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1
Hatayama, Takahide. "Regeneration of gingival tissue using in situ tissue engineering with collagen scaffold." Kyoto University, 2019. http://hdl.handle.net/2433/243271.
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Sulaiman, Nadiah. "Decellularised veins as a scaffold for in situ vascular tissue engineering." Thesis, University of Bristol, 2019. http://hdl.handle.net/1983/71d16a49-8294-4cae-ae98-d16da0ee84c0.
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Development of new arterial-like vascular conduits for coronary and peripheral bypass grafting surgery is desirable to overcome the limitations of currently available biological and/or synthetic grafts; to reduce the incidence of early thrombosis, late intimal thickening and infection. One alternative is to use arterialised decellularised venous scaffolds. The aim of this PhD project was to assess the feasibility and suitability of human saphenous vein (hSV) decellularisation as a way to obtain effective biological cellular scaffold for vascular grafting. We identified the optimal sodium dodecyl sulphate (SDS) concentration needed to decellularise short segments of hSVs (~0.5 cm). Low concentration (0.01%) (w/v) SDS removed most of the nuclei, but this approach was not effective in removing nuclei when using ~4 cm long hSVs. Hence, a modified flow technique of decellularisation was established with successful decellularisation of longer hSV segments. Biocompatibility and integrity of decellularised hSVs were then evaluated. Methylene blue assay detected only trivial residual concentrations of SDS after decellularisation. This was biocompatible as this residual amount of SDS did not affect the viability of porcine carotid artery endothelial cells (pCA ECs) to populate the acellular hSV (AlamarBlue) and to proliferate (EdU proliferation assays). Next, the ECM integrity of acellular hSVs was assessed by quantifying major ECM proteins (collagen, elastin and glycosaminoglycan). Results revealed that decellularisation with ≤0.01% (w/v) SDS did not have a significant impact on ECM content. We then tested the feasibility, safety and capacity of acellular hSVs to arterialise following surgical implant with end-to-end anastomoses in pig without immunosuppression. This pilot study showed that porcine carotid artery xenograft of decellularised hSV was feasible and safe, with 50% graft patency rate at 4 weeks and signs of in situ vascular tissue engineering by host cells. In conclusion, effective decellularisation of hSV is feasible, safe and reproducible as potential acellular vascular scaffolds. Acellular hSVs may be used as vascular acellular scaffolds either for in situ vascular engineering by host cells or following ex vivo manipulation before implantation. However, this approach warrants further investigations.
3
Umeda, Hiroo. "In situ Tissue Engineering of Canine Skull with Guided Bone Regeneration." Kyoto University, 2009. http://hdl.handle.net/2433/124318.
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Glover, Christopher John. "In Situ Polymerizing Collagen for the Development of 3D Printed Tissue Engineering Scaffolds." Thesis, University of Missouri - Columbia, 2019. http://pqdtopen.proquest.com/#viewpdf?dispub=13850736.
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<p> Natural materials have been processed and utilized as scaffold materials in the field of tissue engineering for many years. One natural material often utilized is collagen since it is the main structural protein in mammalian tissues and exhibits microstructures suitable for the survival and proliferation of many different cell lineages. However, a common challenge with fibrillized collagen is the difficulty associated with trying to process it into specific three-dimensional designs for the development of scaffolds aimed at regenerating particular tissue types. This project consists of utilizing a custom platform capable of 3D printing <i>in situ</i> polymerizing collagen into user-defined morphologies for the development of 3D collagen-based scaffolds. Various anti-inflammatory compounds such as gold nanoparticles and curcumin were also incorporated into the scaffolds post printing in order to further tailor the cellular responses to the scaffolds. Scanning electron microscopy and neutron activation analysis were performed to verify and quantify the attachment of the gold nanoparticles, respectively. Differential scanning calorimetry was utilized to examine and optimize the stability of the scaffolds after crosslinking. Lastly, water soluble tetrazolium salt and reactive oxygen species assays were performed to assess the biocompatibility of the scaffolds using L929 murine fibroblasts. The results exhibited the viability of the platform to become an effective technique to manufacture and process custom scaffolds for tissue engineering applications.</p><p>
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Ringe, Jochen. "Differenzierungs- und Migrationspotential mesenchymaler Stamm- und Progenitorzellen für das in situ Tissue Engineering." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=980187052.
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Balaji, Swathi. "In situ tissue engineering using angiogenic peptide nanofibers to enhance diabetic wound healing." University of Cincinnati / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1291151135.
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Ge, Cheng. "Novel technologies for cell culture and tissue engineering." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:ab1014cf-80a4-4675-b607-96dc52c39b17.
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Cell culture has been a fundamental tool for the study of cell biology, tissue engineering, stem cell technology and biotechnology in general. It becomes more and more important to have a well-defined physiochemical microenvironment during cell culture. Conventional cell cultures employ expensive, manually controlled incubation equipment, making it difficult to maximize a cultures yield. Furthermore, previous studies use qualitative methods to assess cell culture proliferation that are inherently inaccurate and labour intensive, thereby increasing the cost of production. In addition, three dimensional cell culture, in scaffold, has been shown to provide more physiological relevant information as it mimic more accurate conditions that are similar to the physiological conditions of the human body compared with two dimension, which has special interest to regenerative medicine. Therefore, a portable and automated total-analysis-system (μTAS) was proposed with microenvironment control and quantitative analysis techniques to monitor cell proliferation and metabolic activity. The automated portable heating system was validated to be capable to maintain a stable physiochemical microenvironment, with little margin of error, for cellular substrate outside of conventional incubation. A standalone platform system was designed and fabricated with accurate temperature control by employing an optically transparent ITO-film with a large heating area. The transparency of the film is critical for continuous in-situ microscopic observation over long-term cell culture process. Previous studies have attempted to use ITO-film as a heating element, but were unable to distribute the heat evenly onto the microbioreactor platform. This nagging problem in the literature was improved through a novel film design. As a result, the ITO-film based heating system was evaluated and constructed successfully to serve as a heating element for long-term static cell culture with facilitated proliferation rate in gas-permeable PDMS microbioreactor outside of conventional incubation. In addition to maintaining a stable microenvironment, a non-invasive in-situ technology for monitoring cell viability and proliferation rate was constructed and developed based on bioimpedance spectroscopy (BIS). It was primarily focused on making decisions for structure and specification of proposed system-on a chip BIS measurement. The miniaturization of BIS system on microbioreactor platform was achieved by utilizing and integrating switching matrix array, impedance analyzer chip with reliable analogue-front-end circuitry. The realized system was verified with the DLD-1 cells and its monitored data were validated with conventional bioassays. Three dimensional cell cultures with scaffold is a key to the success of tissue engineering. Engineered cornea collagen scaffold may be feasible using re-seeding proper human cells onto a decellularized corneal scaffold. The quality of the scaffold and the interaction of the cells are critical to the key function (i.e transparency, haze and total transmittance) of final products. An integrated corneal collagen scaffold quality assessment system, via optical property inspection unit, was innovatively designed and realized with non-invasive and non-destructive characteristics. The H1299 cells were seeded onto inspected corneal scaffold and BIS system, which were realized in the previous chapter, were used to validate its applicability for 3D cell culture. The cell adhesion as an outcome at different scaffolds with different optical properties has revealed the importance of the microstructure of scaffold on the cell functions. The results showed the developed technologies can be used for the quality control of corneal scaffold and the fabricated μTAS not only enabled environmental control but, with BIS-based in-situ assay, it also facilitate the function (i.e adhesion) and viability monitoring with quantitative and qualitative analysis in 3D-alike cell culture. Additionally, by considering its low decontamination and cost-effective nature with compatibility for high-throughput screening applications, the fabricated and integrated systems has significant applications in tissue engineering.
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Ahmed, Shehnaz. "Self-reporting scaffolds for in situ monitoring for regenerative medicine and tissue engineering applications." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/49511/.
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This thesis describes the development and utilisation of a self-reporting scaffold to improve current monitoring methods of the cellular microenvironment. In vitro tissue models hold a lot of promise for regenerative medicine and tissue engineering. However, many models lack the ability to non-invasively monitor in situ cellular responses in a physiologically relevant environment. By development of electrospun self-reporting scaffolds and incorporation of flow culture conditions, this limitation can be overcome. Electrospun matrices have been shown to mimic the structural architecture of the native extracellular matrix, whilst flow conditions have been shown to regulate cellular processes, and enhance mass transport and nutrient exchange throughout polymeric scaffolds. Here we show the development of optically transparent self-reporting electrospun scaffolds that incorporate ratiometric pH-sensitive nanosensors and respond to biological and mechanical cues of the native extracellular matrix through exposure to shear stress. Optically transparent self-reporting scaffolds were fabricated by directly electrospinning pH responsive, ratiometric nanosensors within a gelatin biopolymer matrix. The sensors consist of a porous polyacrylamide matrix which encapsulates pH-sensitive fluorophores that exhibit an additive fluorescent response across the full physiological range between pH 3-8, and a pH-insensitive reference fluorophore. The self-reporting scaffold was able to support cell growth whilst being able to simultaneously monitor local pH changes in real time. A Quasi-Vivo® bioreactor system was also used to generate a flow of cell culture medium and expose cell-seeded scaffolds to a continual shear stress. This novel diagnostic scaffold and the use of flow conditions can help simulate enhance the understanding of in vitro conditions, and generate advanced simulations in vivo to facilitate tissue engineering and regenerative medicine applications.
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Mo, Jingyi. "In-situ analysis of nanoscale deformation mechanism in mutable collagenous tissue." Thesis, Queen Mary, University of London, 2018. http://qmro.qmul.ac.uk/xmlui/handle/123456789/44694.
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Echinoderms, for example sea cucumber, contain a unique collagenous tissue, with special biomechanical properties, which could near-instantly change their mechanical state (going from stiff to soft, and vice versa, in less than a second). However, the structure-function relation has so far not been exploited. Understanding how the material design of mutable collagenous tissue (MCT) enables this remarkable dynamical mechanical behaviour will help enable development of new biomaterials with adaptable mechanical properties. Currently, it is hypothesised that MCT can rapidly form crosslinks between the collagen fibrils and stiffen the interfibrillar matrix under neural control, but this had never been shown directly. In this thesis, we carried out an experimental study of quantifying how the interfibrillar matrix response to stimuli agents, to generate active forces and change conformation using a synchrotron in situ X-ray nanomechanical imaging method. By the uncovering of the mechanisms of active force generation, a valuable guideline, which could be applied in bioinspired constructs that response to external stimuli, can be obtained.
