However, the study does not deliver such a viable substitute as an end product. The primary reason for this outcome is the rapid degradation of the Biofleece scaffold materialLectin Histochemie und Elektronenmikroskopie wurden benutzt, um das Wachstum von humanen respiratorischen Epithelzellen (RECs), welche auf zwei Biomaterialien kultiviert wurden, festzusetzen und ihren Differenzierungsgrad zu bestimmen. Das erste Trägermaterial, welches auf einem Hyaluronsäurederivat basiert, ließ keine Anheftung der RECs zu. Diese fehlende Anheftung ließ sich jedoch nicht zurückführen auf das Vorhandensein der Hyaluronsäure bindenden Domaine auf der Oberfläche isolierter RECs. Das andere Trägermaterial, aus Pferdekollagen hergestellt, zeigte dagegen eine verstärkte Teilungsaktivität und Anheftung der REC. Die positive Ulex Europaeus Agglutinin (UEA) Lectin Färbung dieser Proben ließ die Anwesenheit von mit Zilien versehenen RECs auf der Trägerstoffoberfläche vermuten. Darüber hinaus weist das im Vergleich zu Kontrollkulturen und nativem Gewebe deutliche Nachlassen der positiven Peanut Agglutinin–Färbereaktion auf eine Dedifferenzierung der sekretorischen Zellen in der Monolayer-Kultur hin. Die rasterelektronenmikroskopische Untersuchung der auf dem Kollagenbiomaterial kultivierten RECs bestätigte das Auftreten von Zellen mit Zilien und damit auch die Aussagekräftigkeit der positiven UEA–Färbung. Dies zeigt somit, dass Biomaterialien aus Kollagen für das Tissue Engineering von RECs geeignet sind und dass sowohl die UEA–als auch die PNA–Färbung geeignete Methoden zur Charakterisierung von Zellen darstellen, die auf Biomaterialien kultiviert wurden. Somit helfen sie bei der Identifizierung von Biomaterialien für bestimmte Einsatzgebiete im Tissue Engineering.
Des weiteren wurde die Kultivierung von RECs auf einem Air liquid interface (ALI) untersucht, wobei sowohl der konventionelle ALI–Einsatz als auch das Biovliesmaterial zum Einsatz kamen. Dabei wuchsen die Zellen auf dem konventionellen Einsatz in Multilayern und zeigten nach einem Zeitraum von 10 Tagen einen bestimmten Anteil an Ziliierung. Die Zellen auf dem Biovlies dagegen wurden nekrotisch und gingen schließlich an Nahrungsmangel ein. Deshalb muss der Einsatz von ALI–Kulturtechniken bei Trägermaterialien dementsprechend modifiziert werden, dass eine ausreichende Versorgung der Zellen mit Nährstoffen gewährleistet ist.
Für das in vitro–Tissue Engineering von Knorpel erwies sich das Biovlies jedoch als geeignet. Mit ihm konnten nach zwei Wochen Kulturzeit Konstrukte mit einer knorpelähnlichen Morphologie erzeugt werden. Dabei zeigte sich, dass der Tissue Engineering–Knorpel eine höhere Zellzahl bei reduzierter extrazellulärer Matrix (ECM) aufwies als vergleichbares natives Kontrollgewebe. Dabei wurden Saugtechniken benutzt, um die Verteilung der Zellen im Trägerstoff zu verbessern. Die Alzian – Blau – Färbung (AB) und Papanicolau – Färbung (PN) zeigten bei dem Tissue Engineering–Knorpel zwei unterschiedliche Regionen innerhalb des Konstrukts, nämlich eine knorpelähnliche bereits entwickelte Region und eine sich entwickelnde Region. Bei letzterer dürfte es sich wohl um Gebiete handeln, in denen Zellen noch im Begriff sind, den Trägerstoff vollends umzubauen und ihre eigene „native“ ECM abzulagern. Nichtsdestoweniger büßte das Biovlies während des Tissue Engineering Prozesses über einen Zeitraum von zwei Wochen annähernd 40-50 % seines anfänglichen Volumens ein. Somit übersteigt das Ausmaß der Degradation des Biovlieses das des Heranreifens von Knorpelgewebe in dem Trägermaterial. Diese schnelle Biodegradation ist am ehesten das Ergebnis der Aktivität von Matrixmetalloproteinasen (MMP), insbesondere der Kollagenase, welche von reifenden Chondrozyten produziert wird. Diese Schrumpfung bedeutet also, dass das Biovlies kein geeignetes Material für das Tissue Engineering der Trachea darstellt. Denn ein optimales Biomaterial für das Tissue Engineering der Trachea sollte sich innerhalb derselben Zeit bzw. über einen längeren Zeitraum hinweg abbauen, als innerhalb desjenigen, den die sich in dem Trägermaterial befindlichen Zellen benötigen, um zu funktionalem Gewebe heranzureifen.
