Dissertations / Theses on the topic 'Ex vivo bone'

Limitations in current model systems for researching bone repair have hampered the development of alternative clinical therapies. This thesis aimed to develop and validate an ex vivo rat mandible model, to investigate specific molecular and cellular processes involved in bone repair. Maintenance of cell and tissue architecture and viability was shown within mandible slices cultured for up to 21 days, both intact and fractured. Autoradiographic studies showed that resident cells were actively synthesising and secreting proteins, and cells of the osteoblast lineage were shown to survive throughout the culture period. The model was responsive to exogenously added growth factors TGF-p1 and BMP-2, with increased cellular migration / proliferation and expression of bone matrix proteins observed. A second model system, an in vitro bone slab cell culture system, demonstrated that endogenous growth factors could be released from the matrix of bone by chemicals such as EDTA, calcium hydroxide, and sodium hydroxide. Different growth factor release kinetics were observed with each treatment, and released growth factors were capable of actively influencing the behaviour of osteogenic cells. Pre-treatment of mandible slices with these chemical treatments yielded similar results, with an observed increase in cell number, proliferation, and bone matrix protein expression. The ex vivo mandible model developed within this study may represent an ideal system for investigating specific processes of bone repair, as well as a promising alternative to in vivo testing of novel clinical therapeutics.

Thesis (Ph. D.)--University of Cincinnati, 2006.
Title from electronic thesis title page (viewed Nov. 30, 2006). Includes abstract. Keywords: Ultrasound, hyperthermia, thrombolysis, clotted blood, ischemic stroke. Includes bibliographical references.

The goal of this project was to validate a novel, mechanically loaded culture system, for the maintenance of cancellous bone explants ex vivo. The Zetos system utilised cancellous biopsies (5 mm high, 10 mm diameter) from ovine distal femora, bovine distal metacarpals and human femoral heads loaded daily for 300 cycles, at 1 Hz, giving 4,000 microstrain. Prior to culture, qualitative evaluation of bone density and overall morphology was conducted. These tissues were highly variable with bovine tissue being the most homogenous with regards to density and that each species contained different ratios of red and yellow marrow. The viability of bone cells and matrix synthesis were analysed using a variety of techniques. The outcome of this study was that diffusion constraints were the major limitation of this system. Chamber design was not optimal for bathing the explants, which was inferior to submerged static culture in centrifuge tubes. Harvesting the tissue created damage to the bone core that resulted in a maximal volume loss of 36%, which also encouraged unwanted growth of a fibrous-like tissue over the explant periphery in a wound-like response, possibly enhanced by foetal calf serum in the media. Nevertheless, 3H-glycine incorporation detected proteins synthesised during day 7 and 14 of culture. Collagen was the predominant protein synthesised. Fluorochrome labelling demonstrated human bone apposition during culture, but was unsuccessful with bovine and ovine tissue. Mechanically loaded explants were qualitatively more viable than unloaded disuse explants and submerged static controls. These results demonstrate cell viability at least 15 days post-harvest. If the limitations can be improved, then there is potential for this system to become routinely used in bone research. This system would provide a future means to allow bone-biomaterial interactions and interfaces to be studied, reducing, refining and replacing the need for animal experimentation.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Approximately 25% of all fractures in dogs involve the pelvis, of which 18-46% are iliac fractures. Conservative treatment can be performed in simple cases where minimum displacement occurs fractured fragments. However when there is severe displacement of the fragments, pelvic canal narrowing and involvement of weight bearing, surgical fixation is indicated. A variety of techniques have been described for the iliac fracture fixation. The highest percentage of successful cases can be attributed to the use of plates. Other methods used include pins, cerclage wire and compression screw. This study evaluated biomechanically the use of cortical allografts preserved in honey for the stabilization of transverse osteotomy of the ilium in dogs, as well as the use of hemicerclage wire isolated this cases, and compared the of two methods against the forces of bending. Were prepared cortical bone implants removed from humerus of dogs that eventually died for reasons not related to this research. The implants were preserved in honey for a period between 30 and 128 days. Were tested bilaterally thirteen canines pelves which held the body transverse osteotomy of the ilium. One hemipelvis of each dog was stabilized with a bone graft fixed by two hemicerclage wire and the contralateral hemipelvis with hemicerclage wire alone. To test the strength of flexion was used a manual compression machine where hemipelvis each was mounted on a wooden support. It was established that the time to stop the application of bending force would be when the fissure of the fracture suffer traction until half the width of the ilium (TMLI) or to failure. The strength of flexion needed to TMLI was significantly higher (P = 0.03) for hemipelves stabilized with bone implants (mean ± SD: 16.54 ± 5.29 kg) than for hemipelves stabilized with hemicerclage wire used alone (mean ± SD: 12.54 ± 4.01 kg). The force applied to fail was also statistically higher (P = 0.002) for hemipelves stabilized with bone implants (mean ± SD: 20.16 ± 7.3 kg) than in stabilized with hemicerclage wire used alone (mean ± SD: 12.54 ± 4.01 kg). The results showed that the use of cortical bone implants is a viable alternative for fixing the iliac osteotomy and is more resistant to strength of flexion in relation to the use of hemicerclage wire used in isolation.
Aproximadamente 25% de todas as fraturas em cães envolvem a pelve, sendo que 18-46% são fraturas ilíacas. O tratamento conservador pode ser realizado em casos simples onde ocorre deslocamento mínimo dos fragmentos fraturados. Entretanto, quando há deslocamento grave dos fragmentos, estreitamento do canal pélvico e comprometimento do suporte de peso, a fixação cirúrgica é indicada. Uma variedade de técnicas tem sido descrita para a fixação de fraturas ilíacas. A maior porcentagem de casos de sucesso pode ser atribuída ao uso de placas. Outros métodos usados incluem pinos, cerclagem de fio de aço e parafusos compressivos. Este trabalho avaliou biomecanicamente o uso de um implante ósseo cortical alógeno preservado em mel para a estabilização de osteotomia transversa de ílio em cães, bem como o uso de hemicerclagem de fio de aço isoladamente nestes casos, e comparou os dois métodos de estabilização de ílio frente às forças de flexão. Foram confeccionados implantes ósseos corticais alógenos retirados de úmeros de cães que vieram a óbito por motivos não relacionados com este trabalho. Os implantes foram preservados em mel por um período entre 30 e 128 dias. Foram testadas bilateralmente 13 pelves caninas nas quais se realizou osteotomia transversa do corpo do ílio. Uma hemipelve de cada cão foi estabilizada com o implante ósseo fixado por meio de duas hemicerclagens de fio de aço e a hemipelve contralateral com hemicerclagem de fio de aço isoladamente. Para testar a força de flexão, foi utilizada uma prensa de compressão manual onde cada hemipelve foi montada em um suporte de madeira. Foi estabelecido que o momento de interromper a aplicação da força de flexão seria quando a fenda da fratura sofresse tração até a metade da largura do ílio (TMLI) ou até a falha. A força de flexão necessária para TMLI foi significativamente maior (P=0,03) para hemipelves estabilizadas com implante ósseo (média ± SD: 16,54 ± 5,29 kgf) do que para as hemipelves estabilizadas com hemicerclagem de fio de aço usada isoladamente (média ± SD: 12,54 ± 4,01 kgf). A força aplicada para falhar também foi estatisticamente maior (P= 0,002) para as hemipelves estabilizadas com implante ósseo (média ± SD: 20,16 ± 7,3 kgf) do que nas estabilizadas com hemicerclagem de fio de aço usada isoladamente (média ± SD: 12,54 ± 4,01 kgf). Os resultados demonstraram que o uso de implante ósseo cortical alógeno é uma alternativa viável para a fixação da osteotomia ilíaca e apresenta maior resistência à força de flexão em relação ao uso de hemicerclagem de fio de aço usada isoladamente.