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Coffel, Joel. "Implementation and modeling of in situ magnetic hyperthermia." Diss., University of Iowa, 2016. https://ir.uiowa.edu/etd/2058.
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Health-care associated infections (HAIs) on medical implant surfaces present a unique challenge to physicians due to their existence in the biofilm phenotype which defends the pathogen from antibiotics and the host’s own immune system. A 2004 study in the U.S. showed that 2 to 4% of implanted devices become infected and must be treated via surgical explantation—a process that is both expensive and dangerous for the patient. A potential, alternative strategy to antibiotics and surgery is to use heat delivered wirelessly by a magnetic coating. This thermal treatment strategy has the potential to kill these HAIs directly on the implanted surface and without the patient requiring surgery.
This thesis introduces an iron oxide nanoparticle composite coating that is wirelessly heated using energy converted from an alternating magnetic field. Iron oxide nanoparticle composites are demonstrated to be remotely heated in both hydrophilic and hydrophobic polymer composites. In designing the composite coating, multiple parameters were investigated for how they impact the normalized heating rate of the material. Specifically, the amount of iron in the coating, the coating thickness, the polymer type, and the orientation of the coating relative to the applied magnetic field were investigated. Power output was shown to increase proportionally with iron loading whereas nearly two times the amount of power output was observed for the same coatings positioned parallel to magnetic field lines versus those positioned perpendicular—a result believed to be due to magnetic shielding from neighboring particles.
Microscope slides coated with 226 µm of composite delivered up to 10.9 W cm⁻² of power when loaded with 30.0% Fe and positioned parallel in a 2.3 kA m⁻¹AMF. Pseudomonas aeruginosa biofilms were grown directly on these coatings and heated for times ranging from 1 to 30 min and temperatures from 50 to 80 °C. Less than one order of magnitude of cell death was observed for temperatures less than 60 °C and heat shock times less than 5 min. Up to six orders of magnitude reduction in viable bacteria were observed for the most extreme heat shock (80 °C for 30 min).
Introducing this wirelessly heated composite into the body has the potential to kill harmful bacteria but at the risk of thermally damaging the surrounding tissue and organs if the treatment is not designed and predicted intelligently. Thermal energy will propagate differently depending on the surrounding heat sink, with convective heat sinks (i.e. those due to blood flow) requiring much more power to reach the same surface temperature than a conduction-only heat sink. To study how heat is transferred in biological tissues, a robust, poly(vinyl alcohol) tissue phantom was developed that can be poured to accommodate any geometry, is volume stable in water and under thermal stress, and can be modified with inert particle fillers to adjust its thermal conductivity from 0.475 to 0.795 W m⁻¹°C⁻¹. In vitro heat transfer was measured through this hydrogel tissue phantom with at least 10 °C of temperature rise, penetrating 5 mm of tissue in less than 120 sec for an 80 °C boundary condition.
A computational model was used to solve three-dimensional energy transfer through a combined fluid mimic/tissue mimic heat sink spanning the same surface boundary condition. The model was validated with experimental models using a custom designed heat transfer station. This scenario is applicable in the instance where the same coating is subject to starkly different heat sinks: half subject to convective heat loss, half to conductive heat loss. Based on these conditions, a magnetic coating would need to be designed that has a power gradient up to 15 times larger on the fluid half versus the other.
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Burke, Julie Leigh. "In situ engineering of skeletal tissues using self-assembled biomimetic scaffolds." Thesis, University of Leeds, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.559370.
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The overall aim of this thesis was to establish whether selected Leeds' β-structure self- assembling peptides were suitable candidates for use in skeletal tissue engineering. This was addressed by first carrying out simple cytotoxicity studies on selected SAPs using an osteosarcoma cell line in vitro, followed by assessing extent of bone regeneration / repair and safety in vivo in a rabbit calvarial defect model. In vitro investigations revealed that the SAPs (P11-4, P11-8, P111-12, P11-1S, P11-16 and Pl1- 20) tested were cytocompatible, though the positively charged SAP P11-16 performed less well than other SAPs tested in the contact cytoxicity assay, possibly due to the presence of chemical contaminant introduced during the peptide purification process. Cell proliferation was generally superior on the negatively charged peptides compared with the positively charged peptides and cells appeared to thrive at the lower concentrations (30 mg/mL) of negatively charged SAPs compared with their behaviour on the positively charged peptides at similar concentrations. In vivo, SAPs P11-4, P11-8 and P11-20 were applied to critical size defects in rabbit calvaria. Puralvlatrix' and Bio-Oss" were used as positive controls and no treatment was used as the negative control. Calvaria were harvested after 3 days, 10 days, 28 days and 84 days and subjected to micro-CT. The calvarial samples were then decalcified and evaluated histologically and immunohistochemically. Application of the negatively charged P11-4 to cranial defects resulted in the most bone regeneration over 3 months. The other negatively charged peptide (P11-20) and the positively charged peptide (P11-8) resulted in a similar amount of bone regeneration as the positive control hydrogel, Puralvtatrix, and the empty control defects. The bone regenerated was of normal architecture and exhibited the usual patterns for the mature bone marker, osteocalcin. However, the patterns of bone regeneration associated with the different treatment applications varied, with Pn-4 treated sites showing a distinctive spicule-type bony repair which may reflect its superior mineral nucleating ability in vitro. Immunohistochemistry showed the presence of assembled SAP fibres within the defect sites at 3 days post treatment, with some indication of intracellular- SAP present at day 10. No evidence of fibrillar SAP was present at day 28 for any of the peptides used. All safety parameters investigated in the in vivo model (presence of localised tissue reaction, general well being of the animal, IgG levels, detection of specific anti-peptide antibodies, histological evidence of necrosis/inflammation/foreign body reaction, presence of amyloid deposits) were all negative, suggesting SAPs had low immunogenicity and were safe to use. In conclusion, this is the first time that these self-assembling peptides have been investigated in vivo. The results suggest that they as good as the market leads in inducing bone repair, their biocompatibilitv and low immunogenicity strongly support their further development for use in human tissue engineering applications.
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Jordan, Alex Michael. "FIBER-COMPOSITE IN SITU FABRICATION: MULTILAYER COEXTRUSION AS AN ENABLING TECHNOLOGY." Case Western Reserve University School of Graduate Studies / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=case1467832877.
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Sánchez, Ferrero Aitor. "Biomimetic hydrogels for in situ bone tissue engineering : nature-inspired crosslinking methods as a tool to tune scaffold physical properties." Doctoral thesis, Universitat Politècnica de Catalunya, 2015. http://hdl.handle.net/10803/373909.
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The global incidence of bone fractures, and subsequently that of non-healing ones, is expected to rise in the coming decades, mostly due to an increased risk of age-related conditions. Currently, the biomaterials field is moving towards the design of scaffolds mimicking the cell microenvironment to guide stem cells differentiation and recapitulate the development of target tissues. Biomimicry is a wide concept and several approaches have been adopted to produce cellinstructive scaffolds. Herein, we have explored the use of citric acid and lysyl oxidase, both of them related to bone nanostructure and mechanical performance, to develop scaffolds resembling the extracellular matrix of developing bone.
First, elastin-like recombinamers (ELRs) hydrogels were achieved through a one-step chemical crosslinking reaction with citric acid, a molecule currently considered to be essential for the proper performance of bone tissue. By systematically studying the crosslinking reaction and its contribution to hydrogel properties, we were able to control the architecture and stiffness of citric acid-crosslinked hydrogels while preserving the integrity of adhesion sequences in ELRs.
Interestingly, the use of citric acid conferred so-produced hydrogels the ability to nucleate calcium phosphate. Mechanically-tailored citric acid-crosslinked hydrogels were shown to be able to support the growth of human mesenchymal stem cells and to lead to seemingly biocompatible degradation products. Despite in vitro differentiation studies weren¿t conclusive as to their osteogenic potential, both mechanically-tailored and non-tailored citric acid-crosslinked hydrogels were shown to integrate into bone and to be partially degraded upon implantation in critical size defects in mouse calvaria. Despite cell invasion in mechanically-tailored scaffolds was seemingly lower than in non-tailored counterparts, both types of matrices allowed the formation of bone tissue, by intramembranous ossification, to a similar extent by the end of the study. At the time points selected for the in vivo study, both tailored and non-tailored hydrogels were found to be osteoconductive; osteoinduction was not observed in any of the cases. Mechanically-tailored hydrogels not being seemingly superior to control matrices at selected time points could to be due to (i) a high surface polymer density hindering cell invasion and thus delaying osteoinduction, or to (ii) a non-osteoinductive combination of properties (chemical + physical) despite hydrogels possessing theoretically osteoinductive stiffness.
These results point out that scaffolds must be seen as a whole given the high complexity of the in vivo cell niche, whose signals act synergistically to define cell behavior. Thus, more complex designs are required if recapitulation of bone development is to be targeted.
