Rozprawy doktorskie na temat „Intervertebral Disc Regeneration”
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Ho, Grace, i 何秀慧. "Intervertebral disc regeneration by use of autologous mesenchymal stemcells". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B31541616.
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Ho, Grace. "Intervertebral disc regeneration by use of autologous mesenchymal stem cells". Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B31541616.
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Yang, Fan. "Intervertebral disc regeneration using mesenchymal stem cells a mouse model study /". Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/HKUTO/record/B39556979.
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楊帆 i Fan Yang. "Intervertebral disc regeneration using mesenchymal stem cells: a mouse model study". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39556979.
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Choy, Tsz-hang Andrew, i 蔡子鏗. "Fabrication of a biphasic scaffold for tissue engineering of intervertebral disc". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B49799459.
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Current treatments to intervertebral disc degeneration alter spine biomechanics and have complications. Tissue engineering offers an approach to regenerate a biological disc that provides flexibility and stability to, and integrates with the spine. To date, a scaffold that mimics the extracellular matrix composition and mechanical strength of a native disc is lacked. In this project, a biphasic scaffold was fabricated using glycosaminoglycan (GAG) and collagen, the prevalent ma-trix components in a native disc. It also adapted the structure of the disc, with la-mellae of collagen surrounding a collagen-GAG (CG) core.
The first part of this project studied chemical modification of CG and evaluated the physiochemical and biological properties of modified CGs. As only loosely bound by GAG under physiological environment, collagen was modified by deamination, methylation and amination, and yielded Deaminated, Methylated and Aminated CGs upon co-precipitation with GAG. While GAG was mostly lost within 1 day in Untreated and Deaminated CGs, 20% and 40% GAG was retained after 6 days in Methylated and Aminated CGs respectively. In cell-seeded Aminated CG, over 60% GAG was retained after 8 days. Aminated CG, having the highest GAG/HYP of 4.5, best simulated the GAG-rich nucleus pulposus tissue. In ultrastructural analysis, Aminated CG consisted of abundant granular sub-stances that resembled the nucleus pulposus. Despite the differential initial number adhered to the CG scaffolds, human mesenchymal stem cells (hMSCs) had over 90% viability at all time points. Cell morphology was distinct, being round in Untreated and Methylated CGs but elongated in Deaminated and Aminated ones. The adhesion of hMSCs via collagen receptor, integrin alpha2beta1, was observed in all CG scaffolds, while adhesion via general matrix receptor, integrin alphaV, was extensive in all but Aminated CG. Based on improved GAG incor-poration and retention, which approximate the matrix composition of nucleus pulposus, Aminated CG was chosen as the core of the biphasic scaffold.
The second part of this project studied lamination in biphasic disc scaffold and evaluated its mechanical properties in creep, recovery and dynamic loadings. A process was optimized to encapsulate a CG under physiological condition whilst producing an intact collagen gel, which allowed the CG to retain more GAGs and to be confined by the annulus structurally as was in the disc. This encasing approach was repeated for multiple lamellae, one lamella per day. Scaffolds with more lamellae had increased viscous compliance in creep and recovery, which was explained by the less laminated scaffolds being overloaded. Another lamination approach replaced most encasing lamellae with coiling ones. Despite low sample size, it was shown that this combined approach produced scaffolds with lower elastic and viscous compliances and longer equilibrating time in both creep and recovery, and higher complex modulus under dynamic loading. Full recovery was not achieved by any scaffold.
This study demonstrated that a biphasic disc scaffold, made of GAG and collagen, contained similar matrix components to native disc, was almost mechanically comparable to the disc, and was cyto-compatible. It paved way towards tissue engineering of intervertebral disc and the intervertebral disc motion segment.published_or_final_version
Mechanical Engineering
Doctoral
Doctor of Philosophy
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Khan, Shahnaz. "The effect of the intervertebral disc microenvironment on disc cell and mesenchymal stem cell behaviour : implications for disc degeneration and regeneration". Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/the-effect-of-the-intervertebral-disc-microenvironment-on-disc-cell-and-mesenchymal-stem-cell-behaviour-implications-for-disc-degeneration-and-regeneration(b5629a75-4cb0-45d8-affb-2b936d9408e1).html.