Durch den Einsatz von Matrigel als Ersatz für die Basalmembran konnte eine Kokultur aus RECs und Chondrozyten etabliert werden (wobei anzumerken ist, dass sich direktes Wachstum von RECs auf Knorpelgewebe als problematisch erweist). Die Konstrukte aus Kokulturen waren nicht stabil, da das Biovlies in Anwesenheit beider Zelltypen hochgradig abgebaut wird. Innerhalb von 3–7 Tagen schrumpften die Konstrukte auf ca. 35–50 % ihrer Ausgangsgröße zusammen. Der Grund für diesen beschleunigten Abbau ist unbekannt, jedoch ist am ehesten eine ausgeprägte Produktion von MMP durch die beiden Zellarten anzunehmen, sobald diese in Kombination vorliegen.
Insgesamt lässt sich sagen, dass die Methoden zur Zell- und Gewebecharakterisierung, welche in dieser Studie benutzt wurden (histochemische Färbungen, Fluoreszenzfärbung und Elektronenmikroskopie) sowohl für mit RECs als auch mit Chondrozyten hergestellte Konstrukte für die vorliegende Arbeit als auch zukünftige Studien als geeignet anzusehen sind. Diese Studie hat in vielerlei Hinsicht erfolgreich das Tissue Engineering einer Luftröhre untersuchen können, indem sie im Detail aufzeigt, wie RECs und Chondrozyten auf Biomaterialien kultiviert und für das Tissue Engineering eingesetzt werden können. Trotzdem kann diese Arbeit kein einsetzbares Ersatzmaterial als Endprodukt liefern. Der Hauptgrund für dieses Ergebnis ist in erster Linie in dem schnellen Abbau des Biovlieses als Trägermaterial zu sehen
CONSTRUCTIVE TISSUE REMODELING BY EXTRACELLULAR MATRIX BIOSCAFFOLDS WITHIN THE AGING SKELETAL MUSCLE MICROENVIRONMENT
No full textAdult mammalian skeletal muscle tissue retains inherent regenerative capability in response to injury. This regenerative response is contingent upon an activated progenitor cell population and a temporal transition from pro-inflammatory M1 to immunomodulatory and constructive M2 polarized macrophages at the site of injury. Specifically, pro-inflammatory effector molecules secreted by M1 macrophages promote the expansion of skeletal muscle progenitor cells, while resolution of the inflammatory response and myogenic differentiation of skeletal muscle progenitor cells is dependent upon immunomodulatory effector molecules secreted by M2 macrophages.
The decline in the regenerative capacity of skeletal muscle tissue associated with advanced age is largely a consequence of progenitor cell dysfunction. Furthermore, advanced age is accompanied with immunosenesence of the innate immune system resulting in an impaired macrophage polarization potential.
Biologic scaffold materials composed of mammalian extracellular matrix (ECM) have been successfully used in both pre-clinical animal studies and in human clinical applications to promote constructive tissue remodeling in a variety of anatomic locations including skeletal muscle. The presence of ECM bioscaffolds at sites of skeletal muscle injury has been associated with a predominant M2 macrophage phenotype and downstream site-appropriate or constructive, functional tissue remodeling. The mechanisms responsible for this constructive tissue remodeling response are only partially understood.