Additionally, recombinant lysyl oxidase (LOX) from human aorta was successfully produced in Escherichia coli to high purity. Despite achieving LOX with copper cofactor amounts and activity higher than those found in the literature, overall activity was low and the insolubilization of ELRs was not achieved, suggesting that novel expression and purification systems not compromising enzymatic activity are required if LOX is to be used to produce scaffolds.<br>S'espera que la incidència global de fractures òssies, i per extensió la d'aquelles que no són capaces de consolidar per si soles, augmenti en les pròximes dècades, principalment degut a l'increment del risc de patologies associades a l'envelliment. Actualment, el camp dels biomaterials es mou cap al disseny de bastides que mimetitzen el microambient cel·lular per tal de guiar la diferenciació de cèl·lules mare i recapitular el desenvolupament de teixits diana. El biomimetisme és un concepte ampli i diverses aproximacions han sigut dutes a terme per tal de produir bastides capaces de guiar el comportament cel·lular. En aquesta tesi hem explorat l'ús d'àcid cítric i l'enzim lisil oxidasa, ambdós relacionats amb la nanoestructura i propietats mecàniques del teixit ossi, per desenvolupar bastides que mimetitzin la matriu extracel·lular de l'os en desenvolupament. En primer lloc, es va produir hidrogels basats en polímers recombinants de tipus elastina (Elastin-like recombinamers; ELRs) mitjançant una reacció d'entrecreuament en un pas amb àcid cítric, una molècula actualment considerada una peça essencial per l'adequat funcionament mecànic del teixit ossi. Havent fet un estudi sistemàtic de la reacció d'entrecreuament i de la seva contribució a les propietats dels hidrogels, vam ser capaços de controlar l'arquitectura i la rigidesa de les bastides entrecreuades amb àcid cítric, tot preservant la integritat de les seqüències d'adhesió cel·lular contingudes als ELRs. És interessant remarcar que l'ús d'àcid cítric com agent entrecreuant conferí als hidrogels capacitat de nucleació de fosfats de calci. Es va demostrar que els hidrogels entrecreuats amb àcid cítric i amb propietats mecàniques diana permeten el creixement de cèl·lules mare mesenquimals humanes i donen lloc a productes de degradació aparentment biocompatibles. Tot i que els estudis de diferenciació in vitro no van ser concloents pel que fa al potencial osteogènic d'aquestes bastides, tant les matrius amb propietats mecàniques diana com aquelles control van ser capaces d'integrar-se amb l'os natiu i van ser parcialment degradades un cop implantades en defectes de mida crítica en calotes de ratolí. Tot i que la invasió cel·lular en hidrogels amb propietats mecàniques diana va ser inferior en comparació amb l'observada en bastides control, ambdós tipus de matrius van permetre la formació d'os nou, mitjançant ossificació intramembranosa, en quantitats similars al final de l'estudi. Als punts temporals seleccionats, ambdós tipus de bastides van demostrar ser osteoconductives, però no es va observar evidències d'osteoconducció en cap cas. El fet que les bastides amb propietats mecàniques diana no fossin superiors a les matrius control pel que fa a formació òssia, podria ser degut a (i) a una elevada densitat superficial que hauria dificultat la invasió cel·lular i retrassat l'osteoinducció, o (ii) a una combinació de propietats (químiques + físiques) no osteoinductiva tot i que les bastides posseïen una rigidesa teòricament osteoinductiva. Aquests resultats demostren que les bastides han de ser vistes com un tot donada l'elevada complexitat del nínxol de cèl·lules mare in vivo, les senyals del qual actuen de forma sinèrgica per definir el comportament cel·lular. Així, és necessari assolir bastides amb un nivell de complexitat més elevat per tal de recapitular el desenvolupament ossi. Addicionalment, es va produir lisil oxidasa (LOX) d'aorta humana en forma recombinant a elevada puresa a partir de cultius d'Escherichia coli. Tot i que es va aconseguir produir LOX amb un contingut del cofactor coure i una activitat superiors a aquells trobats en la literatura, l'activitat de l'enzim va ser generalment baixa i no es va assolir la insolubilització d'ELRs. Això evidencia la necessitat de desenvolupar nous sistemes d'expressió i purificació de l'enzim per tal que aquest pugui ser aplicat a la producció de bastides.
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Zhao, Yunqin. "SPECTRAL CALIBRATION FOR SPECTRAL DOMAIN OPTICAL COHERENCE TOMOGRAPHY BASED ON B-SCAN DOPPLER SHIFT WITH IN SITU. TISSUE IMAGES." Miami University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=miami1562594660964602.
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Kinneberg, Kirsten R. C. "Tissue Engineering Strategies to Improve Tendon Healing and Insertion Site Integration." University of Cincinnati / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1307106075.
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SONG, MIN JAE. "Elucidating the Mechanical Milieu of Stem Cells In Situ and Delivering Mechanical Signals to Direct Cell Fate in Tissue Engineering Scaffolds." Case Western Reserve University School of Graduate Studies / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=case1341330220.
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Turner, Gabrielle M. "Development of in situ hybridisation to examine tissue-specific expression patterns of the invertase genes in sugarcane culm." Thesis, Stellenbosch : University of Stellenbosch, 2005. http://hdl.handle.net/10019.1/16621.
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Thesis (MSc)--University of Stellenbosch, 2005.<br>ENGLISH ABSTRACT: The goals of this project were firstly to develop the tissue preparation and in situ
hybridisation protocols for sugarcane culm tissue, and secondly to use the developed
techniques to examine the expression patterns of three invertase isoforms in sugarcane
internodes of various developmental stages. Sugarcane invertases have been the focus of
intense research for many years, yet almost nothing is known of their tissue-specific
distribution. It was thought that by characterising their expression patterns using in situ
hybridisation, more knowledge of their functions and involvement in sucrose
accumulation would be gained.
Although in situ hybridisation is now regularly used to study gene expression in plants,
there is to date only a single publication describing its use on immature sugarcane tissue.
Therefore this technique needed further development, and this was achieved by
comparing different tissue preparation methods, as well as by systematically testing the
various parameters pertaining to each method. The in situ hybridization technique was
also developed by testing and comparing a number of key parameters. It was found that
fixing whole mount tissue for 48 h preserved sugarcane tissue adequately. High
hybridization temperatures and probe concentrations provided the best signal, and
including pre-treatment with HCl and Pronase was essential in sensitizing the tissue to
the probe. A less viscous detection buffer reduced both osmotic effects and time required
for signal detection.
In the second part of this study, the developed method was used to examine the
expression patterns of the three invertase isoforms in young, maturing and mature
internodes of sugarcane, and the results were complemented with Northern blot analysis.
Transcript of all three isoforms was found to be present in the storage parenchyma and in
the phloem tissue. Transcript levels of all three isoforms declined in maturing tissue, with
soluble acid invertase declining sharply and dropping below detection in maturing and
mature tissue. Transcript levels of cell wall invertase and neutral invertase declined only
gradually, and appreciable levels of both were still present in mature tissue. Acid invertase is suggested to be mainly involved in internode elongation, while cell wall
invertase would appear to play important roles in phloem unloading and turgor control.
Neutral invertase is suggested to be involved in either sucrose cycling or maintenance of
hexose pools, however the function of this enzyme remains unclear.
This study has demonstrated the value of in situ hybridization, yet at the same time has
shown its limitations, especially when more traditional biochemical techniques are not
employed to complement the results. Although the precise functions of the invertase
isoforms in sugarcane remain inconclusive, this study has opened up the way for tissuespecific
promoter design and future in situ studies of sugarcane invertases<br>AFRIKAANSE OPSOMMING: Die doel van hierdie projek was tweeledig: eerstens om weefselvoorbereiding en in situhibridisasie-
protokolle vir die stingelweefsel van suikerriet te ontwikkel; en tweedens om
die ontwikkelde tegnieke te gebruik om die uitdrukkingspatrone van drie invertaseisovorme
in die suikerriet-internodes van verskeie ontwikkelingstadia te ondersoek.
Suikerriet-invertases is al vir jare lank die fokus van intense navorsing, maar baie min is
bekend oor hulle weefselspesifieke verspreiding. Die idee was om meer kennis oor
suikerriet-invertases se funksies en betrokkenheid by sukrose-akkumulasie te verkry deur
in situ-hibridisasie te gebruik om hulle uitdrukkingspatrone te karakteriseer.
Alhoewel in situ-hibridisasie deesdae gereeld gebruik word om geenuitdrukking in plante
te bestudeer, is daar tot op datum slegs een publikasie wat die gebruik daarvan in
onvolwasse suikerrietweefsel beskryf. Hierdie tegniek moes dus verder ontwikkel word,
en dit is gedoen deur verskillende weefselvoorbereidingsmetodes te vergelyk en
sistematies die verskillende parameters wat op elke metode van toepassing is te toets.
Die in situ-hibridisasie-tegniek is ook ontwikkel deur die toetsing en vergelyking van 'n
aantal sleutelparameters. Daar is gevind dat suikerrietweefsel voldoende gepreserveer
word deur die intakte gemonteerde weefsel vir 48 uur te fikseer. Hoë hibridisasietemperature
en hoë peilerkonsentrasies het die beste sein gegee; die insluiting van voorbehandeling
met HCl en Pronase was noodsaaklik om die weefsel meer gevoelig vir die
peiler te maak. Osmotiese invloede en die tyd nodig vir seindeteksie is verminder deur
die viskositeit van die buffer te verminder.
In die tweede deel van die studie is die ontwikkelde metode gebruik om die
uitdrukkingspatrone van die drie invertase-isovorme in jong, ontwikkelende en volwasse
internodes te ondersoek en die resultate is deur 'n noordelike oordraganalise
gekomplementeer. Transkripte van al drie isovorme is in die stoorparenchiem en
floëemweefsel gevind. Transkripvlakke van al drie isovorme het afgeneem in
ontwikkelende weefsel, met oplosbare suurinvertase wat skerp afgeneem en tot onder die deteksie-limiet gedaal het in ontwikkelende en volwasse weefsel. Transkripvlakke van
selwandinvertase en neutrale invertase het slegs geleidelik afgeneem en merkbare vlakke
van albei was teenwoording in ontwikkelende en volwasse weefsel. Daar word
voorgestel dat suurinvertase hoofsaaklik betrokke is by internodeverlenging, terwyl
selwandinvertase skynbaar 'n belangrike rol in floëem-ontlading en turgor-beheer speel.
Daar word voorgestel dat neutrale invertase betrokke is óf by die sukrose-sirkulering óf
by die onderhoud van heksose-poele; die funksie van hierdie ensiem is egter steeds nie
duidelik nie.
Hierdie studie het die waarde van in situ-hibridisasie gedemonstreer maar terselfdetyd
ook die beperkinge daarvan uitgewys, veral as meer tradisionele biochemiese tegnieke
nie gebruik word om die resultate aan te vul nie. Alhoewel daar onsekerheid is oor die
presiese funksies van die invertase-isovorme in suikerriet, het die studie die weg gebaan
vir weefselspesifieke promotorontwerp en toekomstige in situ-studies van
suikerrietinvertases.
18
Karadas, Ozge. "Collagen Scaffolds With In Situ Grown Calcium Phosphate For Osteogenic Differentiation Of Wharton." Master's thesis, METU, 2011. http://etd.lib.metu.edu.tr/upload/12612975/index.pdf.