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O uso de materiais produzidos a partir de osso bovino tem sido proposto na confecção de implantes como pinos, placas e parafusos, por promoverem as mesmas funções de um enxerto ósseo, ou seja, serem osteoindutores e osteocondutores. Entretanto, aspectos estruturais e mecânicos devem ser estudados previamente ao uso in vivo de implantes de osso. Portanto, o objetivo desse estudo foi avaliar o comportamento mecânico, por meio do ensaio mecânico de flexão, de placas produzidas a partir osso cortical bovino, no reparo de fratura de tíbia de coelhos ex vivo. Para tal, 26 placas foram confeccionadas a partir de osso cortical bovino e conservadas em solução de sal a 150%. Foram utilizados três grupos para estudo: grupo GP (n=10), composto pelas placas ósseas; grupo GTP (n=16), tíbias de coelhos osteotomizadas e estabilizadas com placas ósseas e quatro parafusos; grupo GT (n=10), tíbias intactas. No ensaio biomecânico de flexão em três pontos, verificou-se a tensão máxima, deflexão máxima e rigidez. Os resultados foram submetidos ao teste de Kruskal-Wallis (p<0,05) e ao teste de Dunn. Comparando GT com o GTP, observou-se redução de 80% na tensão máxima. Também se notou redução de 87% na tensão máxima ao comparar GP com o GTP. Verificou-se que a placa de osso bovino possuiu maior tensão máxima que a tíbia do coelho. Houve redução a 52% na rigidez do GTP em relação ao GT. Não observou-se diferença significativa nesta propriedade entre GPT e GP. Observou-se diferença significativa entre os três grupos com relação à deflexão máxima, onde notou-se aumento de 100% e 30% nos grupos GTP e GP, respectivamente, em relação ao GT. Pode-se concluir que placas ósseas, no reparo de fratura de tíbia de coelhos ex vivo obtiveram propriedades mecânicas inferiores, quando comparada à tíbia intacta.

This investigation was conducted in two phases. The first phase aim was to describe the length, internal and external diameter, cancellous bone volume/extent and cortical bone thickness at predetermined locations in the radius and ulna in a cohort of skeletally mature and disease-free cat cadavers using radiography and computed tomography. This phase provided a morphometric description of normal cat antebrachii and served as reference for implant selection for the second phase. The aim of the second phase was to compare the biomechanical properties of three constructs for dual bone fixation (DBF) in a cat ex vivo antebrachial, fracture-gap model. Twelve cat cadaver antebrachii were radiographed to confirm normal skeletal appearance and maturity. Antebrachii were allocated for application of one of three constructs in an incomplete randomised block design (n=8/group); 10-hole 1.5/2.0mm Synthes® LCP radial plate (P); Plate and 1.2mm ulna intramedullary pin (PI) and Plate with an 8-hole 1.5/2.0mm orthogonal Synthes® ulna LCP plate (PP). Dual bone fixation constructs were PP and PI with P used as a control. Constructs were tested in non-destructive mediolateral and caudocranial four point bending, axial compression and finally axial compression to failure. Dual bone fixation constructs (PI and PP) were significantly stiffer (P< 0.001) than P in axial compression and caudocranial bending. There was no difference between PI and PP in axial compression and caudocranial bending (P=0.28) and no difference between any construct in mediolateral bending (P=0.72). The failure load was significantly greater for PP than PI (P<0.001) and PP and P (P<0.001) respectively. There was no difference between PI and P failure loads (P=0.45). In this cat ex vivo fracture-gap model, DBF (PP and PI) constructs were significantly stiffer in axial compression and caudocranial bending than radial plate alone. Dual plate constructs had a significantly higher failure load than any other construct.

Human mesenchymal stem cells (hMSCs) have been shown to have potential in regenerative approaches in bone and blood. Most protocols rely on their in vitro expansion prior to clinical use. However, several groups including our own have shown that hMSC lose proliferation and differentiation ability with serial passage in culture, limiting their clinical applications. Cellular prion protein (PrP) has been shown to enhance proliferation and promote self-renewal of hematopoietic, mammary gland and neural stem cells. With this work I tested the hypothesis that PrP decreased with cellular ageing of hMSC and was, at least in part, responsible for the loss of proliferation and differentiation seen with expansion in culture. Here I showed, for the first time, that expression of PrP decreased in hMSC following ex vivo expansion. When PrP expression was knocked down, hMSC showed significant reduction in proliferation and differentiation. In contrast hMSC expanded in the presence of small molecule 3/689, which stabilized PrP expression, extended lifespan up to 10 population doublings. These cells showed a 10 fold increase in engraftment levels in bone marrow 5 weeks post-transplant suggesting they were of superior quality. This was due to enhanced protection from DNA damage and enhanced cell cycle progression through upregulation of superoxide dismutase-2 (SOD2). The increase in SOD2 was dependent on PrP expression and suggested increased scavenging of reactive oxygen species as mechanism of action. My data point to PrP as a good target for chemical intervention to delay stem cell ageing.

Tissue engineering has emerged as a promising alternative to conventional orthopaedic grafting therapies. The general paradigm for this approach, in which phenotype-specific cells and/or bioactive growth factors are integrated into polymeric matrices, has been successfully applied in recent years toward the development of bone, ligament, and cartilage tissues in vitro and in vivo. Despite these advances, an optimal cell source for skeletal tissue repair and regeneration has not been identified. Furthermore, the lack of robust, functional orthopaedic tissue interfaces, such as the bone-ligament enthesis, severely limits the integration and biological performance of engineered tissue substitutes. This works aims to address these limitations by spatially controlling the genetic modification and differentiation of fibroblasts into a mineralizing osteoblastic phenotype within three-dimensional polymeric matrices. The overall objective of this project was to investigate transcription factor-based gene therapy strategies for the differentiation of fibroblasts into a mineralizing cell source for orthopaedic tissue engineering applications. Our central hypothesis was that fibroblasts genetically engineered to express Runx2 via conventional and biomaterial-mediated ex vivo gene transfer approaches will differentiate into a mineralizing osteoblastic phenotype. We have demonstrated that a combination of retroviral Runx2 overexpression and glucocorticoid hormone treatment synergistically induces osteoblastic differentiation and biological mineral deposition in primary dermal fibroblasts cultured in monolayer. We report for the first time that glucocorticoids induce osteoblastic differentiation in this model system by modulating the phosphorylation state of a negative regulatory serine residue (Ser125) on Runx2 through an MKP-1-dependent mechanism. Furthermore, we utilized these Runx2-genetically engineered fibroblasts to create mineralized templates for bone repair in vitro and in vivo. Finally, we engineered a heterogeneous bone-soft tissue interface with a novel biomaterial-mediated gene transfer approach. Overall, these results are significant toward the ultimate goal of regenerating complex, higher-order orthopaedic grafting templates which mimic the cellular and microstructural characteristics of native tissue. Cellular therapies based on primary dermal fibroblasts would be particularly beneficial for patients with a compromised ability to recruit progenitors to the sight of injury as result of traumatic injury, radiation treatment, or osteodegenerative disease.