The present dissertation shows that the age of source animals from which ECM bioscaffold materials are harvested represents a determinant factor of the constructive tissue remodeling potential induced by these materials. The present work also shows that degradation products of mammalian ECM promote the constructive M2 macrophage phenotype in both young and age-impaired macrophages. Moreover, effector molecules from macrophages exposed to ECM degradation products are chemotactic and myogenic for skeletal muscle progenitor cells. The present dissertation describes a new rodent model of volumetric muscle loss (VML) and an ECM bioscaffold based approach for tissue replacement associated with modulation of macrophage phenotype and the endogenous recruitment of perivascular stem cells
Supplemental_Material_for_Perfusion_System_by_O_Grady_et_al – Supplemental material for A Customizable, Low-Cost Perfusion System for Sustaining Tissue Constructs
No full textSupplemental material, Supplemental_Material_for_Perfusion_System_by_O_Grady_et_al for A Customizable, Low-Cost Perfusion System for Sustaining Tissue Constructs by Brian J. O’Grady, Jason X. Wang, Shannon L. Faley, Daniel A. Balikov, Ethan S. Lippmann and Leon M. Bellan in SLAS Technology</p
Detection of Mycolactone A/B in Mycobacterium ulcerans-Infected Human Tissue.
No full textBACKGROUND: Mycobacterium ulcerans disease (Buruli ulcer) is a neglected tropical disease common amongst children in rural West Africa. Animal experiments have shown that tissue destruction is caused by a toxin called mycolactone.
METHODOLOGY/PRINCIPAL FINDINGS: A molecule was identified among acetone-soluble lipid extracts from M. ulcerans (Mu)-infected human lesions with chemical and biological properties of mycolactone A/B. On thin layer chromatography this molecule had a retention factor value of 0.23, MS analyses showed it had an m/z of 765.6 [M+Na(+)] and on MS:MS fragmented to produce the core lactone ring with m/z of 429.4 and the polyketide side chain of mycolactone A/B with m/z of 359.2. Acetone-soluble lipids from lesions demonstrated significant cytotoxic, pro-apoptotic and anti-inflammatory activities on cultured fibroblast and macrophage cell lines. Mycolactone A/B was detected in all of 10 tissue samples from patients with ulcerative and pre-ulcerative Mu disease.
CONCLUSIONS/SIGNIFICANCE: Mycolactone can be detected in human tissue infected with Mu. This could have important implications for successful management of Mu infection by antibiotic treatment but further studies are needed to measure its concentration
Synthesis of Nanometer-sized Yttrium Oxide Particles in Diisooctyl Sodium Sulphosuccinate/Isooctane Reverse Micelle Solution
No full textThis thesis describes the synthesis of yttrium oxide nanoparticles in an AOT/isooctane reverse micelle solution. Two synthetic methods are compared. First is the precipitation reaction between yttrium nitrate and ammonia, second is the hydrolysis of yttrium isopropoxide. The effects of annealing of the resulting the yttrium oxide nanoparticles are also described.
The nitrate method produced network-like aggregates of yttrium oxide nanoparticles ranging from 10 nm to 40 nm in diameter. Reaction conditions, including the water/AOT ratio (1 to 15), the nitrate concentration (0.02 M to 1.0 M), the ammonia concentration (2 M to 14.8 M), the AOT concentration (0.1 M and 0.5 M), the aging time (1 h to 5 d), and the washing method, were varied to investigate their influence on nanoparticle formation. The optimized synthetic conditions were: a water/AOT ratio of 7.5, [NO₃⁻] = 0.5 M, [NH₃] = 2 M, and [AOT] = 0.1 M.
The as-prepared yttrium oxide nanoparticles had highly distorted structures related to the cubic Y2O3 phase. Annealing improved the crystallinity of the as-prepared nanoparticle products and led to larger particles. As annealing temperatures increased, the yttrium oxide nanoparticles gradually evolved into the cubic Y₂O₃ phase. However, an unknown intermediate phase was also observed during the annealing process, which disappeared when the annealing temperature was sufficiently high and the annealing time was long enough (>1000 °C and 4 h).