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Intervertebral disc (IVD) degeneration is associated with low back pain (LBP). It has been suggested that changes in the IVD physio-chemical microenvironment (i.e. hypoxia, reduced nutrient and acidic conditions) may lead to disc degeneration. Studying the response of human nucleus pulposus (NP) cells to these conditions could establish the causal relationship between IVD microenvironment and aberrant cellular behaviour, characteristic of disc degeneration. Human bone marrow mesenchymal stem cells (BM-MSCs) are a promising cell population for disc regeneration. However, knowledge of their survival and functioning in the microenvironment of the IVD is still lacking. Moreover, in vitro co-culture model studies that are used to study MSC/disc cell interaction, also need to consider the effect of the microenvironment on cellular responses. BM-MSCs and degenerate NP cells were cultured alone or co-cultured in monolayer under hypoxia (2%O2), reduced nutritional (2% serum or/and 5mM glucose) and acidic (moderate pH 6.8 or severe pH 6.5) conditions alone or in combination for 7 days. Cell viability, proliferation, gene and protein expression was assessed. Degenerate NP cells and BM-MSCs maintained good cell viability under all conditions. Both cell types demonstrated overall similar proliferation and gene and protein responses under the majority of the conditions and combinations studied. Hypoxia promoted aggrecan and versican matrix biosynthesis in both cell types. Nutrient deprived and moderate acidic conditions (pH 6.8) inhibited proliferation of both cell types. Interestingly the combination of hypoxia with these conditions showed a protective effect in modulating cell proliferation. These results imply that hypoxia may be beneficial in some instances. Nutrient deprived conditions had a relatively minor effect on degenerate NP cell gene and protein expression but these conditions specifically inhibited VCAN expression in BM-MSCs. The combination of hypoxia with these conditions increased or restored VCAN expression. Interestingly the combination of hypoxia with reduced glucose conditions increased aggrecan and versican matrix biosynthesis in both NP cells and BM-MSCs. The combination of hypoxia and complete nutrient deprived conditions (both reduced serum and reduced glucose) impaired ACAN, VCAN and PAX-1 gene and aggrecan and versican protein expression in degenerate NP cells implicating disc hypoxia and complete nutrient deprived combined microenvironment in accelerating degenerate changes in NP cells. In contrast, these conditions showed no such detrimental effects on BM-MSC gene and protein expression. pH 6.5 was critical for both cell types proliferation and ACAN and VCAN gene expression suggesting that severe acidic conditions may exacerbate degenerative changes and be inhibitory for implanted MSCs. Finally, a combination of hypoxia, complete nutrient deprived and moderate acidic conditions, reduced cell proliferation without affecting the gene expression profile of both cell types. IVD-like physio-chemical microenvironmental conditions also appeared to influence differentiation of BM-MSC and modulation of degenerate NP cell phenotype observed during co-culture. Noticeably hypoxia, reduced serum or reduced glucose conditions stimulated BM-MSC differentiation and modulation of degenerate NP cell phenotype. Hypoxia also increased or recovered changes at gene expression level in both BM-MSCs and degenerate NP cells under nutrient deprived (reduced serum or/and reduced glucose) conditions during co-culture. Degenerate NP cell and BM-MSC co-culture also showed noticeable increase in aggrecan and versican biosynthesis under hypoxia and reduced glucose combine conditions, implicating these in improving the co-culture responses. Severe pH condition alone, pH 6.8 in combination with hypoxia and finally all IVD-like physio-chemical conditions together compromised co-culture responses. Such results imply that IVD-like physio-chemical microenvironmental conditions may influence MSC based regenerative outcomes. This work has increased our understanding about the influence of disc harsh microenvironment on degeneration and regeneration processes.
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Rodrigues, Pinto Ricardo Pedro Ferreira. "Isolation and phenotypic characterisation of human notochordal cells : implications for the development of cell-based therapies for intervertebral disc degeneration". Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/isolation-and-phenotypic-characterisation-of-human-notochordal-cells-implications-for-the-development-of-cellbased-therapies-for-intervertebral-disc-degeneration(8d5cbfdd-edd0-458c-a048-554f6a2c830b).html.