L'ostéoporose est une maladie du squelette caractérisée par une dégradation de la qualité osseuse conduisant à une majoration du risque fracturaire. Le seul examen permettant actuellement de prédire ce risque est l'ostéodensitométrie à double rayonnement X (DXA) par la mesure de la densité minérale osseuse (DMO). Cependant, la DMO seule ne rend compte que de 40 à 70% de la variation de la résistance mécanique osseuse. Les 3 buts ce travail étaient : 1/ d'évaluer les rôles respectifs de la microarchitecture corticale et trabéculaire dans le comportement mécanique vertébral, 2/ d'évaluer le rôle propre de l'hétérogénéité de la microarchitecture trabéculaire et 3/ de décrire le comportement mécanique vertébral post-fracturaire et d'en identifier les déterminants. Nous montrons que la mesure de l'épaisseur de la corticale antérieure et de son rayon de courbure ainsi que la détermination de la variation régionale de la microarchitecture trabéculaire améliorent significativement la prédiction du risque fracturaire. Il existe une variation marquée du comportement mécanique vertébral après une fracture de grade 1 de Genant. La microarchitecture osseuse, et non la masse osseuse, explique les propriétés mécaniques vertébrales plastiques et élastiques post-fracturaires.

O objetivo do presente estudo foi avaliar, ex vivo, a influência da irradiação do laser Er,Cr:YSGG na resistência de união do material obturador à base de resina epóxica à dentina radicular, por meio do teste de push-out, e na interface dentina/material obturador, por meio de microscopia confocal a laser. Avaliou-se, ainda, a variação de temperatura externa da dentina radicular durante irradiação. Para tanto, 96 caninos foram instrumentados com sistema rotatório K3 até #45/.02 e irrigados com 2 mL de água destilada e deionizada entre os instrumentos utilizados. Os espécimes foram distribuídos em 3 grupos (n=32) em função do protocolo de irrigação final (10 mL): água destilada e deionizada, NaOCl 1% e EDTAC 17%. Em seguida, foram redistribuídos em 4 subgrupos (n=8), de acordo com a irradiação do laser: não irradiado, 2 W/20 Hz, 3 W/20 Hz e 4 W/20 Hz. Durante a irradiação, foram aferidos os valores máximo e mínimo de temperatura na parede radicular externa nos terços cervical, médio e apical e no ápice radicular. Após irradiação, os espécimes foram obturados com cimento AH Plus e guta-percha pela técnica de condensação lateral. Decorridos três vezes o tempo de endurecimento o tempo de do cimento, as raízes foram seccionadas transversalmente em slices de 1 mm de espessura. Dois slices de cada terço foram submetidos ao push-out em máquina universal de ensaios e o tipo de falha foi analisado em lupa estereoscópica e classificado em: adesiva ao material obturador, adesiva à dentina, mista, coesiva no material obturador e coesiva na dentina. O slice restante foi submetido à análise em microscopia confocal a laser, onde avaliou-se a porcentagem de perímetro da secção transversal do canal radicular com tags de cimento e a profundidade dos tags na interface dentina/material obturador de forma quali-quantitativa. Os dados de resistência de união (Mpa) e porcentagem de perímetro com tags foram analisados por meio do teste ANOVA; enquanto os dados da variação de temperatura (ºC) e profundidade dos tags (μm) por meio do teste de Kruskal-Wallis, ambos seguidos de teste de Tukey. A irradiação do laser Er,Cr:YSGG aumentou a resistência de união do cimento obturador à dentina, independente da irrigação final, sendo que os maiores valores foram obtidos para as potência de 3 W (4,02±1,32) e 4 W (4,18±0,98) e os menores valores para o grupo não irradiado (2,64±0,58) (p<0,05). A potência de 2 W (3,28±0,91) apresentou valores intermediários. A irrigação final com EDTAC 17% resultou em maiores valores de resistência de união (4,01±1,02) quando comparado à água destilada (3,11±1,09) e ao NaOCl 1% (3,47±1,18) (p<0,05). Em relação aos terços radiculares, o terço cervical (4,01±1,21) apresentou valor de resistência de união estatisticamente maior que o terço apical (3,04±0,89), enquanto o terço médio apresentou valores intermediários (3,54±1,15) (p<0,05). Em todos os grupos foi observado maior percentual de falhas adesivas e mistas. Nos grupos irradiados com 3 W [21,1 (14,1-27,7)] e 4 W [17,8 (11,9-23,7)] foi observado maior profundidade de tags do material obturador quando comparado ao grupo não irradiado [12,9 (9,0-20,0)]; a potência de 2 W promoveu valores intermediários [15,6 (11,7-23,3)] de profundidade de tags. Os maiores percentuais de perímetro com tags foram observados para os grupos irradiados, não havendo diferença estatística entre eles (p>0,05). O aumento da temperatura foi proporcional ao aumento da potência do laser, não excedendo 3 ºC. Concluiu-se que o laser Er,Cr:YSGG promoveu aumento da resistência de união do cimento à base de resina epóxica à dentina radicular, com maior formação de tags em todas as potências estudadas, principalmente quando associado à irrigação final com EDTAC 17%; o aumento da temperatura durante a irradiação não foi considerado elevado a ponto de prejudicar os tecidos adjacentes.
The aim of this study was to evaluate ex vivo the influence of Er,Cr:YSGG laser irradiation on the bond strength of an epoxy resin-based root canal sealer to root dentin, using the push-out test, and on the dentin/filling material interface, using confocal laser microscopy. The temperature variation on the outer root dentin during irradiation was also evaluated. For this purposes, 96 canines were instrumented with K3 rotary system up to the #45/.02 instrument, irrigating with 2 mL of distilled and deionized water at each change of instrument. The specimens were randomly assigned to 3 groups (n=32), according the final irrigation protocol (10 mL): distilled and deionized water, 1% NaOCl and 17% EDTAC. They were next reassigned into 4 subgroups (n=8), according to the laser irradiation parameters: non-irradiated, 2 W/20 Hz, 3 W/20 Hz and 4 W/20 Hz. During irradiation, the maximum and minimum temperatures were measured on the outer root dentin wall in the cervical, middle and apical thirds as well in the root apex. Following irradiation, the canals were filled with lateral condensation of AH Plus sealer and gutta-percha cones. After a period three times longer than the sealers setting time, the roots were sectioned transversally to obtain 1-mm-thick slices. Two slices from each third were subjected to a push-out test in a universal testing machine and the failure mode was analyzed with stereoscopic lens and classified as: adhesive to the filling material, adhesive to dentin, mixed, cohesive in the filling material and cohesive in dentin. The remaining slice was analyzed by confocal laser microscopy to evaluate the percentage of the perimeter of the root canal cross-section with sealer tags. The depth of tags at the dentin/filling material interface was evaluated in a quali-quantitative manner. Bond strength (MPa) data and the percentage of perimeter with tags were analyzed by ANOVA, while temperature variation (ºC) and tag depth (μm) were analyzed by Kruskal-Wallis test, both followed by Tukeys test. The Er,Cr:YSGG laser irradiation increased sealer bond strength to dentin, regardless of the final irrigation protocol. The highest values were obtained for 3 W (4.02±1.32) and 4 W (4.18±0.98) powers and the lowest for the non-irradiated group (2.64±0.58) (p<0.05). The use of 2 W power (3.28±0.91) resulted in intermediate values. Final irrigation with 17% EDTAC provided higher bond strength (4.01±1.02) compared with distilled water (3.11±1.09) and 1% (NaOCl 3.47±1.18) (p<0.05). Regarding the root thirds, the cervical third (4.01±1.21) presented significantly higher bond strength than the apical third (3.04±0.89), while the middle third presented intermediate values (3.54±1.15) (p<0.05). In all groups, there was a greater percentage of adhesive and mixed failures. In the groups irradiated with 3 W [21.1 (14.1-27.7)] and 4 W [17.8 (11.9-23.7)], it was observed a greater depth of filling material tags compared with the non-irradiated group [12.9 (9.0-20.0)]; 2 W power produced intermediate tag depth values [15.6 (11.7-23.3)]. The greatest percentage of canal perimeter with sealer tags was observed in the irradiated groups, with no statistically significant difference among them (p>0.05). The temperature rise was proportional to the increase of laser power, not surpassing 3 °C. It was therefore concluded that Er,Cr:YSGG laser irradiation increased the bond strength of an epoxy resin-based sealer to root dentin, with greater formation of sealer tags when for all tested powers, especially if combined with final irrigation with 17% EDTAC; temperature rise during irradiation was not considered high enough to cause harmful effects to the adjacent tissues.