As-prepared products from the isopropoxide hydrolysis also contained network-like nanoparticle aggregates. Particle sizes ranged from 10 nm to 20 nm. Some experimental conditions were varied; they were the water/AOT ratio (10 to 40), the isopropoxide concentration (0.0001 M to 0.003 M), the aqueous phase pH (7.0 and 12.0), the aging temperature (room temperature, approximately 25 °C, and refluxing temperature, approximately, 100 °C), and the aging time (1 h to 5 d). Transmission Electronic Micrographs showed that products of desirable morphology could be produced in a much wider range of experimental conditions by this method compared to those produced by nitrate hydrolysis.Master of Scienc
A web-based screening tool for near-port air quality assessments
No full textAuthor(s): Isakov, Vlad; Barzyk, Timothy M; Smith, Elizabeth R; Arunachalam, Saravanan; Naess, Brian; Venkatram, Akul
Supplemental Material - Quality of life of patients with soft tissue sarcoma treated with doxorubicin in the ANNOUNCE phase III clinical trial
No full textSupplemental Material for Quality of life of patients with soft tissue sarcoma treated with doxorubicin in the ANNOUNCE phase III clinical trial by Brian A Van Tine, Anders Krarup-Hansen, Lisa M Hess, Albiruni R Abdul Razak, Victoria Soldatenkova, Jennifer Wright and Se Hoon Park in Rare Tumors</p
Tissue-specific expression profiles of IGF system binding proteins in bovine pre- and postnatal developmental stages.
No full textAbundances of transcripts for IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7 and IGFBP8 were measured in tissues of Day 48 embryos, Day 153 fetuses and 12–14 month-old juveniles. Placental samples were obtained from Day 48 embryos, Day 153 fetuses and term calves born by Caesarean section (C-section) at Day 277/278 of gestation. Means and standard deviations of means for each transcript and tissue were calculated based on triplicate measures of pooled cDNA comprising up to 60 embryonic cDNA samples, 73 fetal cDNA samples, 5 placental cDNA samples of C-section calves and 17 juvenile cDNA samples. Transcript abundances were calculated by the standard curve method and expressed in relative units, and are presented in logarithmic scale. ‘m’ denotes missing tissue such as kidney that is not yet present in embryos, where transcript abundances could not be determined.</p
Tissue-specific expression profiles of IGF system receptors in bovine pre- and postnatal developmental stages.
No full textAbundances of global IR transcript and splice variants IR-A and IR-B, IGF1R and IGF2R were measured in tissues of Day 48 embryos, Day 153 fetuses and 12–14 month-old juveniles. Placental samples were obtained from Day 48 embryos, Day 153 fetuses and term calves born by Caesarean section (C-section) at Day 277/278 of gestation. Means and standard deviations of means for each transcript and tissue were calculated based on triplicate measures of pooled cDNA comprising up to 60 embryonic cDNA samples, 73 fetal cDNA samples, 5 placental cDNA samples of C-section calves and 17 juvenile cDNA samples. Transcript abundances were calculated by the standard curve method and expressed in relative units, and are presented in logarithmic scale. ‘m’ denotes missing tissue such as kidney that is not yet present in embryos, where transcript abundances could not be determined.</p
Analysis of the Changes in the Oxidation of Brain Tissue Cytochrome-c-Oxidase in Traumatic Brain Injury Patients during Hypercapnoea A Broadband NIRS Study
No full textUsing broadband near-infrared spectroscopy (NIRS) and cerebral micro-dialysis (MD), we investigated cerebral cellular metabolism and mitochondrial redox states, following hypercapnoea in 6 patients with traumatic brain injury (TBI). In all patients hypercapnoea increased intracranial pressure and cerebral blood flow velocity measured with transcranial Doppler. Despite the likely increase in cerebral oxygen delivery, we did not see an increase in the oxidation status of cytochrome-c-oxidase [oxCCO] in every patient. Analysis of the NIRS data demonstrated two patterns of the changes; Group A (n = 4) showed an increase in [oxCCO] of 0.34(+/-0.34)mu M and Group B (n = 2) a decrease of 0.40(+/- 0.41)mu M. Although no obvious association was seen between the Delta[oxCCO] and the MD, measured changes in lactate and pyruvate concentrations. Further work using model informed data interpretation may be helpful in understanding the multimodal signals acquired in this heterogeneous patient group
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