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Back pain is a highly prevalent condition whose pathogenesis is associated with intervertebral disc (IVD) degeneration. Degeneration is driven by abnormal cell biology, particularly within the IVD’s inner core, the nucleus pulposus (NP). In recent years, there has been an ever-increasing search for cell-based therapies aimed at correcting the cell biology and thus repairing/regenerating the degenerate IVD. The success of these novel therapies, however, requires a thorough understanding of IVD development and of the phenotype of its cells. The embryonic, foetal and juvenile NP is populated by large vacuolated notochordal cells that with skeletal maturity are replaced by smaller NP cells. Since notochordal cells have been shown to display protective and anabolic roles in the IVD their loss in humans has often been suggested to initiate the degenerative process. As such, a detailed understanding of notochordal cells and their regulatory pathways may help identify factors involved in IVD homeostasis and aid the development of novel cell-based therapies targeting IVD degeneration. The study of human notochordal cells has, however, been hindered by ethical, logistical and technical difficulties in obtaining suitable samples and, as such, the human notochordal cell phenotype is, to date, unknown, constituting a major limitation in the field. The work presented here was conducted with the objective of developing a methodology to isolate human developing notochordal cells (NP progenitors) from adjacent sclerotomal cells (annulus fibrosus and vertebral body progenitors), to characterise the notochordal cell phenotype and identify potential factors involved in notochordal cell biology. Initially, human embryonic and foetal spines were characterised to assess their suitability as a source of notochordal cells and to identify a notochord-specific marker that could be used to isolate notochordal cells for microarray studies. The human developing spine contained large vacuolated notochordal cells in all stages analysed (3.5-18 weeks post-conception (WPC)) that specifically expressed KRT8, KRT18 and KRT19 at all stages and CD24 between 5.5-18 WPC. KRT18 and CD24 were independently used to label notochordal cells (7.5-14 weeks post-conception) and separate them from sclerotomal cells. Methodologies were developed to allow extraction of RNA of sufficient quality for microarray analysis from fixed, permeabilised (in the case of KRT18) and/or, labelled and sorted cells (CD24). Microarray analysis identified and real-time qPCR and, for some markers, immunohistochemistry, validated GRB14, SLC19A1, FGF10, ADORA3, TBXA2R, CDH6, ANPEP, CD69, CD24, RTN1, PRPH, MAP1B, ISL1 and CLDN1 as human notochordal cell markers. Ingenuity pathway analysis was performed to investigate the pathways/networks and upstream regulators and downstream effectors of notochordal cells. Inhibition of inflammation and angiogenesis were identified as relevant to notochordal cell biology, function and, possibly, to the known protective and anabolic role notochordal cells display in the IVD. Notochordal marker gene expression was identified in adult NP tissue, and negatively correlated with degeneration. Proteins encoded by ADORA3 and MAP1B were expressed by a proportion of adult NP cells, suggesting the presence of notochord-derived cells in the adult NP.Importantly, this is the first study to detail a methodology and successfully isolate human notochordal cells. Such methodology has the potential to be used to culture and investigate the biology of viable human notochordal cells (CD24+ve). Future studies aimed at developing cell-based therapies for IVD degeneration could also use these identified markers to assess appropriate stem cell differentiation to notochordal cells.
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Strassburg, Sandra. "An In Vitro Culture System to Study Human Mesenchymal Stem Cell / Nucleus Pulposus Cell Interactions : Implications for Intervertebral Disc Regeneration". Thesis, University of Manchester, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.521587.
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Aladin, Kaderbatcha Darwesh Mohideen. "Nanoscale structure-property and macroscale biomechanical function of nucleus pulposus in health, disease and regeneration". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45197143.
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Figueiró, Manuela. "Análise histológica, imunohistoquímica e isolamento de células-tronco mesenquimais adultas do disco intervertrebal degenerado aplicadas a medicina regenerativa". reponame:Repositório Institucional da UCS, 2014. https://repositorio.ucs.br/handle/11338/915.
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Fotticchia, Andrea. "Design and development of anisotropic laminate scaffolds of electrospun polycaprolactone for annulus fibrosus tissue engineering applications". Thesis, Loughborough University, 2016. https://dspace.lboro.ac.uk/2134/21407.
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In several cases, current therapies available to treat a large number of musculoskeletal system diseases are unsatisfactory as they provide only temporary or partial restoration of the damaged or degenerated site. In an attempt to maintain a high standard of life quality and minimise the economic losses due to the treatments of these frequently occurring ailments and subsequent lost working days, alternative therapies are being explored. Contrary to the current treatments, tissue engineering aims to regenerate the impaired tissue rather than repair and alleviate the symptoms; thus offering a definitive solution. The annulus fibrosus (AF) of the intervertebral disc (IVD) is a musculoskeletal system component frequently subjected to degeneration and rupture, characterised by predominance of anisotropically arranged collagen fibres. In the present thesis, electrospinning technology is used to fabricate polycaprolactone (PCL) scaffolds intended to replicate the anisotropic structure of the AF.
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Frayssinet, Antoine. "Hydrogels composites collagène/acide hyaluronique cellularisés et biomimétiques pour la régénération du Nucleus Pulposus". Electronic Thesis or Diss., Sorbonne université, 2020. http://www.theses.fr/2020SORUS312.