O objetivo deste estudo foi avaliar ex vivo a resistência de união de pinos de fibra de vidro (FV) e pinos de fibra de carbono (FC) nos terços cervical, médio e apical do canal radicular, cimentados com dois tipos de cimentos resinosos: auto-adesivo (RX) e convencionais (CP). Quarenta caninos superiores foram separados em 4 grupos (n = 10) de acordo com o cimento e pino utilizado. Após a secção transversal dos dentes em fatias de 2mm de espessura, o teste de resistência adesiva (0,5mm/min) foi realizado nos terços cervical, médio e apical de cada espécime. Cinco espécimes de cada grupo foram analisados em microscopia eletrônica de varredura para observar o tipo de fratura. Os dados foram submetidos à 2-way ANOVA (Bonferroni, p< 0,05). Os resultados mostraram que os pinos de FV apresentaram os maiores valores de resistência adesiva, tanto cimentados com RX quanto com CP. Analisando especificamente o tipo de cimento, pinos cimentados com RX (FV e FC) apresentaram maiores valores de resistência adesiva (p<0,05) que CP. Para todos os grupos em estudo, a resistência de união foi maior no terço cervical, seguido pelo terço médio e apical. A análise de fratura mostrou predominância de fratura coesiva de pino para RX, sendo que para os pinos cimentados com CP houve predominância de fratura adesiva entre dentina/cimento e mista. Pode-se concluir que a resistência de união foi significativamente afetada pelo tipo de pino e também pelo tipo de cimento utilizado, sendo que os mais altos valores de resistência de união foram encontrados com a utilização de pinos de FV e cimento RX.
The objective of this study was to evaluate ex vivo the Bond strength of glass-fiber posts (GF) and carbon-fiber posts (CF) to cervical, medium, and apical thirds of root canals after luting with two types of resinous cements: Self-adhesive (RX) and conventional (CP). Forty maxillary canines were divided into 4 groups (n=10) according to the luting cement and fiber post used. After roots were perpendicularly sectioned in 2mm-thick slices, bond strength teste (0.5mm/min) were perform in coronal, midlle and apical thirds. Five specimens were selected from each group for analysis in Scanning Electron Microscopy to observe the type of fracture. The data were analyzed by 2-way ANOVA (Bonferonis test, p<0.05). The results showed that highest bond strength values to GF, for both luting cements (RX and CP). Regard the type of luting cement, posts (GF and CF) luting with RX had the best performance (p<0.05) that CP. For all groups, bond strength values were higher at cervical third, followed by midlle and apical thirds. The failure analysis demonstrated a predominance of post-cohesive failure for RX, and dentin-adhesive-cement and mixed failure for CP. It was concluded that bond strength was affected by the type of fiber post and type of luting cement, where the highest bond strength values were showed by GF-post and RX-luting cement.

O presente estudo teve como objetivo avaliar, por meio da análise da microdureza dentinária, teste push-out e microscopia confocal a laser, a influência da irrigação final do canal radicular com quitosana 0,2% e EDTA 15% sobre a adesão e penetração do cimento endodôntico através dos túbulos dentinários. Selecionaram-se 50 caninos superiores humanos, os quais após a remoção das coroas foram instrumentados com Reciproc® e hipoclorito de sódio 1%. Os espécimes foram distribuídos em 5 grupos (n=10), conforme protocolo de irrigação final: G1- irrigação convencional com EDTA 15%; G2- EndoVac com EDTA 15%; G3- irrigação convencional com quitosana 0,2%; G4- EndoVac com quitosana 0,2%; G5- grupo controle (sem irrigação final). As raízes foram obturadas pela técnica da condensação lateral com AH Plus e seccionadas transversalmente. O primeiro slice de cada terço foi destinado à resistência de união por cisalhamento por extrusão (RU), e o segundo slice às análises de microdureza dentinária e microscopia confocal a laser. Os dados obtidos foram submetidos à análise estatística descritiva com números absolutos, médias e desvios-padrões e análise de variância de Kruskal-Wallis seguido de teste de Tukey, com significância de 5%. Os resultados mostraram que em todos os grupos houve redução da microdureza dentinária de forma significantemente diferente ao controle (p=0,001). Na comparação intragrupos entre G1, G2 e G3 a penetração de cimento endodôntico no terço cervical foi maior que no apical (p<0,005). No G4 não houve diferença significante entre os três terços (p=0,481). Para a RU, considerando os terços cervical e apical, houve semelhança entre todos os protocolos de irrigação, sendo esses diferentes estatisticamente do grupo controle (p<0,001). Em geral, a resistência de união observada no terço cervical foi estatisticamente superior aos demais terços (p<0,001). No terço cervical dos espécimes em geral, houve predominância de falhas mistas (55,4%) e de falhas adesivas à obturação nos terços médio (58,8%) e apical (56,8%). Concluiu-se que o uso das soluções estudadas, independentemente do protocolo, reduziram a microdureza nos três terços radiculares, favoreceram a penetração do cimento através dos túbulos e aumentaram a adesão do cimento às paredes dentinárias em relação ao controle.
This study aimed to assess, through dentin hardness analysis, push-out tests and laser confocal microscopy, the influence of root canal final irrigation with 0.2% chitosan and 15% EDTA among endodontic cement adhesion and penetration through the dentinal tubules. Fifty human upper canines were selected. After crowns cut off and removal, roots were instrumented with Reciproc® and 1% sodium hypochlorite. The specimens were divided into 5 groups (n = 10) according to final irrigation protocol: G1- conventional irrigation with 15% EDTA; G2-EndoVac use + 15% EDTA; G3- conventional irrigation with 0.2% chitosan; G4-EndoVac use with 0.2% chitosan; G5- control group (without final irrigation). Roots were filled by lateral condensation technique with AH Plus and sectioned transversely. The first slice of each third was reserved for bond strength shear extrusion (SE) tests, and the second slice for dentin microhardness and laser confocal microscopy analysis. Data were submitted to descriptive statistical analysis with absolute numbers, means, standard deviations and Kruskal-Wallis variance analysis followed by Tukey\'s test. A significance level of 5% was considered. Results showed that in all groups there was a reduction in dentin microhardness, with significantly difference if compared with the control group (p=0.001). In intra-groups comparisons between G1, G2 and G3, cervical third sealer penetration was higher than apical third (p<0.005). In the G4 there was no significant difference between thirds (p=0.481). In the SE cervical and apical thirds comparisons, showed to be similar among all irrigation protocols, with statistically difference from the control group (p<0.001). The bond strength observed in the cervical third was statistically higher to the other thirds (p<0.001). In specimens cervical third, there was a predominance of mixed failures (55.4%) and adhesive failures on obturation in the middle (58.8%) and apical (56.8%) thirds. It can be concluded that regardless of the protocol, the studied solutions reduced the microhardness in the three root thirds, favored tubules cement penetration and increased dentin walls cement adhesion if compared to the control group.