Full textAbstract:
COLLAGEN IN SITU GROWN CALCIUM PHOSPHATE SCAFFOLDS
FOR OSTEOGENIC DIFFERENTIATION OF WHARTON&rsquo<br>S JELLY AND
MENSTRUAL BLOOD STEM CELLS
Karadas, Ö<br>zge
M.Sc., Department of Biotechnology
Supervisor : Prof. Dr. Vasif Hasirci
Co-Supervisor: Assoc. Prof. Dr. Gamze Torun Kö<br>se
February 2011, 91 pages
The importance of developing new techniques for the treatment of bone
and joint diseases is increasing continuosly together with the increase of human
population and the average life span. Especially bone fractures as a result of
osteoporosis are often seen in humans older than 50 years old. The expenses of
bone and joint disease operations are very high and the duration of recovery is
long. Because of these reasons World Health Organization, The United Nations
and 37 countries announced that the years 2000-2010 is the Bone and Joint
Decade. Tissue engineering is an alternative approach to clinically applied
methods. In this study collagen scaffolds crosslinked with genipin, to improve the
stability of foams in culture media, were prepared by lyophilization. To mimic the
natural bone structure calcium phosphate mineral phase in the foam was formed
by wet chemical precipitation. Collagen concentration (0.75% and 1%, w/v),
freezing temperature (-20 oC and -80 oC) of the collagen solution before
lyophilization and immersion duration (2x4 h and 2x48 h) of the foams in calcium
and phosphate solutions for wet chemical precipitation were changed as process
v
parameters of foam production. Pore size distribution and porosity analysis as
well as compression test were performed for characterization of the scaffolds. The
foam with 1% w/v collagen concentration, frozen at -20 oC before lyophilization
and immersed for 2x4 h in calcium and phosphate solution was chosen for in vitro
cell culture studies. The defined foam had 70% porosity and pore sizes varying
between 50 and 200 &mu<br>m. The elastic modulus and compressive strength of the
foam was calculated as 127.1 kPa and 234.5 kPa, respectively.
Stem cells isolated from Wharton&rsquo<br>s jelly (WJ) and menstrual blood (MB)
were seeded to foams to compare their osteogenic differentiation. Both cells are
isolated from discarded tissues and used in this study as an alternative to the
commonly used cells which are isolated by invasive techniques such as bone
marrow stem cells. Cells were seeded to collagen foams with and without calcium
phosphate (CaP). It was observed that WJ cells proliferated during 21 days on
collagen foams without CaP, but MB cell number decreased after day 14.
Collagen foams with CaP supported the alkaline phosphate (ALP) activity
compared to tissue culture polystyrene (TCPS) and foams without CaP. Contrarily
lower cell numbers achieved on CaP containing collagen foams, possibly because
of the calcium and phosphate concentration changes in the medium and as the
result of osteogenic differentiation. ALP activity of both cell types increased
almost 10 times and specific ALP activity (activity per cell) increased 40 times
and 150 times for WJ and MB cells, respectively on the CaP containing foams
compared to TCPS.
Therefore, in this study it was shown that in situ CaP formed collagen
foams induce osteogenic differentiation of WJ and MB cells, and these cells
isolated from discarded tissues can be used as alternative cell sources in bone
tissue engineering applications.
19
Gedamu, Abraham. "Magnetic resonance intensity standardization for multi-site tissue classification of brains with Multiple Sclerosis: a comparative analysis." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=21939.
Full textAbstract:
Tissue classification on multi-centre MR data for the purpose of studying brain pathology is becoming more prevalent. Classification algorithms rely on features in the image to be able to separate the different tissue types. An important feature in MR is the intensity. However, MR intensity is a relative value and there are intensity variations for any given tissue type with data from a single scanner. In addition, there are intensity variations between scanners. We have developed an intensity standardization technique based on non-brain tissue and compared our intensity standardization technique to a method developed by Nyul et al. The goal is to determine the effectiveness of the two standardization techniques when applied prior to a tissue classification technique designed to identify multiple sclerosis (MS) lesions in the cerebral white matter.<br>Dans l'étude des pathologies cérébrales, l'utilisation de la classification des tissus sur des images de résonance magnétique provenant de plusieurs sites différents est de plus en plus prévalente. Les algorithmes de classification se basent sur des caractéristiques de l'image afin de pouvoir discerner différents types de tissus. Une caractéristique importante en RM est l'intensité. Cependant, l'intensité du signal est une valeur relative, et les données provenant d'un seul scanner sont parfois variables, même pour le même tissu. De plus, il y a des variations d'intensité entre chaque appareil IRM. Nous avons développé une technique adapteé à des tissus non cérébraux pour standardiser l'intensité et nous avons comparé notre technique avec la méthode développée par Nyul et al. L'objectif est de déterminer l'efficacité des deux méthodes de standardisation lorsqu'elles sont appliquées préalablement à une technique automatisée de classification des tissus cérébraux pour la détection de lésions de sclérose en plaques dans la matière blanche cérébrale.
20
Meng, Linghui. "Polymer Biomaterial Constructs For Regenerative Medicine and Functional Biological Systems." Case Western Reserve University School of Graduate Studies / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=case1327682278.
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21
Ramoutar, Rakeshnie. "The development of an in situ hybridisation technique to determine the gene expression patterns of UDP-Glucose dehydrogenase, pyrophosphate-dependent phosphofructokinase and UDP-Glucose pyrophosphorylase in sugarcane internodal tissues." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/49795.
Full textAbstract:
Thesis (MSc)--University of Stellenbosch, 2003.<br>ENGLISH ABSTRACT: The cellular expression of the enzymes implicated in regulating sucrose metabolism and
accumulation in sugarcane is poorly understood. The present study was therefore aimed at the
development of an in situ hybridisation (ISH) technique to study differential gene expression
among the various cell types of the sugarcane culm. This technique in conjunction with
northern and western blotting was then used to determine the sites of cellular and tissue
specific expression of the cytosolic enzymes, UDP-Glc dehydrogenase, pyrophosphate
dependent phosphofructokinase and UDP-Glc pyrophosphorylase, involved in sucrose
metabolism.
This study revealed that the determination of the influencing parameters associated with the
development of an ISH protocol was essential for the successful detection of the endogenous
RNA sequences in sugarcane internodal tissues. The parameters that were investigated
included the type of embedding medium, duration of fixation period, pre-treatment procedures
and hybridisation temperature. It further revealed that fresh internodal tissue sections, fixed
for a period of 24 h and thereafter exposed to pre-treatment and hybridisation, facilitated the
analysis of cytological gene expression at all stages of sugarcane development.
The second part of this study revealed very localised transcript expression for UDP-Glc DH,
PFP and UGPase in the different internodal tissue and cell types. The UDP-Glc DH and
UGPase transcripts were localised to the phloem elements, whilst xylem tissue only expressed
the UDP-Glc DH transcript. Transcripts of UDP-Glc DH, PFP and UGPase were all
expressed in the parenchyma cells that were associated with the vascular bundles and the stem
storage compartment, suggesting that the parenchyma cells distributed throughout the stem in
the different tissue types complement each other in function for the purposes of phloem
loading, unloading and assimilate transport processes.
Complimentary northern and western hybridisations demonstrated that internode 7 represents
a shift in the sink from utilisation to storage. This is evident by the observed decline in both
the relative transcript and protein abundances of UDP-Glc DH, PFP and UGPase at this stage
of development. The relative mRNA and protein abundances for the three enzymes showed a
similar trend. Higher levels of the gene transcripts and translated products were observed in
the younger sucrose importing tissues, than in the older sucrose accumulating internodes. At
a cellular level, it was found that the sites of cellular UDP-Glc DH, PFP and UGPase
expression differed marginally. Whilst UDP-Glc DH was expressed in the phloem, xylem and parenchyma cells of the vascular complex and in storage parenchyma cells, PFP was
expressed exclusively in parenchyma cells that were associated with the vascular bundles and
those serving a storage function in the stem pith and UGPase was found to be localised in the
phloem and parenchyma of the vascular bundles and the storage parenchyma cells. Such
findings have demonstrated an increase in resolution with which gene expression can be
examined at a cellular level. Hence, the results from this study have demonstrated that the
knowledge of metabolic compartmentation between different tissue and cell types is a
requisite to understanding the function(s) of individual enzymes within complex structures
such as the sugarcane culm.<br>AFRIKAANSE OPSOMMING: Die sellulêre lokalisering van die ensieme wat geïmpliseer word in die regulering van sukrose
metabolisme is onbekend. Met dit in gedagte, was hierdie studie gefokus op die ontwikkeling
van 'n in situ hibridisasie (ISH) tegniek om differensiële geenuitdrukking in die verskillende
seltipes van die suikerrietstingel te ondersoek. Hierdie tegniek, tesame met RNA-en proteïen
gel blots, is volgens aangewend om die areas van sellulêre-en weefselspesifieke uitdrukking
van die sitosoliese ensieme UDP-glukose dehydrogenase, pirofosfaat-afhanklike
fosfofruktokinase en UDP-glukose pirofosforilase, wat almal betrokke is by
sukrosemetabolisme, te bepaal.
Dit het duidelik geword gedurende die studie dat die bepaling van die optimale parameters
van die ISH protokol vir suikerriet van deurslaggewende belang sou wees vir die opsporing
van endogene RNA volgordes. Die parameters wat ondersoek is het ingesluit die tipe
inbeddingsmedium, die tydsduur van fiksering, vooratbehandelings- en hibridisasiemetodes.
Dit het duidelik geword dat vars internodale weefselsnitte wat vir 24 h gefikseer is en daarna
voorafbehandeling en hibridisasie ondergaan het, die bepaling van geenuitdrukking tydens
alle fases van suikkerrietontwikkeling moontlik gemaak het.
Die tweede fase van hierdie studie het aangetoon dat al drie ensieme spesifiek gelokaliseerde
uitdrukkingspatrone gehad het in verskillende internodale weefsels en seltipes. Al drie gene is
konstitutief uitgedruk in internodes. Die UDP-glukose dehydrogenase en UDP-glukose
pirofosforilase transkripte is gelokaliseer na die floeëm elemente, terwyl xileem slegs die
UDP-glukose dehydrogenase transkripte bevat het. Al die gene is in die parenchiemselle
uitgedruk wat geassosieer is met die vaatbondels en die stingel stoorkompartement, wat
moontlik beteken dat die parenchiem selle wat deur die stingel versprei is 'n sentrale netwerk
vorm wat direk of indirek koolstofassimileringsprosesse beïnvloed.
RNA-en proteïen gel blots op dieselfde internodes het gewys dat internode sewe 'n
verskuiwing, van koolstofverbruik na berging, verteenwoordig. Dit word gerllustreer deur die
afname in beide transkrip en proteïen vlakke van die drie ensiem in hierdie stadium van
ontwikkeling. Alhoewel beide mRNA en proteïen vlakke vir al die ensieme 'n soortgelyke
tendens getoon het, het die sellulêre uitdrukking van die ensieme volgens ISH verskil, wat die
krag van die tegniek illustreer. Die resultate van hierdie studie het gedemonstreer dat begrip
van die kompartementalisasie van metabolisme tussen verskillende weefsel-en seltipes 'n voorvereiste is om die funksie/s van individuele ensieme in komplekse strukture soos die
suikerrietstingel te bepaal.