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50 % des douleurs dorsales chroniques sont associées à une dégénérescence du disque intervertébral (IVD). Nous avons émis l'hypothèse qu'un hydrogel biomimétique favoriserait la régénération du Nucleus Pulposus, la partie centrale de l’IVD, en fournissant des signaux physiques adéquats à des cellules souches mésenchymateuses (MSC) pour se différencier en nucléopulpocytes. Avec des teneurs différentes en acide hyaluronique fonctionnalisé (HA-Tyr), des hydrogels composites Collagène/HA-Tyr ont été synthétisés et caractérisés par microscopies électroniques à balayage et à transmission, rhéologie, DSC, par test de dégradation enzymatique in vitro accélérée et par tests de capacité à absorber l’eau. Des MSC ont ensuite été incorporées dans les composites Col/HA-Tyr, puis cultivées pendant 28 jours. La viabilité cellulaire a été évaluée, et leur différenciation en nucléopulpocytes analysée par PCR quantitative et par immunohistochimie indirecte. La présence de plusieurs marqueurs de différenciation des nucléopulpocytes, tels que le Collagène de type II, l'Agrécane et le KRT 18 a été suivie. Le processus de fabrication a permis la génération d'hydrogels hautement hydratés (> 90%), mécaniquement biomimétiques, résistants à la dégradation enzymatique et dans lesquels la fibrillogenèse du collagène a été préservée. En absence de facteur de différenciation, l’élasticité et la structure des hydrogels composites Col/HA-Tyr semble suffire pour induire la différenciation des MSC incorporées en nucléopulpocytes. Développée suivant une approche biomimétique, cette plateforme d'hydrogels composites Col/HA-Tyr parait donc prometteuse pour la régénération du disque intervertébralHalf of chronic back pain is associated with intervertebral disc (IVD) degeneration. We hypothesized that a biomimetic hydrogel would promote the regeneration of the Nucleus Pulposus, the central part of IVD. Hydrogels will provide cues to incorporated mesenchymal stem cells (MSC) to in situ differentiate into nucleopulpocytes. With different contents of functionalized hyaluronan (HA-Tyr), Collagen/HA-Tyr hydrogels were produced and characterized using scanning and transmission electron microscopy, rheology, DSC, accelerated in vitro enzymatic degradation and tested for their ability to absorb water. MSC were then incorporated within Col/HA-Tyr composites and cultured over 28 days. Cell viability was assessed and cell differentiation was analysed by quantitative PCR and indirect immunohistochemistry. The presence of several nucleopulpocytes differentiation markers, such as type II Collagen, Aggrecan and KRT 18 was monitored. The manufacturing process allowed the generation of highly hydrated hydrogels (> 90%), mechanically biomimetic, resistant against enzymatic degradation, in which collagen fibrillogenesis was preserved. Without any differentiation factor, both elasticity and structure of the Col/HA-Tyr composite hydrogels seems to be sufficient to induce the differentiation of the incorporated MSCs into nucleopulpocytes. In addition, the presence of collagen was necessary for an adequate cell adhesion. Developed according to a biomimetic approach, this platform of Col-HA-Tyr hydrogels appears promising for the intervertebral disc repair
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Halimi, Célia. "Conception et élaboration d’une solution de chitosane injectable : application en ingénierie tissulaire pour la régénération du derme et du disque intervertébral". Thesis, Lyon, 2016. http://www.theses.fr/2016LYSE1074.
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Le travail présenté dans le manuscrit concerne la conception d'un dispositif médical de classe III pour des applications en ingénierie tissulaire du derme et du disque intervertébral.Il s'agit d'une solution aqueuse de chitosane, stérilisée par autoclave, et dont le pH et l'osmolarité ont été ajustés selon un procédé de dialyse classique. Cette solution possède des propriétés de gélification in situ innovantes, ce qui lui confère de très bonnes propriétés mécaniques quelques minutes après l'injection, sans l'utilisation d'agents de réticulation mais de façon modulable en fonction de la concentration en polymère. De plus, cette solution possède une bonne injectabilité favorisant le développement d'une technique d'implantation mini-invasive pour la régénération du derme et du disque intervertébral. Un critère de performance rhéologique a notamment été mis au point afin de relier l'injectabilité aux propriétés mécaniques de l'injectât (dermal filler).Les propriétés mécaniques de l'injectât gélifié doivent être comparables au tissu natif en particulier pour le dermal filler mais aussi pour le disque intervertébral. Le comportement mécanique viscoélastique du disque intervertébral a été évalué par des essais de relaxation de contraintes et modélisé avec un modèle de Maxwell solide à trois branches. Ces essais ont été conduits sur des disques sains, ayant subi une altération de structure (fenestration) et après l'injection de biopolymères.La solution de chitosane a été injectée (i) dans le tissu cutané de deux modèles animaux : le porc et le rat et (ii) dans le disque intervertébral de deux modèles animaux : le porc et le lapin. La biocompatibilité ainsi que la réponse biologique de solutions/gels physique de chitosane in vivo ont été validées pour tous ces modèles animauxThis work deals with the conception of a class III medical device for applications in tissue engineering of dermis and intervertebral disc.This device consists in an aqueous chitosan solution, sterilized by autoclaving, with pH and osmolality adjusted by a dialysis process. This chitosan solution shows in situ gelation ability with a post-injection increase of mechanical properties. This feature is related to polymer concentration, gelation time, and is performed without external cross-linking agent. In addition, the solution exhibits a good injectability allowing the development of minimally invasive techniques to treat dermis and intervertebral disc diseases. A rheological performance criterion was defined linking injectability to mechanical properties of the implant (dermal filler).Mechanical properties of gel implant formed in contact with body fluids, in situ, have to be similar to that of native tissues. The viscoelastic behavior characterization of intervertebral disc was performed using stress relaxation and was modeled using a generalized solid Maxwell model (composed of three Maxwell elements). The tests were performed on healthy disc, fenestrated discs and after biopolymers injection.Chitosan solutions were injected into (i) porcine and rat cutaneous tissue and (ii) porcine and rabbit intervertebral discs. The biocompatibility and biofunctionality of chitosan solutions and physical hydrogels was evidenced in vivo for all animals
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Castro, André. "Development of a biomimetic finite element model of the intervertebral disc diseases and regeneration". Doctoral thesis, 2013. http://hdl.handle.net/1822/28831.
