In scaffold-based tissue engineering, control of scaffold degradation turns out to be a critical issue for reliable clinical applications. Degradation in this thesis refers to mass loss. Most of the present control methods take the approach of scaffold material modification and/or scaffold work environment adjustment to address this issue. The latter can easily get to its limit, and the former is not promising in the in-vivo implementation. This thesis proposed a new approach to control of scaffold degradation, that is, closed-loop and real-time control. To realize this approach, this thesis has tackled three important problems, namely (1) effects on degradation, (2) modeling of degradation, and (3) real-time measurement of degradation. This thesis is grounded to a biomaterial called chitosan, as it is widely used for building scaffolds. For the first problem, a statistical experiment was designed and a factorial analysis was conducted. For the second problem, a combined empirical-based and probabilistic-based approach was taken. For the third problem, a prototype of a sensor, which is based on the concept of carbon nanotube (CNT) conductive polymer, was built and tested. This thesis concludes (1) a joint thermal and enzymatic effect is significant on chitosan degradation, (2) the model for chitosan degradation is accurate, and (3) real-time measurement of mass loss of scaffold by means of carbon nanotube film is feasible. The major contributions of this thesis are (i) the proposal of the concept of the closed-loop control of degradation, (ii) a finding that there is a significant conjoint thermal and enzymatic effect on chitosan degradation in terms of mass loss, and (iii) a prototype of the novel CNT (carbon nanotube) chitosan film sensor for real-time measurement of mass loss of the scaffold. The significance of these contributions is that they give us confidence to a full development of the closed-loop and real-time degradation control approach. This approach appears promising to bring forth a transformative impact to clinic applications of scaffold-based tissue regeneration.

A limitation to engineering viable thick tissues (greater than a few hundred microns in thickness) has been the lack of vascularisation and a vascular supply. A key element in engineering such tissues is the generation of a supporting scaffold with a defined and wellcharacterized architecture. To date relatively little attention has been paid to characterization. The objective of this research was to develop well-characterized structures which will inform the rational design of the next generation of engineered thick tissues. Specifically, this research aimed to test combinations of various culturing environments, cell mono- and co-cultures, and scaffold architectures; develop improved imaging techniques and structural/spatial analytical methods to characterise porous polymer scaffolds; and use various spatial and morphological measures to quantify the relationships between scaffold geometric structure and cell distribution. Isotropic and anisotropic pore scaffolds were manufactured and then processed with nondestructive and destructive imaging methods, and characterised using image analysis methods to measure geometric parameters such as the degree of anisotropy/isotropy, porosity, and fractal parameters of pore and strut networks. Cells were introduced into scaffolds using a range of seeding methods and cultured in static and hydrodynamic environments. Quantification of the spatial cell distribution in cell-seeded scaffolds was done with first-order spatial statistics and fractal analysis. Findings comparing various destructive and non-destructive imaging methods found that cryotape cryohistology was the most accurate method for processing bare polymer scaffolds and eliminated histological artefacts common to other techniques. It was found with the various image analysis methods, surface and internal scaffold geometric architectures were strongly isotropic for porogen-fused porogen-leached scaffolds and anisotropic for TIPS scaffolds. For both isotropic and anisotropic pore scaffolds, collagen hydrogel infusion and droplet methods gave the highest cell seeding efficiencies (at 100% efficiency). The key finding in this study was that first-order spatial statistics and fractal analysis of cell distribution revealed that the geometric structure of the scaffolds had the strongest effect on spatial cell infiltration and distribution compared to the influence of culture environment or mono- and co-culture. Isotropic pore scaffolds had a higher level of cell distribution. Further work with optimizing the growth environment parameters, and utilizing collagen-infused cell-seeded scaffolds, may assist in achieving better cell growth. The work presented therefore provides the analytical basis for the rational design of tissue engineering scaffolds.

A limitation to engineering viable thick tissues (greater than a few hundred microns in thickness) has been the lack of vascularisation and a vascular supply. A key element in engineering such tissues is the generation of a supporting scaffold with a defined and wellcharacterized architecture. To date relatively little attention has been paid to characterization. The objective of this research was to develop well-characterized structures which will inform the rational design of the next generation of engineered thick tissues. Specifically, this research aimed to test combinations of various culturing environments, cell mono- and co-cultures, and scaffold architectures; develop improved imaging techniques and structural/spatial analytical methods to characterise porous polymer scaffolds; and use various spatial and morphological measures to quantify the relationships between scaffold geometric structure and cell distribution. Isotropic and anisotropic pore scaffolds were manufactured and then processed with nondestructive and destructive imaging methods, and characterised using image analysis methods to measure geometric parameters such as the degree of anisotropy/isotropy, porosity, and fractal parameters of pore and strut networks. Cells were introduced into scaffolds using a range of seeding methods and cultured in static and hydrodynamic environments. Quantification of the spatial cell distribution in cell-seeded scaffolds was done with first-order spatial statistics and fractal analysis. Findings comparing various destructive and non-destructive imaging methods found that cryotape cryohistology was the most accurate method for processing bare polymer scaffolds and eliminated histological artefacts common to other techniques. It was found with the various image analysis methods, surface and internal scaffold geometric architectures were strongly isotropic for porogen-fused porogen-leached scaffolds and anisotropic for TIPS scaffolds. For both isotropic and anisotropic pore scaffolds, collagen hydrogel infusion and droplet methods gave the highest cell seeding efficiencies (at 100% efficiency). The key finding in this study was that first-order spatial statistics and fractal analysis of cell distribution revealed that the geometric structure of the scaffolds had the strongest effect on spatial cell infiltration and distribution compared to the influence of culture environment or mono- and co-culture. Isotropic pore scaffolds had a higher level of cell distribution. Further work with optimizing the growth environment parameters, and utilizing collagen-infused cell-seeded scaffolds, may assist in achieving better cell growth. The work presented therefore provides the analytical basis for the rational design of tissue engineering scaffolds.