22
Fisher, Paul. "Additives to Control Mechanical Properties and Drug Delivery of Injectable Polymeric Scaffolds." UKnowledge, 2014. http://uknowledge.uky.edu/cbme_etds/25.
Full textAbstract:
In situ forming implants (ISIs) are popular due to their ease of use and local drug delivery potential, but they suffer from high initial drug burst, and release behavior is tied closely to solvent exchange and polymer properties. Additionally, such systems are traditionally viewed purely as drug delivery devices rather than potential scaffold materials due to their poor mechanical properties and minimal porosity. The aim of this research was to develop an injectable ISI with drug release, mechanical, and microstructural properties controlled by micro- and nanoparticle additives.
First, an injectable ISI was developed with appropriate drug release kinetics for orthopedic applications. Poly(β-amino ester) (PBAE) microparticles were loaded with simvastatin or clodronate, and their loading efficiency and drug retention after washing was quantified. Drug-loaded PBAE microparticles and hydroxyapatite (HA) microparticles were added to a poly(lactic-co-glycolic acid) (PLGA)–based ISI. By loading simvastatin into PBAE microparticles, release was extended from 10 days to 30 days, and burst was reduced from 81% to 39%. Clodronate burst was reduced after addition of HA, but was unaffected by PBAE loading. Scaffold mass and porosity fluctuated as the scaffolds swelled and then degraded over 40 days.
Next, the mechanical properties of these composite ISIs were quantified. Both micro- and nanoparticulate HA as well as PBAE microparticle content were varied. Increasing HA content generally improved compressive strength and modulus, with a plateau occurring at 30% nano-HA. Injectability remained clinically acceptable for up to 10% w/w PBAE microparticles. Ex vivo injections into trabecular bone improved both strength and modulus.
Lastly, HA-free ISIs were investigated for drug delivery into the gingiva to treat periodontitis. Doxycycline and simvastatin were co-delivered, with delivery of doxycycline over 1 week accompanied by simvastatin release over 30 days. PBAE-containing ISIs exhibited higher initial and progressive porosity and accessible volume than PBAE-free ISIs over the course of degradation. Additionally, PBAE-containing ISIs provided superior tissue retention within a simulated periodontal pocket. The ISIs investigated here have a wide range of potential applications due to their flexible material and drug release properties, which can be controlled by both the chemistry and concentration of various particulate additives.
23
Breidenbach, Andrew P. "Translating Mechanisms of Tendon Development to Improve Adult Tendon Repair." University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1406809080.
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24
Kretschmer, Simon [Verfasser], and Hans [Akademischer Betreuer] Zappe. "Multi-modal endoscopic probes for in situ tissue inspection." Freiburg : Universität, 2019. http://d-nb.info/119221529X/34.
Full text
25
Dawson, Jennifer Elizabeth. "Cardiac Tissue Engineering." Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/20071.
Full textAbstract:
The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.
26
Somasundaram, Murali. "Intestinal tissue engineering." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:54e0f17f-fe04-4012-b0d3-04f436e9af9a.
Full textAbstract:
Tissue engineering (TE) principles have been successfully clinically applied to treat disease affecting specific organs (e.g. trachea) but developments in some organs has lagged behind. The inability to repair or replace significantly damaged intestinal tissue remains a barrier to improving patient outcomes and the promise of Tissue Engineered Intestine (TEI) that was first made more than 20 years ago, is yet to be realised. This work explored the potential of TEI and literature review formed a basis for developing a clinically transferrable experimental model. It was hypothesised that, porcine large intestine could be retrieved from pigs and decellularized to create a biological scaffold that demonstrated favourable properties for TE, including potential for vascular perfusion and cell engraftment. Novel experiments were performed in intestinal retrieval and decellularization, resulting in scaffolds characterised by a number of methods (e.g. histology, immunohistochemistry). Assessment of the scaffold's ability to support cell engraftment required development of protocols for isolation and culture of appropriate progenitors, including adipose/bone marrow derived mesenchymal stromal cells and intestinal organoid units. Finally, in-vitro cultures combining scaffolds and cells were used to assess the ability of scaffolds to promote tissue regeneration. Perfusion decellularization methods proved effective in creating biological scaffolds that retained radiologically demonstrated vascular perfusion networks, permitting a future route for recellularization and/or transplantation. Scaffolds demonstrated retention of essential extracellular matrix components (e.g. glycosaminoglycans, collagen) and an absence of cell nuclei. Mesenchymal stem cells were isolated, cultured and combined in-vitro with scaffolds in an attempted scaled-down seeding model. Control of culture conditions was challenging and results inconclusive with respect to the scaffold's regenerative potential. The work demonstrates an exciting prospect for biological scaffold development for a clinically transferrable, semi-xenogeneic transplant or drug delivery model but further experiments in scaffold seeding are required to assess the full potential.
27
HajMohammadi, Sassan. "Development of FISH technology in pathological tissue." Thesis, University of Southampton, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.284578.
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Alves, Karla Shangela da Silva. "Effect of tissue-tolerable plasma on oral biofilms formed in situ." Universidade Federal do CearÃ, 2016. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=17913.
Full textAbstract:
CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior<br>The Low temperature plasma is a promising technology that has being studied in dentistry for its various properties, among them, for their ability to destroy bacteria present in mature biofilm and also destroying the polymeric matrix of oral biofilms. The aim of this study was to evaluate the antimicrobial effect of argon plasma on oral biofilms formed in situ and also verify if this treatment damages the structure of the treated enamel. For that, twenty-two volunteers used palatine intraoral devices containing 6 to bovine enamel slabs that were dripped sucrose 10 times a day with 10% sucrose. The intraoral period was 7 days. At the 7th day, enamel slabs were treated for 5 minutes with plasma, argon flow, 0.12% chlorhexidine, or 0.89% NaCl solution. Thus, biofilms samples were collected, weighed, serial diluted and plated in culture medium for the growth of total microorganisms, total streptococci, mutans streptococci and lactobacilli. In order to analyze whether the treatments damaged the enamel structure, the slabs were analyzed by scanning electron microscopy (SEM) and Raman spectroscopy. Low temperature plasma treatment showed a significant reduction in the viability of the total microorganisms (p <0.001), total streptococcus (p = 0.037) and mutans streptococci (p = 0.004). Argon flow also significantly reduced the mutans streptococci counts. The plasma treatment did not influence on the viability of the lactobacilli under the conditions tested (p = 0.497). SEM revealed smooth and homogeneous regions on the enamel surface, unobserved topographical differences between the glazes in biofilms subjected to different treatments. In Raman spectroscopy, we identified four major bands present in the inorganic phase: 324 (V1), 582 (V2), 960 (V3) e 1045 (V4) cm-1 and other 4 identified in the organic layer 1448 (V5), 1465 (V6), 1653 (V7) 2943(V8) cm-1. No statistical differences in the Raman spectra were observed for enamel of different treatments. In conclusion, the plasma was effective in reducing viable bacteria present in mature oral biofilms produced in situ and not alter in the enamel surface treated biofilms.<br>O plasma de baixa temperatura à uma tecnologia promissora que vem sendo estudada na odontologia por suas vÃrias propriedades, dentre elas, por sua capacidade de destruir bactÃrias presentes em biofilme maduro destruindo a matriz do biofilme oral. O objetivo deste estudo foi avaliar o potencial antimicrobiano do plasma de argÃnio de baixa temperatura em biofilmes orais formados in situ e verificar se o tratamento proposto danifica a estrutura do esmalte tratado. Para tanto, vinte e dois voluntÃrios utilizaram dispositivos intraorais palatinos contendo 6 blocos de esmalte bovino que foram gotejados sacarose 10 vezes ao dia uma soluÃÃo de sacarose a 10%, durante 7 dias. Ao final do perÃodo intraoral, os biofilmes formados sobre os blocos de esmalte receberam tratamento durante 5 minutos com plasma de argÃnio, gÃs argÃnio, clorexidina a 0,12%, ou soluÃÃo salina de NaCl a 0,89%. As amostras dos biofilmes foram coletadas, pesadas, diluÃdas de forma seriada e plaqueadas em meio de cultivo para o crescimento de microrganismos totais, estreptococos totais, estreptococos do grupo mutans e lactobacilos. A fim de analisar se os tratamentos danificaram a estrutura do esmalte, os blocos foram analisados por microscopia eletrÃnica de varredura (MEV) e por espectroscopia Raman. O tratamento do plasma mostrou uma reduÃÃo significativa na viabilidade dos microrganismos totais (p <0,001), estreptococos totais (p = 0,037) e estreptococos do grupo mutans (p = 0,004). O gÃs de argÃnio tambÃm reduziu significativamente as contagens de estreptococos do grupo mutans. O tratamento com plasma nÃo demonstrou influÃncia sobre a viabilidade dos lactobacilos nas condiÃÃes testadas (p = 0,497). A MEV revelou regiÃes lisas e homogÃneas na superfÃcie do esmalte, nÃo sendo observadas diferenÃas topogrÃficas entre os esmaltes sob biofilmes submetidos aos diferentes tratamentos. No Raman, foram identificadas quatro bandas principais presentes na fase inorgÃnica: 324 (V1), 582 (V2), 960 (V3) e 1045 (V4)cm-1 e outras 4 identificadas na fase orgÃnica 1448 (V5), 1465 (V6), 1653 (V7) 2943(V8) cm-1, nÃo ocorrendo diferenÃas estatÃsticas entre os espectros do Raman nos blocos de esmaltes dos diferentes tratamentos. Em conclusÃo, o plasma foi eficaz na reduÃÃo de bactÃrias viÃveis presentes em biofilmes orais maduros produzidas in situ e nÃo alterou a superfÃcie do esmalte sob biofilme tratado.
29
Rouwkema, Jeroen. "Prevascularized bone tissue engineering." Enschede : University of Twente [Host], 2007. http://doc.utwente.nl/57929.
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30
Mirsadraee, Saeed. "Tissue engineering of pericardium." Thesis, University of Leeds, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.426783.
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Tseng, Yuan-Tsan. "Heart valve tissue engineering." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:e67c780d-d60f-42e7-9311-dd523f9141b3.