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Tese de doutoramento do Programa Doutoral em Engenharia BiomédicaDegenerative Disc Disease is one of the largest health problems faced worldwide, based on lost working time and associated costs. This is the driving force for the development of a biomimetic Finite Element (FE) model of the Intervertebral Disc (IVD), which is a multiphasic and highly inhomogeneous structure. A great amount of experimental and numerical works have studied the IVD and proven that it presents osmo-poro-hyper-visco-elastic behavior, with high influence of the anisotropic behavior of collagen fibers. Poroelastic models of the IVD are mostly implemented in commercial FE-packages, which means that the accessibility to the source algorithm is often circumscribed. In order to approach to the biomechanical behavior of the IVD in the Human spine with higher flexibility and accuracy, an innovative poroelastic formulation implemented on a home-developed open-source FE solver is addressed and validated throughout this work. Numerical simulations were mostly devoted to the analysis of the non-degenerated Human IVD time-dependent behavior, using a geometrically accurate FE model of a full motion segment (MS), constructed with quadratic 27 nodes hexaedral elements. The results of the tests performed for creep assessment were inside the scope of the experimental and numerical literature data, with remarkable improvements of the numerical accuracy when compared with some previously published results obtained with the commercial FE-package ABAQUS®. Previously unpublished experimental data from the research partners at VUmc (Amsterdam, The Netherlands) were also analyzed and compared with the MS FE model, which proved to reproduce satisfactorily to the physiological and non-physiological conditions of those experimental tests. The IVD biomechanical behavioral is complex and dependent on multiple factors. The numerical simulations with the present MS FE model, using the home-developed open-source FE solver, demonstrated potential to biomimitize the IVD and thus contribute to the advance of the knowledge on its biomechanics.
A Doença Degenerativa dos Discos é um dos maiores problemas de saúde enfrentados a nível mundial, a nível de tempo de trabalho perdido e custos associados. Esta é a motivação para o desenvolvimento de um modelo biomimético de Elementos Finitos (EF) do Disco Intervertebral (DIV), que é uma estrutura multifásica e altamente heterogénea. Um grande número de trabalhos, experimentais e numéricos, estudou o DIV e provou que este apresenta comportamento osmo-poro-hiper-visco-elástico, com influência significativa do comportamento anisotrópico das fibras de colagénio. Os modelos poroelásticos do DIV têm sido frequentemente implementados em programas comerciais de EF, o que significa que o acesso ao algoritmo-fonte é circunscrito. Para obter uma aproximação mais flexível e rigorosa ao comportamento biomecânico do DIV, uma formulação poroelástica inovadora foi implementada num programa de EF de acesso livre, desenvolvido internamente. Esta formulação é descrita e validada ao longo do presente trabalho. As simulações numéricas foram quase totalmente dedicadas à análise do comportamento do DIV Humano não-degenerado, que se sabe ser fortemente dependente do factor tempo. Para esse feito, foi utilizado um modelo de EF geométrico correcto de um segmento móvel (SM) completo, construído com elementos quadráticos hexaédricos de 27 nós. Os resultados dos testes levados a cabo para análise do comportamento do DIV em termos de fluência ficaram dentro do espectro dos resultados experimentais e numéricos disponíveis na literatura. Foram, inclusivé, registadas melhorias notáveis em relação a alguns trabalhos que utilizaram ABAQUS®, um programa de EF comericalmente disponível. Foram também analisados dados experimentais não publicados dos parceiros de investigação da VUmc (Amesterdão, Holanda). A comparação com o modelo EF do SM demonstrou que este modelo reproduz satisfactoriamente as condições dos testes experimentais, sejam elas condições fisiológicas ou não-fisiológicas. O comportamento biomecânico do DIV é complexo e dependente de múltiplos factores. As simulações numéricas levadas a cabo com o modelo EF do SM, utilizando o programa de EF de acesso livre desenvolvido internamente, demonstraram potencial para biomimetizar o DIV e assim contribuir para o avanço do conhecimento da sua biomecânica.