OBJECTIVE: To determine the effects of vitamin C and insulin on osteoblasts harvested from neonatal mouse calvaria. To determine the effects of experimental media (vitamin C and insulin and a combination) on the ex-vivo live bone organ culture model and explore the capacity of addition of osteoblasts to allow for bone formation within a critical defect. To use the chick chorioallantoic membrane (CAM) model to explore bone formation within critical bone defect. METHODS: Osteoblasts were harvested from neonatal mice were tested using four types of experimental media: control DMEM, media prepared with 150 μg/ml vitamin C, 10 nM media, or a combination of both vitamin C, insulin, and a combination of vitamin C and insulin media. Cell were cultured for 18 days at 37°C. Neutral red was done to identify cellular activity and silver nitrate to detect calcium deposits. Two types of scaffolds were inserted into the defect: collagen membrane scaffold and NuOss (xenograft) with collagen scaffold. After 30 days the samples were collected for histological analysis. Neonatal mouse calvaria were harvested and a 2mm critical defect made on each calvaria. Each calvaria received a scaffold of collagen or NuOss with or without osteoblasts with one of three experimental media within the CAM model. After 7 days, the amnion membrane of the egg was dropped and a window was made. The calvaria with the scaffold samples were placed on the amnion membrane. The eggs were incubated for 10 days then the experiment was terminated. Calvaria were collected and processed for histological evaluation. RESULTS: Neutral red and silver nitrate of 2D in-vitro cells revealed calcium deposits in culture well using vitamin C media, cell cultured with insulin media showed calcium deposits and cell morphological change, and cell cultured with a combination of vitamin C and insulin media showed the most calcium deposits and morphological changed. Ex-Vivo samples with collagen scaffold had bone thickening but not enough nutrients for bone regeneration, despite the addition of cells. The collagen scaffold is a more suitable material than xenograft due to particle size. The CAM model showed new bone formation and new vessels were most abundant in areas closest to lining cells in collagen samples. Samples with additional osteoblasts added showed greater results. NuOss scaffold samples did not show the same bone formation or vessel growth. CONCLUSIONS: The results indicate and confirm the basic principles of tissue engineering. In order to have bone regeneration more cells allow for better results. The quality of the scaffold is important and should have stability as well as enough space for cellular migration and recruitment for new blood vessel to support regeneration of bone to its original state.

Parkinson's Disease (PD) is a devastating neurodegenerative disease affecting up to 1% of persons over age 60. PD is marked by a loss of dopaminergic neurons in the substantia nigra pars compacta and clinical symptoms that include bradykinesia, tremor at rest, and rigidity. Although the disease course is progressive, almost all patients experience a normal life span. The mainstay of therapy for PD is oral administration of L-3,4-dihydroxyphenylalanine (L-DOPA), the precursor compound for dopamine. However, the efficacy of L-DOPA therapy decreases with time, presumably due to fluctuations in L-DOPA levels within the central nervous system Several strategies for improving treatment for PD involve implantation of therapeutic cells in the brain, including allogeneic and xenogeneic fetal dopaminergic neurons and L-DOPA/Dopamine-producing cells. An alternative donor cell source for neural transplantation is adult stem cells from bone marrow stroma. Marrow stromal cells (MSCs) are multipotential stem cells that can differentiate into osteoblasts, adipocytes, chondrocytes, and myoblasts. Recent evidence suggests that MSCs are also stem cells for non-mesenchymal tissue. For example, human and rat MSCs express markers indicative of neurons when incubated with anti-oxidants in the absence of serum. Isolated simply by their adherence to plastic, human MSCs (hMSCs) can be easily obtained from the PD patient's iliac crest. They are then expanded and engineered in culture, and finally transplanted autologously into the patient We thus propose that MSCs can be used as cellular therapeutic vehicles for treatment of PD when transplanted into the brain. For treatment of PD, undifferentiated MSCs were gene engineered to produce L-DOPA by retroviral transduction to act as a 'biological pump' in the brain. In an alternate strategy, the potential of MSCs to develop into neuronal cells was studied both in culture and in the adult damaged and undamaged rat brain. The overall behavior and immunogenicity of MSCs in the rat brain was explored. We thus performed experiments both in the tissue culture dish and in animal models for PD to determine whether MSCs could be used for cell and gene therapy in PD
acase@tulane.edu

Osteocytes plays an important role in controlling and determining the function of the other cell types in bone, especially in response to mechanical loading and biochemical changes. The available data are still incomplete and much information derives from models that do not emulate the three dimensional structure of living bone. The use of the second generation Zetos™ system that enables long-term culture of trabecular bone ex vivo, together with the more traditional in vitro and in vivo models, will enable us to answer some of these knowledge gaps. A better understanding of osteocyte function specifically, and bone response to mechanical loading in general, can be obtained. In the first study, the second generation Zetos™ system was optimised and its software betatested in a series of trial experiments. The methodology and workflow used to prepare twenty four (24) ex vivo trabecular bone cores from a single bovine sternum, the maximum for each experiment, was described in great detail. Half of these bone cores had their marrow removed in the described experiment. Three (3) groups of four (4) bone cores, both with and without marrow, were loaded dynamically, with a load of 2,000 μstrain at 1 Hz, either for 100 cycles three times a day (100x3) or 300 cycles once a day (300x1) or not loaded (UL) respectively for 10 consecutive days. The bone cores without marrow showed significantly better increase in stiffness and response of their μCT histomorphometric parameters to loading over time, while their media have a consistently higher pH and lower ionic calcium (Ca²⁺) levels than that of the bone cores with marrow. Taken together, they indicated that removal of marrow in the bone cores was beneficial to the study of osteocytes in their native bone matrix environment using this Zetos™ system. This was possibly due to the improved fluid flow within the culture chambers when bone marrow was removed. In the second study, the use of the second generation Zetos™ system to investigate the application of a nano-engineered implant for local drug delivery in trabecular bone was described. The results demonstrated a consistent gradual release of a model drug, with a characteristic 3-dimensional distribution pattern, from the implant into the surrounding bone, over a 5-day period. The parameters that significantly affect the drug distribution were the flow rate of the bone culture medium, trabecular bone microarchitecture and mechanical loading of the bone core samples. This study demonstrated the usefulness of this system for drug release studies in ex vivo bone, which can be used to assist in the design of new drug delivery systems, and the optimisation of specific therapy delivery, in bone. The third study described the effect of exogenous human recombinant sclerostin (rhSCL) on the osteocytes of trabecular bone that were mechanically loaded ex vivo using this system. Bovine trabecular bone cores without marrow were subjected to daily episodes of dynamic loading and compared to unloaded bone cores or with the addition of exogenous rhSCL. Loaded bone showed an increase in apparent stiffness, calcein uptake (as a surrogate for Ca²⁺ influx), the Ca²⁺ and COOH-terminal telopeptide of type I collagen (β-CTX) levels in the perfusate, the mean osteocyte lacunar size (indicative of osteocytic osteolysis), and the expression of catabolic genes. These results indicated the direct contribution of osteocytes to bone mineral accretion and mechanical properties of trabecular bone, and support the concept that sclerostin acts directly on osteocytes to inhibit these effects via modulation of the osteocytic osteolysis process. In conclusion, the work done in this thesis was able to optimise the use of the second generation Zetos™ system for relevant bone and osteocyte-related research. This includes the investigation of drug delivery implants in large animal trabecular bone and as an additional tool for studying the osteocyte within its native lacuno-canalicular network. Further work using this system can assist in filling up important knowledge gaps on osteocytes mechanobiology and its role in the various metabolic functions of bone.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2019