Full textAbstract:
Since current prosthetic heart valve replacements are costly, cause medical complications, and lack the ability to regenerate, tissue-engineered heart valves are an attractive alternative. These could provide an unlimited supply of immunological-tolerated biological substitutes, which respond to patients' physiological condition and grow with them. Since collagen is a major extra cellular matrix component of the heart valve, it is ideal material for constructing scaffolds. Collagen sources have been shown to influence the manufacturing of collagen scaffolds, and two commercial sources of collagen were obtained from Sigma Aldrich and Devro PLC for comparison. Consistencies between the collagens were shown in the primary and secondary structures of the collagen, while inconsistencies were shown at the tertiary level, when a higher level of natural crosslinking in the Sigma collagen and longer polymer chains in the Devro collagen were observed. These variations were reduced and the consistency increased by introducing crosslinking via dehydrothermal treatment (DHT). Collagen scaffolds produced via freeze-drying (FD) and critical point-drying with cross-linking via DHT or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide /N-hydroxysuccinimide (EDC/NHS) were investigated. All the scaffolds were compatible with mesenchymal stem cells (MSCs) according to the proliferation of the cells and their ability to produce ECM, without differentiating between osteogenic, chondrogenic or endothelial lineages. The FD EDC/NHS scaffold demonstrated the most suitable physical property of all. This result illustrates that FD EDC/NHS crosslinking is the most suitable scaffold investigated as a start for heart valve tissue engineering. To prepare a scaffold with a controlled local, spatial and temporal delivery of growth factor, a composite scaffold comprising poly (lactic-co-glycolic acid) (PLGA) microspheres was developed. This composite scaffold demonstrated the same compatibility to the MSCs as untreated scaffold. However, the PLGA microspheres showed an increase in the deterioration rate of Young's modulus because of the detachment of the microspheres from the scaffold via cellular degradation.
32
Getgood, Alan Martin John. "Articular cartilage tissue engineering." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608764.
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Al-Zahrani, Al-Husain Jaber. "Detection of Hepatitis C virus in liver tissue by in situ PCR." Thesis, University of Manchester, 2000. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.488358.
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Shazly, Tarek (Tarek Michael). "Tissue-material interactions : bioadhesion and tissue response." Thesis, Massachusetts Institute of Technology, 2009. http://hdl.handle.net/1721.1/54577.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2009.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (p. 159-162).<br>Diverse interactions between soft tissues and implanted biomaterials directly influence the success or failure of therapeutic interventions. The nature and extent of these interactions strongly depend on both the tissue and material in question and can presumably be characterized for any given clinical application. Nevertheless, optimizing biomaterial performance remains a challenge in many implant scenarios due to complex relationships between intrinsic material properties and tissue response. Soft tissue sealants are clinically-relevant biomaterials which impart therapeutic benefit through adhesion to tissue, thus exhibiting a direct functional dependence on tissue-material reactivity. Because adhesion can be rigorously quantified and correlated to the local tissue response, sealants provide an informative platform for studying material properties, soft tissues, and their interplay. We developed a model hydrogel sealant composed of aminated polyethylene glycol and dextran aldehyde (PEG:dextran) that can possess a wide range of bulk and adhesive properties by virtue of constituent polymer modifications. Through comparison to traditional sealants, we established that highly viscoelastic adhesion promotes tissue-sealant interfacial failure resistance without compromising underlying tissue morphology.<br>(cont.) We analyzed multiple soft tissues to substantiate the notion that natural biochemical variability facilitates the design of tissue-specific sealants which have distinct advantages over more general alternatives. We confirmed that hydrogel-based materials are an attractive material class for ensuring sealant biocompatibility, but found that a marked reduction in adhesive strength following characteristic swell can potentially limit clinical efficacy. To mitigate the swell-induced loss of hydrogel-based sealant functionality, a biomimetic conjugation strategy derived from marine mussel adhesion was applied to PEG:dextran and shown to favorably modulate adhesion. In all phases of this research, we defined material design principles that extend beyond the immediate development of PEG:dextran with potential to enhance the clinical performance of a range of biomaterials.<br>by Tarek Shazly.<br>Ph.D.
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Le, Thua Trung Hau. "Multimodality Treatment of Soft Tissue and Bone Defect: from Tissue Transfer to Tissue Engineering." Doctoral thesis, Universite Libre de Bruxelles, 2015. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/220961.
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In the first part of these studies, we have performed standard microsurgical procedures provide a solution for long standing bone and soft tissue defects, even in cases of longstanding osteomyelitis of long bones. When long bony segments are missing, the microvascular bone transfer provides a reliable method. In smaller soft tissue and bone defects, the application of a descending genicular osteomyocutaneous flap provides an option with low donor site morbidity. In the second part, we have focussed on reducing the donor site morbidity and expanded on the application of tissue engineering methods. MSCs derived from bone marrow can be injected percutaneous or be combined with an autologous bony scaffold for treatment of delayed union and nonunion. The outcome of our studies, however, limited in number of patients, clearly showed the possibilities and advantages of this new approach. A multimodality approach is essential, but it can provide promising solutions. Well-established microvascular and modern biotechnology methods will improve patient satisfaction and functional recovery in severe limb trauma, often the result of high-energy motorcycle accidents.<br>Doctorat en Sciences médicales (Médecine)<br>info:eu-repo/semantics/nonPublished
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Ghezzi, Chiara Elia. "Dense collagen-based tubular tissue constructs for airway tissue engineering." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114489.
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To date, only engineered tissues of planar geometry, such as epidermal and dermal layer substitutes, have successfully reached the market, mainly due to their relative low complexity and simple geometry. In contrast, the mechanical and functional requirements of tubular tissues are more stringent compared to planar tissues. Tubular tissues, which are the main components of several biological systems (e.g. circulatory, urinary or respiratory), not only present an increased complexity in geometry and tissue architecture, they are also populated by mixed cell types. In addition, these are continuously exposed to cyclic mechanical stimuli, which modulate cellular responses and ultimately the functionality of the tissues. Therefore, the understanding and the ability to reproduce physiologically equivalent environments are critical to generate mechanically and biologically functional neo-tissues or tissue models. The aim of this doctoral research was to produce and characterize 3D DC-based tubular constructs as tissue models for airway tissue engineering in physiologically relevant culture conditions. The first objective was to develop DC-based constructs and evaluate, in real-time, the responses of seeded fibroblasts to PC and to culturing with the DC environment; the fabrication and characterization of mesenchymal stem cell (MSC) seeded multilayered DC-SF-DC hybrids; and to evaluate the differentiation of MSCs cultured within multilayered DC-SF-DC hybrids.The second objective was to develop and characterize cell-seeded tubular dense collagen constructs (TDCCs) with bioinspired mechanical properties.The third objective was to implement tubular dense collagen-based constructs as an airway tissue model through the evaluation of airway smooth muscle cell (ASMC) responses within TDCC under physiological mechanical stimuli, and the development of a multilayered tubular dense collagen-silk fibroin construct (TDC-SFC) that mimicked airway tract architecture in order to study MSC responses under physiological mechanical stimulation.By providing ASMCs with a physiologically equivalent niche, and through pulsatile flow stimulation, in vitro, ASMCs exhibited their native orientation, maintained their contractile phenotype and enhanced the mechanical properties of the TDCC through matrix remodelling. The ability of TDC-SFC to transfer physiological pulsatile stimulation to resident MSCs resulted in native-like cell orientation (i.e. parallel to circumferential strain), and induced MSC contractile phenotype expression.In conclusion, the tubular dense collagen-based constructs developed and implemented, in this doctoral dissertation, effectively provided an in vitro airway tissue model for potential preclinical studies to mimic physiological and pathological conditions (e.g. inflammatory and degenerative diseases) in a relevant biomechanical environment, as alternatives to simple tissue culture techniques or complex animal models.<br>À ce jour, seuls les tissus synthétisés de forme plane, comme les substituts dermiques et épidermiques, ont réussi à percer le marché, surtout en raison de leur complexité relativement faible et de leur géométrie simple. À l'opposé, les exigences mécaniques et fonctionnelles des tissus tubulaires imposent un plus grand nombre de contraintes que les tissus planaires. Principales composantes de plusieurs systèmes biologiques (circulatoire, urinaire ou respiratoire), les tissus tubulaires sont non seulement plus complexes sur le plan de la géométrie et de l'architecture tissulaire, mais ils sont aussi composés de cellules de différents types. De plus, ils sont continuellement exposés à des stimuli mécaniques cycliques. Voilà pourquoi il est essentiel de comprendre les milieux physiologiquement équivalents et de pouvoir les reproduire si on veut obtenir des néotissus ou des modèles tissulaires fonctionnels sur le plan mécanique et biologique.La présente recherche de doctorat visait donc à produire et à caractériser des constructions tubulaires 3D à base de CD, les tissus des voies respiratoires dans des conditions de culture physiologiquement pertinentes. Le premier objectif était de concevoir des constructions à base de CD et d'évaluer la réaction des fibroblastes ensemencés à la CP et à la culture dans un milieu à base de CD; de fabriquer et de caractériser des hybrides multicouches CD-fibroïne-CD ensemencés de cellules souches mésenchymateuses (CSM); et d'évaluer la différenciation.Le deuxième objectif de la présente recherche était de concevoir et de caractériser des constructions tubulaires faites de collagène dense (CTCD). Le troisième objectif était d'implanter des constructions tubulaires à base de CD comme modèle tissulaire des voies respiratoires par l'évaluation de la réponse des cellules musculaires lisses (CML) des voies respiratoires dans les CTCD en présence de stimuli mécaniques physiologiques.En leur fournissant une niche physiologiquement équivalente, et grâce à la stimulation de l'écoulement pulsatoire, in vitro, les CML des voies respiratoires ont pris leur orientation naturelle, maintenu leur phénotype contractile et amélioré les propriétés mécaniques de la CTCD grâce au remodelage matriciel. La capacité de la CTCD à transférer la stimulation physiologique pulsatile aux CSM résidentes a donné une orientation des cellules s'apparentant à leur orientation naturelle et induit l'expression phénotypique.En conclusion, les constructions tubulaires à base de collagène dense qui ont été développées et implantées sont parvenues à fournir in vitro un modèle tissulaire des voies respiratoires pour d'éventuelles études précliniques visant à reproduire les conditions physiologiques et pathologiques.
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Chik, Tsz-kit, and 戚子傑. "Fabrication of multi-component tissue for intervertebral disc tissue engineering." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B47849447.
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Intervertebral disc tissue engineering is challenging because it involves the
integration of multiple tissues with distinct structures and compositions such as
lamellar annulus fibrosus, gel?like nucleus pulposus and cartilage endplate. Each
of them has different compositions and different structures. It is hypothesized
that integration of tissues can be enhanced with appropriate mechanical and
biological stimuli. Meanwhile, effect of torsional stimulus on cell re?orientation
in mesenchymal stem cell?collagen tubular constructs is investigated in this study.