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LIU, KENG-CHANG, i 劉耿彰. "Effects of Platelet-Rich Fibrin (PRF) on Regeneration of Muscle, Bone, and Intervertebral Disc". Thesis, 2019. http://ndltd.ncl.edu.tw/handle/p8z6hd.
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博士東海大學
畜產與生物科技學系
107
Platelet concentrates including platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are becoming innovative tools of regenerative medicine. Autologous PRP is derived from an individual's whole blood then centrifuged to remove red blood cells and leucocytes. The remaining plasma has a 5- to 10-fold higher concentration of growth factors than whole blood. These growth factors have been found to promote natural healing responses, and hence PRP is widely used in orthopedic and sports medicine to relieve pain in musculoskeletal injuries. Platelet-rich fibrin (PRF) is a newer generation of platelet concentrates and has being used as an adjunctive autologous biomaterial to promote bone and soft tissue healing and regeneration. This thesis included an in vitro study and 3 clinical trials, and the objectives were to investigate the roles of PRF on regeneration of muscle, tendon, bone, and intervertebral disc (IVD). In vitro study, we investigated the biologic effects of pig PRF on the proliferation of different cells, including rat L8 myoblasts, mouse RAW 264.7 monocytic cells which are precursors of osteoclasts, mouse L929 fibroblasts, and porcine endothelial cells (PEC). L8 cells and L929 cells cultured with PRF showed a decrease in cell number when compared to the control group in which horse serum was used as a control. RAW 264.7 cells and PEC cultured with PRF, on the other hand, increased cell number. These results suggested that PRF had negative effect on the proliferation of L8 and L929 cells, however, expressed positive regulation on RAW 264.7 cells and PEC. In clinical trials, we investigated the clinical effects of PRF on bone fusion in spinal fusion surgery and IVD regeneration after endoscopic discectomy. Posterior lumbar decompression and 2 transforaminal interbody fusion (TLIF) is a safe and effective surgery for degenerative spinal diseases. Several options of bone graft are available but autologous iliac crest bone grafting (ICBG) is still the gold standard. However, some morbidity is associated with ICBG harvesting such as infection, fracture, or donor site pain. To avoid donor site morbidity, various allograft materials have been used as a substitute but are still questionable as to the effectiveness and safety. In our study, allograft combined with autogenous bone marrow and with/without PRF was used to compare with autologous ICBG in TLIF surgery. After at least 12 months follow-up, the fusion rate in single level TLIF was 100% in both autograft group and allograft with PRF group, while the fusion rate of allograft without PRF was 77.8%. The results demonstrated positive effect of PRF on bone fusion in TLIF. Discogenic low back pain (DLBP) is associated with degeneration of IVD and usually is a complex problem regrading to diagnosis and treatment. It is suspected that the pain source of DLBP comes from the torn lesion of annulus fibrosus (AF) which is located on the outer surface of IVD. We applied percutaneous endoscope to treat torn AF in patients with DLBP and the satisfactory outcomes were more than 85% in selected patients at two years follow-up. The procedure is effective and minimally invasive because only inflammatory tissues on AF and loose nucleus debris are removed under clear endoscopic vision. To investigate the effect of PRF on regeneration of IVD, we inserted PRF into IVD in 6 patients who were diagnosed with DLBP and had undergone endoscopic treatment. Autogenous PRF was inserted into nucleus at the end of endoscopic procedure. At 12 months follow-up, 5 of 6 patients showed excellent results and MRI T2-weighted signal of involved IVD did not show further degenerative change. This adjunctive biomaterial expressed beneficial effects on clinical outcomes and radiologic results.
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Windisch, Leah Marianne. "Design and Evaluation of a Disulphide-crosslinked Hyaluronan Hydrogel for Regeneration of the Intervertebral Disc". Thesis, 2008. http://hdl.handle.net/1807/17236.
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A cysteine-containing elastin-like polypeptide (ELP2cys) was successfully synthesized and purified, and was shown to behave in a similar fashion to other well-characterized ELPs. Incorporating the ELP2cys as a crosslinking agent into a solution of sulphated hyaluronan (CMHA-S) not only decreased the gelation time of the solution but also increased the crosslinking density of the resultant hydrogel, in turn increasing both the resiliency and stiffness
of the construct. Preliminary in vitro work involved culture of human disc cells, followed by their encapsulation within the hydrogel. Unfortunately the results were inconclusive, although it appeared as though the addition of ELP2cys to the matrix did not negatively affect the viability
of the cells, as compared to hydrogels with CMHA-S only. This study showed that ELP2cys is a valuable addition to the family of recombinant elastin-like polypeptides, and shows promise as a crosslinking agent in the formation of hyaluronan hydrogels.