碩士
國立清華大學
生物技術研究所
91
Stem cells hold considerable promise for the treatment of a number of diseases, although availability of stem cells must be overcome before their therapeutic potential can be realized. Human bone marrow mesenchymal stem cell (hMSC) has multi-potency of osteogenic, chondrogenic and adipogenic differentiation. It has limited life span and the source of bone marrow is difficult to obtain. Therefore, we design a bicistronic retroviral plasmid construct for reversible immortalization by a combination of SV40 T antigen and hTERT (human telomerase reverse transcriptase) genes with Cre-loxP recombination system. An immortalized cell line of hMSC was failed to establish possibly due to the number of drug-resistant clones selected was not enough. Because genes carried by a retroviral vector integrate into the chromosome randomly so that it may interrupt the genomic structure to lose some functionality of genes. Previous studies have revealed that cell proliferation and cytokine secretion were increased by low level light irradiation. Here, the light emitting diodes (LED) was used to study the effect of light stimulation on cell proliferation and gene expression of hMSC. We found that the rate of hMSC proliferation and CFU-F (colony forming unit-fibroblast) were significantly improved when cells were stimulated by first day irradiation only or daily irradiation at total energy density of 1.5 J/cm2 and 2.5 J/cm2, respectively. Numbers of cell proliferation and CFU-F stimulated by daily light irradiation were significant higher than that by light irradiation on day 0 only. The gene expression of cytokines, extracellular matrix and cell cycle regulatory protein in hMSC were analyzed by RT-PCR. The results showed that no significant change was observed in flt3, GM-CSF and collagen type I expression after LED irradiation. We found that c-fos and cyclin D1 expression were slightly decreased 30 min after LED irradiation, and increased 1 hour after irradiation. 24 hours after light irradiation no effect on c-fos, cyclin D1, and c-myc was seen. There was no difference on cell cycle with or without LED irradiation. The study suggested that low level light stimulation could enhance replicative potential of hMSC. However, the mechanism of light stimulation on the regulation of hMSC proliferation remains unsolved.

碩士
國立成功大學
醫學工程研究所碩博士班
90
Traditional bone fracture therapy requires an external fixation with bone mud at the side of wound. The disadvantages of this type of therapy is poor healing and heavy framing and assembling at wound side. With the advancement of tissue engineering and gene therapy today, bone fracture repair has become a very normative therapeutic technique, and it has ensured a rapid, effective and functional healing of complex fractures in the bone. In this study, we combined both techniques to develop an ex vivo gene therapeutic model to aid bone tissue repair. 　　A chemical vector, polyethylenimine (PEI) was used to carry bone morphogenetic protein-2 (BMP-2) gene into human neuro-teratocarcinoma (hNT) cell. BMP-2 can stimulate the regeneration of bone. In this study, immunocytochemistry and confocal microscopy were combined to examine the results after BMP-2 transfection. The fluorescent images showed that a great amount of bone morphogenetic protein existed in the cells. Western blotting was used to analyze the protein capacity in the cell culture medium. The results indicated that hNT cells can express BMP-2 protein in cytosol and generate BMP-2 in the cell culture medium. 　　In this study, a bone repair therapeutic system by ex vivo gene therapy was developed. The production of BMP-2 from hNT cells by non-viral gene-modification method was successfully demonstrated. In future, biodegradable matrix with hNT cells after transfection treatment will be implanted into bone fracture site to aid tissue regeneration and speed up the healing process.

BACKGROUND: Cells, growth factors and scaffold are the 3 fundamental factors currently proposed necessary for tissue regeneration. The use of these components has to be orchestrated precisely for ideal functional tissue formation. Growth factors enhance cellular activities that may lead to angiogenesis, cell proliferation and extracellular matrix biosynthesis. Due to the complexity of biochemical reactions a single growth factor may have limited effect. In order to explore a mixed profile of growth factors, a new biomaterial containing multiple growth factors derived from human Amniotic Membrane was chosen to compare with a known single growth factor (rhPDGF-BB). AIM: To compare the potential for enhanced bone formation by a morselized amniotic membrane suspension (AmnioSpark) with a known single cytokine PDGF-BB (GEM21,Lynch) under ex-vivo calvaria culture conditions. MATERIALS AND METHODS: 45 Calvaria from 7-9 day neonatal CD-1 mice were surgically harvested under sterile conditions. The calvaria were split through the mid sagittal suture to create 90 test specimens. A 2mm diameter critical size defect was created by biopsy punch thru the center of each calvarial specimen. This defect was bridged with a non-crosslinked type I collagen membrane of the same diameter to act as a scaffold. To compare AmnioSpark (AGF) potential for tissue regeneration against a known single cytokine PDGF-BB, the calvarial specimen were divided into six experimental groups: 1) Defect only, 2) Defect + scaffold, 3) Defect + scaffold + a single dose of (rhPDGF-BB ) a known bone stimulant, 4) Defect + Scaffold + 4 doses (day 0,3,5,7) of ( rhPDGF-BB), 5) Defect + scaffold + a single dose of (AGF) and 6) Defect + scaffold + 4 doses (day 0,3,5,7) of (AGF). Each test group had (N=5). A unique static tissue culture method was used with DMEM medium supplemented with ascorbic acid (150 ug/ml) and bovine serum albumin (5 mg/ml) without fetal calf serum to enhance bone formation for up to 7 weeks. Culture medium was changed every 2 days after day 3 and the harvested media was used for the following analyses: A) Alkaline phosphatase (ALP) as an osteoblastic activity indicator, and B) Tartrate Resistant Acid phosphatase (TRAP) as an osteoclastic bone remodeling activity indicator1. Macro photography and Scanning electron microscope (SEM) image analysis at different magnifications was performed to evaluate surface conditions. Histological analysis was performed with light microscope images on standard 4 um sections using H&E, Tri chrome, Picrosirius red and a fluorescence stain for RUNX2 as an osteoblast marker. RESULTS: With a single dose of test material ALP activity in the AGF group was significantly higher at 5 and 7 days. In addition ALP activity was significantly higher compared to all groups for up to 3 weeks post-application in the multiple dose AGF group (P<0.05). In contrast there was a dramatic decline in ALP in all other groups within the first week. TRAP activity was not detectable in any group. SEM images showed that osteoblast like cells accumulated and new tissue formation occurred over the surface of the scaffold obliterating the defect/membrane interface at 21 days with the AGF stimulus while in the PDGF-BB group the scaffold was still distinguishable from surrounding bone with no new tissue formation or cell migration . Histologic images confirmed an organized distribution of cells along the surface of the scaffold and new bone formation around the periphery of the defect in the AGF group (FIG42), while no bone formation or cell migration occurred in PDGF-BB group (FIG 35-38). Further diagnostic stains confirmed the presence of active osteoblasts (RUNX2)and the production of collagens I and II ( Masson Tri Chrome and PSR). CONCLUSION: Our results indicate that growth factors from amniotic extract (AGF) have the potential to enhance calvarial bone regeneration under an ex-vivo culture condition. These findings suggest that AGF could be a candidate for use as a new type of therapeutic material for regenerative medicine.

OBJECTIVE: To determine the effect of anti-inflammatory drugs (4- methoxybenzophenone) and (kavain) on bone formation and bone resorption models using a three-dimensional (3D) live calvarial bone METHODS: Utilizing neonatal mouse calvarial bones on a grid system with media designed to cause bone formation or resorption. These models were used to study the effect of 4- methoxybenzophenone and kavain on bone remodeling. The spent media were evaluated by quantitative analysis of tartrate resistant acid phosphatase (TRAP), alkaline phosphatase (ALP) activity and calcium release. The calvaria were stained with neutral red and silver nitrate for histological analysis. RESULTS: Kavain and 4-methoxybenzophenone affected the calvarial bone remodeling by inhibiting resorption. Kavain stimulated bone formation as shown by ALP activity and prevented the transformation of macrophages into osteoclasts as shown by neutral red staining. Kavain prevented resorption as shown by silver nitrate staining. Histological examination of kavain treated bone showed new osteoid formation. The other factor, 4-methoxybenzophenone, had inconsistent effects on bone. Neutral red staining showed no osteoclast differentiation and silver nitrate staining showed no resorption. However calcium release increased in the resorption model and calcium uptake increased in the formation model. There was no effect on ALP or trap activities in either model.