Furthermore, it is proposed that these findings can be used to fabricate a multicomponent
unit for intervertebral disc tissue engineering. It has been
demonstrated that mechanical and biological stimuli can stabilize the interface
between osteogenic and chondrogenic differentiated constructs with enhanced
ultimate tensile stress while the phenotype of osteogenic and chondrogenic
differentiated constructs were maintained. Scanning electronic microscopic
images have shown aligned collagen fibrils and presence of calcium at the
interface, indicating the possibility of the formation of a calcified zone. In
addition, it is proven that torsional stimulus triggered re?orientation of
mesenchymal stem cells in collagen lamellae towards a preferred angle. Cell
alignments were confirmed by using a MatLab?based program to analyze the
actin filament and the cell alignment via Phalloidin and Hematoxylin staining,
respectively. Cells and actin filaments were inclined around 30o from the vertical
axis, while cells and filaments in the control group (static loading) aligned along
the vertical axis. Furthermore, a double?layers bioengineered unit was fabricated,
with intact osteogenic differentiated parts at both ends. Comparatively higher
cell density was observed at the interface between layers, demonstrating the
interactions between layers, while the phenotype of each part was maintained in
14 days culture. This study concludes that a multi?components bioengineered
unit with preferred cell alignments can be fabricated. This provides new insights
to future development of bioengineered spinal motion segment for treating late
stage disc degeneration.<br>published_or_final_version<br>Mechanical Engineering<br>Doctoral<br>Doctor of Philosophy
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Kamei, Yuzuru, Kazuhiro Toriyama, Toru Takada, and Shunjiro Yagi. "Tissue-Engineering Bone from Omentum." Nagoya University School of Medicine, 2010. http://hdl.handle.net/2237/14172.
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Hussain, Timon. "Tissue Engineering mit porösen Polyethylenimplantaten." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-162027.
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Hintergrund: Eine Verbesserung der Biokompatibilität von porösen Polyethylenimplantaten könnte postoperative Komplikationsraten senken und das klinisches Anwendungsgebiet des Biomaterials erweitern. Im Rahmen dieser Studie wurde untersucht, ob eine „Vitalisierung“ von porösen Polyethylenimplantaten mit dermalen Fibroblasten möglich ist und ob hierdurch die mikrovaskuläre Integration sowie die immunologische Reaktion des Wirtsorganismus beeinflusst werden konnte.
Material und Methoden: Poröse Polyethylenimplantate wurden in vitro mit GFP-markierten dermalen Fibroblasten kultiviert. Die auf dem Biomaterial adhärenten Zellen wurden vor Implantation in dorsale Rückenhautkammern an C57/Bl6 Mäusen mittels konfokaler Mikroskopie quantifiziert, ebenso nach Explantation. Native Implantate dienten als Kontrolle. Angiogeneseprozesse sowie die Leukozyten-Endothelzellinteraktion im Implantatmaterial wurden wiederholt mittels in vivo Fluoreszenzmikroskopie analysiert. Abschließend wurde die dynamische Desintegrationskraft quantifiziert und eine Analyse immunmodulatorischer Zytokine durchgeführt.
Ergebnisse: Poröse Polyethylenimplantate konnten nachhaltig mit dermalen Fibroblasten „vitalisiert“ werden. Mikrozirkulatorische Parameter nahmen während des Beobachtungszeitraums zu, allerdings konnten keine signifikanten Unterschiede zwischen den Gruppen festgestellt werden. Einzelne immunmodulatorische Zytokine waren in „vitalisierten“ porösen Polyethylenimplantaten tendenziell erhöht, eine signifikante Beeinflussung der Immunantwort des Wirtsorganismus war jedoch nicht festzustellen.
Schlussfolgerung: Eine „Vitalisierung“ von porösen Polyethylenimplantaten mit dermalen Fibroblasten ist nachhaltig durchführbar, beeinflusst jedoch die mikrovaskuläre Integration in vivo sowie die Immunreaktion des Wirtsorganismus nicht signifikant. Somit sind möglicherweise zusätzliche Maßnahmen im Sinne eines Tissue Engineerings erforderlich, um die Biokompatibilität von porösen Polyethylenimplantaten mittels dieser vielversprechenden Zellquelle zu verbessern.
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Sodian, Ralf. "Tissue-Engineering von kardiovaskulären Geweben." [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=974660175.
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Cooper, Leanne Jane. "Tissue engineering of the cornea." Thesis, Lancaster University, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.421611.
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Schrader, S. "Tissue engineering for conjunctival reconstruction." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1348133/.
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Reconstruction of the conjunctiva is an essential part of ocular surface regeneration, especially if an extensive area or the whole ocular surface is affected, such as in patients with ocular cicatricial pemphigoid, Stevens- Johnson syndrome or chemical/thermal burns. However, there is a lack of suitable donor tissue for conjunctival replacement, especially when large grafts are required and it is important that new materials and methods are developed for conjunctival reconstruction. The aims of this thesis were; to characterise the conjunctival epithelial cell population and to improve the maintenance of the epithelial progenitor cells during in vitro expansion in order to produce conjunctival epithelial cells suitable for therapeutic use. The final aim was to transfer these cells to compressed collagen matrices and amniotic membrane and test the properties of these cell-matrix constructs. Experiments showed that cryopreservation does not to alter the proliferative potential of conjunctival epithelial progenitor cells. It was also demonstrated that the maintenance of conjunctival epithelial progenitor cells during cell expansion can be improved by mimicking an environment in vitro, which is more similar to the stem cell niche in vivo and that this is accompanied by downregulation of key genes in the wnt signaling pathway. The final experimental series showed that after in vitro expansion, conjunctival epithelial cells can be successfully transferred and cultured on amniotic membrane and compressed collagen gels. In conclusion these studies highlighted the complexity of tissue engineering ocular surface substitutes and provided further clues for the goal to obtain a stable conjunctival substitute, suitable for transplantation.
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Sodian, Ralf. "Tissue Engineering von kardiovaskulären Geweben." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2005. http://dx.doi.org/10.18452/13965.
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Beim Tissue Engineering werden Erkenntnisse aus der Medizin, Biologie und Chemie mit Methoden der Ingenieurwissenschaften kombiniert, um biologische Ersatzgewebe herzustellen. Das Konzept besteht darin, aus körpereigenen Zellen einen vitalen und funktionalen Gewebeersatz zu fertigen. Hierbei werden körpereigene Zellen auf ein resorbierbares Gerüst transplantiert, in vitro zu einer stabilen Struktur gefestigt, um letztendlich ein vitales Ersatzgewebe implantieren zu können. Die Konstrukte für die menschliche Herzchirurgie sollten in das umgebende Gewebe einwachsen und haben das Potential sich wie gesundes Gewebe zu entwickeln und mitzuwachsen.<br>Tissue engineering combines knowledge from the fields of medicine, biology and chemistry with the methods of engineering to create artificial tissue. The concept is to produce vital and functional tissue from endogenous cells. These are seeded on to an absorbable scaffold and consolidated to form a stable structure in vitro, with the aim of eventually being able to produce substitute tissue for implantation. The constructs for human cardiac surgery need to embed into the surrounding tissue and, just like natural tissue, to have the potential to grow and develop.
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Wung, Nelly. "Tissue engineering of the liver." Thesis, University of Bath, 2017. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.715264.
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Currently, the only cure for liver failure is orthotopic liver transplantation. However, there are insufficient donor organs available to treat every patient on the transplant list and many die before they are able to receive a liver transplant. The bioartificial liver (BAL) device is a potential extracorporeal treatment strategy utilising hepatocytes or hepatocyte-like cells (HLCs) within a bioreactor to recapitulate normal liver function and therefore ‘bridge’ a patient with liver failure until they receive a transplant. The work in this thesis utilised tissue engineering methods to develop novel approaches to BAL device design through development and characterisation of a polymer membrane scaffold (“PX”) for hollow fibre bioreactor (HFB) culture and a HLC source generated from the transdifferentiation of pancreatic AR42J-B13 (B13) cells. A flat sheet membrane model was used for the development of asymmetrical, hydrophobic polystyrene (PS) phase inversion membranes. Oxygen plasma significantly increased PS membrane surface wettability through addition of oxygen functional groups to create an environment conducive for cell culture. The treated membrane was henceforth referred to as “PX”. The culture medium HepatoZYME+ was investigated for its ability to induce transdifferentiation of B13 cells to HLCs and maintain the hepatic phenotype. Overall, HepatoZYME+-cultured cells experienced viability loss. A diluted version, “50:50”, showed induction of the hepatic markers carbamoylphosphate synthetase-1 (CPS-1) and HNF4α, as well as a change towards a HLC morphology. When using 50:50 as a maintenance medium, transdifferentiated HLCs retained loss of pancreatic amylase and also induction of hepatic markers, with comparable serum albumin secretion to the established Dex + OSM treatment. However, culture viability in 50:50 was still compromised. Therefore, HepatoZYME+ based media were deemed unsuitable for induction and maintenance compared to Dex-based protocols. PX flat sheet membranes were able to support culture of B13 cells and also the human osteosarcoma cell line, MG63, demonstrating improved cell attachment over non-surface treated PS membranes. PX membranes supported transdifferentiation of B13 cells to HLCs, presenting with loss of pancreatic amylase, induction of the hepatic markers transferrin, GS and CPS-1 and serum albumin secretion. Furthermore, PX showed no change in mass or loss of culture surface area over 15 days in culture conditions. Together, the novel membrane material and the media formulation and feeding regime developed have strong potential to be translated to a HFB setting and guide future BAL device design.
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Mehrban, Nazia. "Tissue engineering a ligamentous construct." Thesis, University of Birmingham, 2011. http://etheses.bham.ac.uk//id/eprint/2989/.
