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. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Thesis (Ph.D)--Chemical Engineering, Georgia Institute of Technology, 2010. / Committee Chair: Dr. J. Carson Meredith. Part of the SMARTech Electronic Thesis and Dissertation Collection.

<p>Uobičajeni pristup i testiranju biolo&scaron;kog pona&scaron;anja materijala je da se počne sa jednostavnim in vitro testovima baziranim na ćelijskim kulturama. In vitro testovi citotoksičnosti su danas jedan od osnovnih načina za procenu biolo&scaron;kog odgovora na materijal jer su brži, lak&scaron;i za ponavljanje, ocenjivanje i jeftiniji u odnosu na eksperimente koji se izvode na životinjama i ljudima. Koriste se kao neka vrsta skrining testova za procenu biolo&scaron;ke sigurnosti materijala. Za razliku od ćelijskih kultura, istraživanja koja uključuju eksperimentalne životinje pružaju bolji uvid u biokompatibilnost materijala, zbog mogućnosti praćenja kompleksnog imunolo&scaron;kog odgovora živog organizma. Smatraju se neophodnim za ocenu bilo&scaron;kih odgovora na novi materijal, pre nego &scaron;to se on upotrebi na ljudima. Mnogi aspekti biolo&scaron;kog odgovora ne mogu biti reprodukovani in vitro testovima uključujući krvne interakcije, zarastanje rana, reakcije preosetljivosti, karcinogenezu, hroničnu inflamaciju. Eksperimenti na životinjama pružaju informacije o ovim tipovima efekata bez izlaganja ljudi riziku. Cilj ovog istraživanja bio je da se oceni biokompatibilnost objekata od polimera na bazi epoksi smole Accura&reg; ClearVue&trade; (hemijski sastav: 4,4&rsquo;- izopropilidendicikloheksanol, produkti oligomerne reakcije sa 1-hlor-2,3- epoksipropanom(40-65%), sme&scaron;а triaril-sulfonijum soli (50% propilen-karbonata i 50% triaril-sulfonijum heksafluoroantimonatnih soli) (1-10%) i 3-etil-3hidroksimetil-oksetan(10-20%). U oceni citotoksičnosti materijala Accura&reg; ClearVue&trade; kori&scaron;ćeni su agar diguzioni i MTT test. Oba testa rađena sun a ćelijskim kulturama L929 (mi&scaron;iji fibroblasti) i MRC-5 (humani fibroblasti). Ocena biokompatibilnosti testiranog materijala vr&scaron;ena je na osnovu urađenog testa iritacije oralne mukoze na modelu bukalne kesice hrčka, &scaron;to je definisano standardom ISO 10993-10:2010. Biokompatibilnost materijala ispitana je i implantacijom uzoraka u potkožno tkivo dorzuma pacova soja Wistar.</p> / <p>Uobičajeni pristup i testiranju biolo&scaron;kog pona&scaron;anja materijala je da se počne sa jednostavnim in vitro testovima baziranim na ćelijskim kulturama. In vitro testovi citotoksičnosti su danas jedan od osnovnih načina za procenu biolo&scaron;kog odgovora na materijal jer su brži, lak&scaron;i za ponavljanje, ocenjivanje i jeftiniji u odnosu na eksperimente koji se izvode na životinjama i ljudima. Koriste se kao neka vrsta skrining testova za procenu biolo&scaron;ke sigurnosti materijala. Za razliku od ćelijskih kultura, istraživanja koja uključuju eksperimentalne životinje pružaju bolji uvid u biokompatibilnost materijala, zbog mogućnosti praćenja kompleksnog imunolo&scaron;kog odgovora živog organizma. Smatraju se neophodnim za ocenu bilo&scaron;kih odgovora na novi materijal, pre nego &scaron;to se on upotrebi na ljudima. Mnogi aspekti biolo&scaron;kog odgovora ne mogu biti reprodukovani in vitro testovima uključujući krvne interakcije, zarastanje rana, reakcije preosetljivosti, karcinogenezu, hroničnu inflamaciju. Eksperimenti na životinjama pružaju informacije o ovim tipovima efekata bez izlaganja ljudi riziku. Cilj ovog istraživanja bio je da se oceni biokompatibilnost objekata od polimera na bazi epoksi smole Accura&reg; ClearVue&trade; (hemijski sastav: 4,4&rsquo;- izopropilidendicikloheksanol, produkti oligomerne reakcije sa 1-hlor-2,3- epoksipropanom(40-65%), sme&scaron;a triaril-sulfonijum soli (50% propilen-karbonata i 50% triaril-sulfonijum heksafluoroantimonatnih soli) (1-10%) i 3-etil-3hidroksimetil-oksetan(10-20%). U oceni citotoksičnosti materijala Accura&reg; ClearVue&trade; kori&scaron;ćeni su agar diguzioni i MTT test. Oba testa rađena sun a ćelijskim kulturama L929 (mi&scaron;iji fibroblasti) i MRC-5 (humani fibroblasti). Ocena biokompatibilnosti testiranog materijala vr&scaron;ena je na osnovu urađenog testa iritacije oralne mukoze na modelu bukalne kesice hrčka, &scaron;to je definisano standardom ISO 10993-10:2010. Biokompatibilnost materijala ispitana je i implantacijom uzoraka u potkožno tkivo dorzuma pacova soja Wistar.</p> / <p>The usual approach in testing biological behavior of materials is to start with simple in vitro tests based on cell cultures. In vitro cytotoxicity tests are one of the basic methods of assessing the biological response to material because they are faster, cheaper, easier for repeating and evaluating compared to experiments carried out on animals and humans. They are used as a kind of screening test for evaluating the biosafety of materials. Unlike cell culture, studies involving experimental animals provide better insight into the biocompatibility of materials due to the possibility of monitoring the complex immune response of a living organism. They are considered necessary for assessing the biological response to new material before it is used on humans. Many aspects of a biological response cannot be reproduced with in vitro tests, including blood interaction, wound healing, hypersensitivity reactions, carcinogenesis, chronic inflammation. Animal experiments provide information about these types of effects without exposing humans to risk.&nbsp; The aim of this study was to evaluate the biocompatibility of polymer objects on the basis of epoxy resins Accura&reg; ClearVue &trade; (chemical composition: 4.4&#39; Isopropylidenedicyclohexanol, oligomeric reaction products with 1-chloro-2.3-epoxypropane (40-65%), a mixture of triaryl sulfonium salt (50% propylene carbonate and 50% of a triarylsulfonium hexafluoroantimonate salt) (1- 10%) and 3-ethyl-3-hydroxymethyl-oxetane (10-20%). In the assessment of the cytotoxicity of materials Accura&reg; ClearVue &trade; agar diffusion and MTT tests were used. Both tests were conducted on cell cultures L929 (mouse fibroblasts) and MRC-5 (human fibroblasts). An assessment of the biocompatibility of the tested material was done on the basis of an oral mucosa irritation test on a hamster cheek pouch as defined by ISO 10993-10: 2010. The biocompatibility of the material was also tested with the implantation of a samples into the dorsal subcutaneous tissue of a Wistar rats. The subcutaneous implantation test, as one of the most reliable methods for assessing the biocompatibility of dental materials, is defined by ISO 10993-6: 2010. The study was conducted on 30 rats which were sacrificed in groups</p>