Tibial plateau fractures are associated with reduced lower limb function and poor patient reported outcomes. Allowing earlier postoperative weight bearing in could improve these outcomes. The central aim of this thesis was to improve upon methods for modelling the local mechanical environment in surgically repaired tibial plateau fractures to further understand the mechanics associated with fracture recovery, and to investigate long-term patient responses to immediate postoperative weight bearing. To achieve this, a combination of in silico, ex vivo, and in vivo studies were undertaken. Neuromusculoskeletal methods for estimating knee joint loads were improved and validated using data from instrumented knee replacements. These methods could be applied to tibial plateau fracture patients to better estimate the load applied to their fractures when walking immediately postoperative. Micro-CT imaging during concurrent mechanical loading methods were developed for determining the internal mechanical environment of the proximal tibia. Using these methods, the internal strains calculated using digital volume correlation were compared to subject specific finite element models of the same tibias. The strains within the proximal tibia were within the expected physiological region (200-3000 μϵ) and showed similar median strains to the FE models (error less than 35%). The experimental methods developed were applied to split tibial plateau fractures, showing that, under three bodyweights of load, there is little fracture fragment displacement (<0.3 mm) measured using image correlation. These results suggest that, with adequate mechanical fixation, it is unlikely that simple split fracture fragments would significantly displace from loads applied during basic activities of daily living. Two-year longitudinal patient responses to immediate postoperative weight bearing identified that patients showed similar joint kinematics to immediate postoperative weight bearing when compared to TPF patients who were not prescribed weight bearing. The work presented in this thesis enables further computational and cohort studies of TPF mechanics to assess the safety of postoperative weight bearing.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2022

No
Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) play roles in embryonic development and postnatal remodelling of the skin. Many indications suggest that BMP signalling regulates keratinocyte proliferation and differentiation. Chronic wounds have been shown to exhibit high levels of BMP ligands; however, the effect of BMP pathway modulation on human skin healing remains undefined. A human ex vivo skin wound healing model was used to analyse the expression of BMP signalling pathway components during healing and to investigate the effects of BMPs and the BMP antagonist Noggin on skin repair. Additionally, the effects of BMP signalling on keratinocyte proliferation, apoptosis and migration were tested using in vitro flow cytometry and ‘scratch’ migration assays, respectively. BMP receptor-1B (BMPR-1B) and downstream signalling protein phosphorylated-Smad-1/5/8 were highly expressed in healing epidermis. Treatment of human skin with exogenous BMPs impaired wound closure by reducing keratinocyte proliferation and increasing apoptosis. The BMP antagonist Noggin negated the inhibitory effects of BMP ligands, and when used alone, Noggin reduced keratinocyte apoptosis in the wound bed. In vitro, BMP ligands suppressed keratinocyte proliferation whilst Noggin stimulated proliferation. Keratinocyte migration was slowed following BMP treatment; in contrast, migration was significantly accelerated due to inhibition of BMP activity by either Noggin or BMPR-1B silencing. BMP signalling is inherently involved in wound healing. BMPs slow skin repair by suppressing keratinocyte proliferation and migration. Thus, modulation of BMP signalling using BMP inhibitors such as Noggin may serve as a new approach to promote cutaneous wound repair. Level of evidence: Not ratable.

碩士
國立東華大學
生物技術研究所
90
Umbilical cord blood (UCB) has been considered as an alternative source of primitive hematopoietic stem/progenitor cells to cure several malignant and nonmalignant diseases in clinic. However, the ex vivo expansion of cord blood hematopoietic progenitor cells may be necessary to engraft adult patients. Through secreted extracellular matrices and cytokine environment, bone marrow stromal cells can promote and regulate the self-renewal, differentiation, and proliferation of stem/progenitor cells. This study is designed to prepare different stromal-free condition media sourced from various stromal culture systems and to compare their effects on the ex vivo expansion of cord blood hematopoietic progenitor cells. The murine bone marrow stromal cell line, AC6.21, is a fibroblast-like attachment cell. In the LIF-treated AC6.21 stromal cell condition media（SCM-LIF）, several hematopoietic growth factors, including stem cell factor, interleukin-6 and FLT-3 ligand, were detected. It was found that Cytodex 1, as a carrier in the spinner flask culture system, could support the growth of AC6.21 and then the higher cell density was achieved. In the CB ex vivo expansion experiments, the total cell number was expanded 4.2 fold within 21 days. The AC6.21 SCM-LIF supported hematopoietic progenitor cells differentiating to lymphoid lineage. The human bone marrow stromal HS-5 cell line is a shear-sensitive fibroblast-like attachment cell, which can secrete several hematopoietic factors such as GM-CSF, SCF, IL-3, IL-6, and IL-8 without additional cytokine stimulation. The specific growth rate was 0.004 hr-1 and the glucose uptake rate was 5.5×10-8 mg/hr per cell in the T-flask culture system. Compared to the T-flask culture system, 2D MicroHex carriers in spinner flask culture system could result in increased cytokine production. In the ex vivo expansion experiments, the HS-5 condition medium supported the differentiation of hematopoietic progenitor cells to myeloid lineage.

碩士
國立陽明大學
口腔生物研究所
97
Bone grafting is often used clinically for treating osseous defects. In addition to many inherent limitations for bone grafting, its regenerative effect in large osseous defects are still less than ideal. Tissue engineering, by using scaffold, cell and growth factors, is an emergent regenerative technique with great potential. Several special concerns exist when applying tissue engineering for treating large defects. The scaffold needs sufficient mechanical strength to maintain space for regeneration. Cells with high regenerative capacity such as stem cells should be used. Gene therapy may be used for long term delivery of growth factors. The combination of proper components of tissue engineering may be needed for maximizing the regenerative effect. Previously we have developed a poly (lactide-co-glycolide) (PLGA)/collagen composite graft with good mechanical strength. Recent reports suggested that grafting with allogenic bone marrow stem cells may not induce immune and rejection response. Furthermore, the reparative and regenerative effects of the allogenic bone marrow stem cells were comparable to those of autogenic cells. Bone morphogenetic protein 2 (BMP2) is a powerful osteogenic factor. Lentiviral gene therapy has been used for long term expression of factors of interest. The purpose of this study was to establish a combined tissue engineering treatment system in preparation for the future testing in canine model with large osseous defects. The work in this study included preparation of PLGA/collagen composite scaffold, isolation canine bone marrow stem cells with stem cell phenotype, as well as construct lentiviral vector encoding BMP2 with osteoinductivity. Finally, the osteogenic effect of the established bone marrow stem cell-mediated ex vivo BMP2 lentiviral gene therapy system was proved in a SCID mice subcutaneous ectopic bone formation model. PLGA/collagen composite scaffold was used for the first time in ex vivo viral gene therapy in this study. The isolated canine bone marrow stem cells may be used in canine studies involve the using of allogenic bone marrow stem cells. The BMP2 lentiviral gene therapy system may be used for sustained delivery of BMP2 in future studies. Furthermore, the combined tissue engineering system established in this study may be used and tested in canine models with large osseous defect in the future.