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Tendon and ligament damage causes extreme pain and decreased joint functionality. Current repair methods cannot restore original joint biomechanics nor promote regeneration of native tissue. Recent advances in tendon and ligament repair have involved engineering tissue using cell-seeded scaffolds. Self-aligned cellular structures, similar to those in ligaments and tendons, have been successfully formed, albeit with weak attachment between construct and bone. Calcium phosphates form an intimate bond with both soft and hard tissues and have successfully been used in tissue engineering bone, whilst hydrogels have often been used as cellular scaffolds. This thesis explores agarose, gelatin, carrageenan and fibrin hydrogels as potential soft tissue scaffolds. Fibrin gel exhibited high cellular compatibility with highest metabolic activity on day 14. Although the cellular gel contracted significantly, it was found that the dry weight remained stable in both the acellular and cellular forms. 3D powder printed calcium phosphate scaffolds remained structurally stable after immersion in cell culture media with immersion in protein-rich sera promoting tenocyte attachment. Bracket designs were developed to enhance grip of the cell-seeded fibrin. Ligament constructs were selfsupporting and exhibited structural characteristics similar to native connective tissue. Tenocyte density peaked on day 14, with added L-proline and ascorbic acid inducing a constant level of glycosaminoglycans and 7.4 ± 1.5 % w/w collagen. This research may significantly enhance the clinical application of tissue engineered ligaments and tendons.
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Qiu, Yiwei. "In vitro tendon tissue engineering." Thesis, University of Oxford, 2010. http://ora.ox.ac.uk/objects/uuid:10d2b8fe-c485-44e4-ba03-abdad0da48f7.
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Tendon, ligament, and joint capsular injuries represent 45% of the 32 million musculoskeletal injuries each year in the United States. Tendon injuries are especially common, requiring surgical repair for the shoulder’s rotator cuff tendons (51,000 per year), the Achilles tendon (44,000 per year), and the patellar tendon (42,000 per year). Tissue engineering provides an alternative in the treatment of tendon lesions through replacement of an injured tendon segment. The purpose of this study was to develop a tendon construct in vitro for clinical reconstructive surgery. Human tenocytes were isolated from hamstring tendons of patients who had undergone anterior cruciate ligament (ACL) surgeries. These tenocytes were cultured with culture media (α-MEM) supplemented with various concentrations of foetal bovine serum (FBS) (0%, 1%, 5% and 10%) and in the presence of different growth factors such as PDGFBB (0, 5, 10 and 50ng/ml), basic FGF (0, 5, 10 and 50ng/ml), IGF-1 (0, 10 and 50ng/ml) and TGFβ-3 (0, 1 and 10ng/ml). Fractional factorial design was utilized to select the combinations of growth factors that supported the following criteria: (1) the maximal cell proliferation with a minimum differentiation of the tenocytes in the presence of the least concentration of FBS possible and (2) maintaining cell survival and promoting tenocyte differentiation in FBS free culture media. The results have shown that: (i) The tenocyte cell number when cultured for 14 days in media supplemented with 1% FBS, 50ng/ml PDGFBB and 50ng/ml bFGF matched that of the positive control (10% FBS-treated cells). Not only was the collagen synthesis significantly reduced in these growth factor-treated cultures compared to positive control tenocytes, but also a significant inhibition of the mRNA expression of various tenocyte differentiation markers (Scleraxis, Tenomodulin, Collagen type I and Decorin) was evident. IGF-1 did not promote significant cell proliferation under low serum conditions but did induce tenocyte differentiation in vitro. Examination of the cell morphology confirmed that tenocytes were capable of less differentiation when cultured with 1% FBS, 50ng/ml PDGFBB and 50ng/ml bFGF, this culture condition was termed “the expansion phase”; (ii) The cell survival was maintained for up to 14 days in serum free culture media supplemented with 50ng/ml IGF-1 and 10ng/ml TGFβ-3 whilst cell differentiation was enhanced and evident by the increase in collagen synthesis and cell morphology. Furthermore, mRNA expression of the aforementioned cell differentiation markers were also significantly increased, this culture condition was termed “the differentiation phase”; (iii) By combining the culture condition optimized for the expansion and differentiation phase sequentially, it was possible to maintain a long term 2-D tenocyte culture in vitro for up to 28 days. In these cultures, the presence of dense collagen formation was clearly evident whereas in positive control group (10% FBS group) such observation was not noted even after prolonged culturing period of up to 45 days. These results suggested that the sequential treatment of tenocytes with growth factors identified for the expansion and differentiation phases was significantly more superior than the standard 10% FBS treatment; (iv) By combining the expansion and differentiation phases optimized for the 2-D cultures, it was possible to maintain human tenocytes in a 3-D scaffold (Bombix silk) for up to 28 days. The tendon like constructs that were formed, macroscopically and microscopically resembled the human hamstring tendon. This observation was confirmed by using H&E staining, scanning electron microscopy and by detecting collagen type I immunohistochemically; (v) It was possible to further validate these findings using in vivo animal models. This was undertaken by implanting the tenocytes cultured sequentially in the defined culture media described above, into the quadriceps of Balb/c nude male mice for up to 30 days. The nature and specificity of the tendon like structure that was formed after this implantation was investigated by H&E staining and immunohistochemistry. It was revealed that the culture conditions that were optimized during the expansion and differentiation phases were suitable for generating a human tendon reconstruct; a finding which is of significance due to its potential for tendon reconstructive surgery.
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Muhamad, Farina. "Electrospun scaffolds for tissue engineering." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/14577.
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A critical challenge in designing materials for tissue engineering (TE) is to provide essential cues that can control cellular behaviour and promote tissue regeneration. TE with fibrous scaffolds by using electrospinning is emerging as a major research area in the field of regenerative medicine. This thesis presents the development of novel electrospun fibrous acrylate scaffolds for bone TE. Acrylate fibrous scaffolds were developed by electrospinning photocrosslinkable and low molecular weight acrylate monomers, methyl acrylate (MA) and diethylene-glycol dimethacrylate (DEGMA). Photocrosslinked fibres were successfully produced by electrospinning different MA and DEGMA compositions and post-UV crosslinking. The ability to produce topologically and mechanically diverse fibrous scaffold materials was demonstrated. Varying MA and DEGMA composition affected overall fibre morphology, swelling and mechanics of the fibrous scaffold. An assessment of biological activity of the acrylate fibrous scaffold was performed to evaluate the effect of varying ratios of MA/DEGMA of the fibrous scaffold on the viability of two different cell types, osteosarcoma-derived osteoblastic cells (Saos-2) and mesenchymal stromal cells (hMSCs). The potential of MA/DEGMA fibrous scaffolds to support Saos-2 cell viability and proliferation was demonstrated. However, the considerable increase in apoptosis of hMSCs cultured on both fibrous and flat samples suggested a lower potential of the MA/DEGMA scaffolds to support hMSCs cell attachment and viability. The fibrous scaffolds were immobilized with synthetic peptides utilizing cysteine-functionalized RGD or DGEA peptide sequences in combination with MA/DEGMA monomers and by employing a photoinitiated mixed-mode thiol-acrylate polymerization mechanism. Cysteine-functionalized DGEA and RGD peptides were incorporated efficiently in the synthesized acrylate scaffold. The peptide-conjugated fibrous scaffolds showed increased hMSCs adhesion and viability. Through cell adhesion and soluble peptide competition assays, the bioactivity and specificity of each peptide conjugated to the scaffold was confirmed. Finally, hMSCs cultured on DGEA conjugated scaffolds exhibited the activation of osteogenic differentiation markers, alkaline phosphatase (ALP) and osteocalcin (OCN). The results presented in this thesis strongly suggest the potential of the acrylate fibrous scaffold for bone TE.
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Place, Elsie Sarah. "Bioactive hydrogels for tissue engineering." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/7106.
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Modern tissue engineering (TE) scaffolds are expected to actively promote tissue repair as well as meeting the traditional requirements of non-toxicity, degradability and structural integrity. This thesis presents two novel bioactive hydrogel systems for bone and cartilage TE. A series of alginate hydrogels were developed in which all or a fraction of the calcium normally used for crosslinking alginate was replaced by bioactive strontium and/or zinc ions. Strontium was chosen for its ability to stimulate bone formation, while zinc is essential for alkaline phosphatase activity. Due to an interaction between the crosslinking ion and alginate type, the hydrogel properties could be tailored independently of the crosslinking ion used – meaning that varying biological and materials requirements can be accommodated. Strontium release from alginate gels was of a physiologically relevant magnitude, and alkaline phosphatase protein activity in Saos-2 cells was highest in strontium gels. Secondly, a biomimetic strategy for transforming growth factor beta (TGF-β) presentation and release was evaluated. TGF-β in vivo is secreted as part of an inactive latent complex, which is sequestered in a stable form within extracellular matrix until released by cells. TGF-β was therefore incorporated into poly(ethylene glycol)-hyaluronic acid hydrogels in its latent form. When compared to free TGF-β, advantages were demonstrated in terms of lower protein adsorption to tissue culture plastic and relative biological inactivity. The latter implies that high doses may be loaded into TE scaffolds without exposing cells to excessive quantities of active growth factor, with TGF-β bioavailability then being controlled by gradual activation by cells. Increased metabolic activity and ECM deposition by bovine chondrocytes were seen after almost five weeks in culture with a single initial loading of LTGF-β. These innovations correspond to current TE trends, which seek to use biomimetic principles to evoke regenerative responses from transplanted or host cells, but to do so using technically and commercially feasible means.
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Czechura, Pawel. "Saturated neoglycopolymers for tissue engineering." Thesis, University of Ottawa (Canada), 2006. http://hdl.handle.net/10393/27121.
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Norbornene monomers bearing carbohydrate groups of relevance for tissue engineering were synthesized via the norbornene acid chlorides, transformed into their ROMP polymers, and reduced to yield saturated neoglycopolymers. These materials bear either O-glycoside groups designed to cross-link collagen via reaction of the ring-open sugar with free NH2 groups of lysine, or C-glycoside groups. The latter are intended for use as the central block in triblock copolymers terminated with blocks capable of crosslinking: they serve only as potentially biocompatible "spacers" to help span the interlamellar distance in collagen. A third ROMP monomer bearing a succinimide group as an alternative crosslinking agent was also prepared and polymerized. The O-glycoside monomer, bis(1,2;3,4-di-O-isopropylidene-D-galactopyranos-6-O-yl) 5-norbornene-2,3-dicarboxylate 9, and the succinimide monomer, 5-norbornene-2-carboxylic acid N-hydroxysuccinimide ester 11, were prepared by the Diels-Alder synthesis of 5-norbornene-2,3-dicarbonyl chloride 7 and 5-norbornene-2-carboxylic acid chloride 8, and subsequent nucleophilic substitution of the acid chlorides. (Abstract shortened by UMI.)
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Starly, Binil Sun Wei. "Biomimetic design and fabrication of tissue engineered scaffolds using computer aided tissue engineering /." Philadelphia, Pa. : Drexel University, 2006. http://hdl.handle.net/1860/1114.
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