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Francisco, Aubrey Therese. "Laminin-Functionalized Polyethylene Glycol Hydrogels for Nucleus Pulposus Regeneration". Diss., 2013. http://hdl.handle.net/10161/8204.
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Intervertebral disc (IVD) disorders and age-related degeneration are believed to contribute to low back pain. There is significant interest in cell-based strategies for regenerating the nucleus pulposus (NP) region of the disc; however, few scaffolds have been evaluated for their ability to promote or maintain an immature NP cell phenotype. Additionally, while cell delivery to the pathological IVD has significant therapeutic potential for enhancing NP regeneration, the development of injectable biomaterials that retain delivered cells, promote cell survival, and maintain or promote an NP cell phenotype in vivo remains a significant challenge. Previous studies have demonstrated NP cell - laminin interactions in the NP region of the IVD that promote cell attachment and biosynthesis. These findings suggest that incorporating laminin ligands into biomaterial scaffolds for NP tissue engineering or cell delivery to the disc may be beneficial for promoting NP cell survival and phenotype. In this dissertation, laminin-111 (LM111) functionalized poly(ethylene glycol) (PEG) hydrogels were developed and evaluated as biomaterial scaffolds for cell-based NP regeneration.
Here, PEG-LM111 conjugates with functional acrylate groups for crosslinking were synthesized and characterized to allow for protein coupling to both photocrosslinkable and injectable PEG-based biomaterial scaffolds. PEG-LM111 conjugates synthesized using low ratios of PEG to LM111 were found support NP cell attachment and signaling in a manner similar to unmodified LM111. A single PEG-LM111 conjugate was conjugated to photocrosslinkable PEG-LM111 hydrogels, and studies were performed to evaluate the effects of hydrogel formulation on immature NP cell phenotype in vitro. When primary immature porcine NP cells were seeded onto PEG-LM111 hydrogels of varying stiffnesses, softer LM111 presenting hydrogels were found to promote cell clustering and increased levels of sGAG production as compared to stiffer LM111 presenting and PEG-only gels. When cells were encapsulated in 3D gels, hydrogel formulation was found to influence NP cell metabolism and expression of proposed NP phenotypic markers, with higher expression of N-cadherin and cytokeratin 8 observed for cells cultured in softer (<1 kPa) PEG-LM111 hydrogels.
A novel, injectable PEG-LM111 hydrogel was developed as a biomaterial carrier for cell delivery to the IVD. PEG-LM111 conjugates were crosslinked via a Michael-type addition reaction upon the addition of PEG-octoacrylate and PEG-dithiol. Injectable PEG-LM111 hydrogel gelation time, mechanical properties, and ability to retain delivered cells in the IVD space were evaluated. Gelation occurred in approximately 20 minutes without an initiator, with dynamic shear moduli in the range of 0.9 - 1.4 kPa. Primary NP cell retention in cultured IVD explants was significantly higher over 14 days when cells were delivered within a PEG-LM111 hydrogel carrier, as compared to cells in liquid suspension.
The studies presented in this dissertation demonstrate that soft, LM111 functionalized hydrogels may promote or maintain the expression of specific markers and cell-cell interactions characteristic of an immature NP cell phenotype. Furthermore, these findings suggest that this novel, injectable laminin-functionalized biomaterial may be an easy to use and biocompatible carrier for delivering cells to the IVD.
Dissertation
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Liao, Shao-Yu, i 廖紹妤. "Applications of Low-intensity Pulsed Ultrasound Stimulation on Human Annular Fibrosus Regeneration for Intervertebral Disc Degenerative Disease". Thesis, 2012. http://ndltd.ncl.edu.tw/handle/69486434387688660369.