There is a pressing need for better assessment of bone strength as current clinical tools do not directly measure bone mechanical properties, but offer only surrogate measures of bone strength. We conducted an ex-vivo study of emu bones to examine how two investigative devices, hr-pQCT and MRTA, compare to current clinical tools (DXA and QUS) in predicting true bone mechanical properties. We found that hr-pQCT parameters were able to assess bone strength as well as DXA and better than QUS, while MRTA was able to predict bone strength well in low-density but not high-density bones. Our results suggest that both hr-pQCT, which has the unique ability to specifically assess the various determinants of bone strength, and MRTA, which measures a bone mechanical property (stiffness), have great potential for use as clinical tools that can assess various components of bone strength not measured by current devices.

Bone metastases occur in more than 75% of patients with advanced breast cancer. Cancer in bone is associated with bone destruction and is responsible for high levels of morbidity and mortality but is notoriously difficult to treat. Bone destruction is also the primary cause of morbidity in patients with primary bone cancer, such as osteosarcoma, with metastatic spread to the lungs correlating with poor survival. Therefore, clearly new therapies are desperately required to target cancers in the bone. This study explored the therapeutic potential of gamma delta (Vγ9Vδ2) T cell based adoptive transfer using animal models of osteolytic breast cancer and osteosarcoma. Cytotoxic Vγ9Vδ2 T cells were expanded ex vivo from peripheral blood using IL-2 and zoledronic acid (ZOL). In vitro, expanded Vγ9Vδ2 T cells were cytotoxic against a panel of breast cancer and osteosarcoma cell lines and pre-treatment with ZOL sensitised all cancer cells to rapid killing by Vγ9Vδ2 T cells. Adoptive transfer of fluorescently labelled ex vivo expanded Vγ9Vδ2 T cells into NOD/SCID mice localised to cancer lesions in bone. Multiple infusions of Vγ9Vδ2 T cells reduced breast cancer growth, but had no effect on osteosarcoma growth in the bone marrow. However, in both cases, ZOL pre-treatment potentiated the anti-cancer efficacy of Vγ9Vδ2 T cells in bone, protected the bone from cancer-induced osteolysis and decreased the incidence of pulmonary metastases. Collectively these studies suggest this treatment regimen to be an effective immunotherapeutic approach for the treatment of primary and metastatic bone cancers.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2017

碩士
中原大學
生物醫學工程研究所
98
This study was undertaken to investigate the effects of serum metabolites of the Pueraria lobata (SMP) and diabetes on rat bone marrow-derived mesenchymal stem cells (rbMSCs). Type I diabetes rat model with pancreas β cell dysfunction was established by using alloxan. Bone mineral content, bone mineral volume, and bone mineral density of tibias and femurs in diabetic rats were decreased compared to those of normal rats as shown by Dual energy X-ray absorptiometry. The effect of diabetes on rbMSCs was explored and the results showed that hyperglycemia led to an inhibition on cell proliferation by 5~30 % and the number of colony forming-fibroblast with diameter greater than 0.6 cm. Intracellular ATP level of diabetic rbMSCs was lower than that of control group. Lactate dehydrogenase (LDH) release analysis showed that hyperglycemia did not seem to affect membrane integrity. No apparent LDH release was observed, which suggested that hyperglycemia did not cause necrosis. The effect of hyperglycemia on rbMSCs apoptosis was then investigated. The phenomena of apoptosis such as DNA fragmentation and chromatin condensation were found in cells under high glucose condition. Hyperglycemia was associated with 38~40 % increase in reactive oxygen species (ROS) level, and had significantly down-regulated the activities of superoxide dismutase (SOD) and catalase (CAT), which suggested that ROS might be responsible for apoptosis. The effects of hyperglycemia and high glucose (HG) treatment on both osteogenic and adipogenic differentiation potentials of MSCs were next detected. The results showed that hyperglycemia and HG treatment caused increased mRNA expression of PPARγ and triacylglycerol accumulation in rbMSCs during adipogenesis, and reduced expression of RUNX2 mRNA and alkaline phosphatase (ALP) activity during osteogenesis. The effects of SMP on diabetic rbMSCs were further investigated. Treatment of rbMSCs with SMP was associated with 11 % increase of control group and 13% increase of diabetes group in cell proliferation. SMP treatment increased the activities of the anti-oxidative enzymes, SOD and CAT, and decreased apoptosis as well as intracellular ROS level induced by hyperglycemia and HG treatment in rbMSCs. SMP treatment also decreased PPARγ mRNA expression during adipogenesis and increased RUNX2 and BMP-2 mRNA expression during osteogenesis in rbMSCs. In conclusion, diabetes rbMSCs exhibited the inhibitory effects on cell growth and osteogenic ability. The apoptosis and adipogenic capability of rbMSCs were increased by hyperglycemia. Furthermore, SMP treatment enhanced cell proliferation, antioxidant enzyme acitivities and osteogenic ability of diabetic rbMSCs. SMP treatment also decreased intracellular ROS level, apoptosis, and adipogenic capability of rbMSCs with HG treatment.

碩士
國立東華大學
生物技術研究所
93
Bone marrow mesenchymal stem cells secrete cytokines and play an important role in supporting hematopoiesis. In this study we firstly examined the effect of shear stress on the cytokine production of the bone marrow mesenchymal stem cells (BMSC), and then investigated the application of the conditioned media prepared from BMSC culture to the ex vivo proliferation and differentiation of hematopoietic stem/progenitor cells. The results indicated that shear stress led to significant increase in the production of the cytokines which were related to the hematopoiesis. The cytokines measured included interlukin-3, interlukin-6, interlukin-7, interlukin-8, interlukin-11, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, stem cell factor, and thrombopoietin. In the ex vivo expansion experiments, CD34+ cells were cultured with the conditioned media prepared from BMSC culture under various shear stresses. It was shown that, after 15 days’ expansion culture, the number of total cells, CD34+, CD33+ and Thy-1+ cells were increased several folds respectively and the hematopoietic stem/progenitor cells tended to differentiate to myeloid lineage. Based on the colony forming unit assay, it was observed that cells harvested from 15 days’ expansion culture still had the potential of differentiating to more mature cells.

博士
國立陽明大學
口腔生物研究所
98
Abstract Cell-based therapies using bone marrow-derived mesenchymal stem cells (MSCs) demonstrate great potential in bone regenerative therapies. Ex vivo expansion of MSCs is often required to generate adequate cell numbers in clinical applications. Senescence of MSCs occurs along with ex vivo passages and results in lower proliferation rate, loss of stemness and compromised therapeutic potential. However, currently no effective and safe method is available to solve the senescence problem. Previous studies indicated that a cell cycle regulator, p21, may be associated with cell senescence. We hypothesized that p21 may play an important role in the senescence of bone marrow-derived MSCs. The purpose of this research was to determine the role of p21 expression in the senescence of human bone marrow-derived MSCs. The results indicated that MSCs increased in p21 expression and became senescent along with ex vivo expansion. Lentiviral transduction of senescent MSCs with p21 shRNAs was able to increase their proliferation capacity, expression of stemness markers, and osteogenic potential in vitro. More importantly, the reduction of p21 expression enhanced the bone repair capacity of senescent MSCs in a mouse calvarial defect model. The p21-knockdowned MSCs showed increased telomerase activity and telomere length but maintained normal chromosome integrity and did not acquire tumorigenic potential. In conclusion, p21 plays an important role in senescence of human bone marrow-derived MSCs. The knockdown of p21 may become an effective and safe strategy to prevent or reduce the senescence of MSCs during ex vivo expansion.