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碩士國立陽明大學
醫學工程研究所
100
The low intensity plus ultrasound (LIPUS) is forms of physical stimulations, ultrasound have been found to be effective non-invasive treatments for bone healing and tissue repair in clinical. Despite the efficacy exhibited in clinical application, molecular evidence of ultrasound therapeutic effects and biochemical mechanisms on intervertebral disc remain limited. The therapeutic effects of ultrasound could be explained by a propagation of acoustic waves that transfers mechanical energy into tissues. The mechanical forces could serve as extracellular information, which may induce certain intracellular signaling pathway and regulate cell growth, function and differentiation. In this study, the aim is elucidate the effects of physical stimulations on annulus fibrosus cells (AF) mechanotransduction and regulation of extracellular matrix metabolisn in vitro. First, we investigate the effects of ultrasound on cultured AF cell and the molecular and biochemical mechanisms by which ultrasound promote AF cell proliferation and regulate extracellular matrix metabolism. Next, we investigate mechanotransduction pathways of AF cell, and crosstalk mechanisms between transforming growth factor-β (TGF-β) and ultrasound stimulation during mechanotransduction. In this study, the best parameters of ultrasound was treat by 0.5W/cm2 for 5 min for 5 days. The result shows that the cell viability and total GAGs contents has significantly enhanced after treatment, and, the transforming growth factor beta1 (TGF-β1) also activited . Therefore, the synthesis of collagen type I content was increased, otherwise, matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-13 (MMP-13) decreased. Furthermore, ultrasound induces Extracellular signal-regulated kinases (ERK) pathway, which doesn’t relate on cell viability. This study revealed that ultrasound stimulation regulates extracellular matrix metabolism through the crosstalk between TGF-β and ultrasound-induced mitogen-activated protein kinases (MAPKs) signaling pathways. Physical stimulus induced the synthesis and release of transformation growth factor-β1, which plays an important role in the synthesis of extracellular matrix catabolism, and the present experimental results show that ultrasound stimulation can indeed enhance the repair of the annulus fibrosus cells.
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Chen, Wei-Hong, i 陳韋弘. "Chondrogenesis in Intervertebral Disc and Articular Cartilage Regenerations". Thesis, 2008. http://ndltd.ncl.edu.tw/handle/03385247164239325186.
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博士臺北醫學大學
醫學科學研究所
96
In this thesis we studied the chondrogenesis mechanisms in intervertebral disc (IVD) and articular cartilage regenerations. Human IVD degeneration often initiated from the human nucleus pulposus (hNP) with aging leading to IVD destruction and extracellular matrix (ECM) depletion. Previously, we have successfully employed transforming growth factor-??1 (TGF-??1) to promote chondrogenesis of mesenchymal progenitor cells (MPCs) and immortalized human mesenchymal stem cells. In the first part of this thesis, we examined the role of TGF-??1 in platelet-rich plasma (PRP) on disc regeneration, including proliferation, redifferentiation, and the reconstitution of tissue-engineered NP. hNP cells were isolated from volunteers with different ages and cultured in the presence of PRP. We found that the most effective concentration for hNP proliferation was designated as 1 ng/ml TGF-??1 in PRP, which was further applied in the following experiments. hNP cell proliferation in all age groups were increased time-dependently by PRP and cell morphologies showed aggregation. The mRNA of Sox9, type II collagen, and aggrecan were all significantly upregulated by PRP through RT-PCR. Glycosaminoglycan (GAG) accumulation reached the highest value at day 7 and continued to day 9 culture. PRP promoted NP regeneration via the Smad pathway was also determined and highly activated p-Smad2/3 at 30 min and continuously sustained to 120 min. Immunostaining of type II collagen indicates that PRP participates in chondrogenesis of tissue-engineered NP with collagen scaffolds. We concluded that growth factors in PRP can effectively react as a growth factor cocktail to induce hNP proliferation and differentiation, and also promote tissue-engineered NP formation. These findings are the first to demonstrate that PRP might be a therapeutic candidate for prevention of disc degeneration. We then investigated the chondrogenesis stages of MSCs committed by articular chondrocytes. Osteoarthritis (OA) is an imbalance in cartilage homeostasis which could potentially be corrected by mesenchymal stem cells (MSCs)-based therapies. However, unexpected results were examined in undifferentiated MSCs after in vivo implantation. To facilitate this, we created a co-culture preconditioning system to improved chondrogenic potentials and to study the detailed chondrogenesis stages of human MSCs (Kp-hMSCs) committed by human chondrocytes (hPi-GFP). We demonstrated that chondrocytes induced commitment to chondrocytic differentiation of hMSCs, based on expressions of chondrogenic (Sox9, type II collagen and aggrecan) but not osteogenic (ALP, osteopontin and osteocalcin) markers, as well as RUNX2. During chondrocytic differentiation of hMSCs, we have more accurately delineated the chondrogenesis stages and have defined the molecular markers. MSCs chondrogenesis stages initiated with highly activated chondrogenic adhesion molecules (ICAM-1) and integrins in committed Kp-hMSCs. Growth factors that promoting chondrogenesis, including TGF-?? superfamily and their downstream regulators SMADs as well as EGF, FGF, IGF and VEGF, were stimulated. An increased accumulation of Col II and glycosaminoglycan (GAG) in committed Kp-hMSCs was detected by immunofluorescent and Alcian blue staining. Furthermore, committed Kp-hMSCs acquired neocartilage forming potentials within the collagen scaffold. Our results first indicate that hMSCs were committed to chondroprogenitor stage by chondrocytes pass through detailed chondrogenesis stages. In addition, the in vitro chondrogenesis of MSCs represents an advance in cell-based transplantation for future clinical use.