Human induced pluripotent stem cells (hiPSCs) have two main properties: pluripotency and self-renewal. Physical cues presented by biomaterial scaffolds can stimulate differentiation of hiPSCs to neurons. In this work, we develop and analyze a mathematical model of aggregate growth and neural differentiation on melt electrospun biomaterial scaffolds. An ordinary differential equation model of population size of each cell state (stem, progenitor, differentiated) was developed based on experimental results and previous literature. Analysis and numerical simulations of the model successfully capture many of the dynamics observed experimentally. Analysis of the model gives optimal parameter sets, that correspond to experimental procedures, to maximize particular populations. The model indicates that a physiologic oxygen level (~5%) increases population sizes compared to atmospheric oxygen levels (~21%). Model analysis also indicates that the optimal scaffold porosity for maximizing aggregate size is approximately 63%. This model allows for the use of mathematical analysis and numerical simulations to determine the key factors controlling cell behavior when seeded on melt electrospun scaffolds. / Graduate

Made available in DSpace on 2016-06-02T19:02:41Z (GMT). No. of bitstreams: 1 3965.pdf: 3863042 bytes, checksum: f995d87531cc46a186a0c566c0be57ea (MD5) Previous issue date: 2011-12-02 / This study aimed to evaluate the effects of low intensity laser therapy (LLLT) (830nm, 120J/cm2, 100mW) and implantation of Biosilicate® scaffolds, associated or not, in histological aspects, biomechanical properties of the bone callus and immunoexpression of proteins, growth and transcription factors related to different stages of bone repair, at 15, 30 and 45 days after surgery of bone defects induced in the tibia.of rats. For this, three studies were performed in which a total of one hundred and twenty male Wistar rats (3 months ± 280 g) were submitted to bilateral tibial defects and randomly distributed in four experimental groups with 30 animals each. In the first study the effects of the implantation of Biosilicate® scaffolds in bone defects of rats were investigated in two groups: bone defect group (GC) and bone defect treated with Biosilicate® scaffold group (GB). The implantation of the scaffold was performed subsequent to surgery of bone defect. Histological analysis revealed that animals of GB showed newly formed bone better organized at 30 and 45 days after surgery. The immunohistochemical analysis demonstrated that the Biosilicate® scaffold promoted a higher expression of COX-2 on days 15 and 30 after surgery, immunostaining positive of RUNX-2 in all periods, increased expression of RANKL on day 15 and positive immunoexpression of BMP-9 on the 45th day. However, the Biosilicate® scaffold did not increase the mechanical properties of bone callus. Thus, the implantation of Biosilicate® scaffold was effective in stimulating the repair of tibial defects, however, was not able to improve their mechanical properties. In the second study, the spatialtemporal changes in the process of bone healing in defects treated with LLLT were evaluated in two groups: GC and bone defect treated with laser group (GL). The laser treatment started immediately following the surgery of bone defects and have been 8, 15 or 23 sessions with an interval of 48 hours between them. The histological and morphometric analysis revealed that the GL showed better tissue organization at 15 and 30 days after surgery, and biggest area of newly formed bone at day 15. The immunohistochemistry showed that the LLLT promoted higher expression of COX-2 at day 15, immunostaining of RUNX-2 positive in all periods, higher immunoexpression of BMP-9 on day 30 and higher immunoreactivity of RANKL at day 15. However, the LLLT did not increase the biomechanical properties of bone callus. Thus, the LLLT improved the process of bone healing, but was unable to improve its biomechanical properties. In the third study the effects of the association of LLLT with implants of Biosilicate® scaffolds in bone healing were investigated in three experimental groups: GC, GB and Biosilicate® scaffold irradiated with laser group (GBL). The implantation of the scaffold was performed following the surgery of bone defect. The laser treatment started immediately after surgery and were performed 8, 15 or 23 sessions with an interval of 48 hours between them. At 15 days after surgery, the histological analysis revealed granulation tissue and newly formed bone juxtaposed to the surface of scaffolds in GB and GBL. Thirty days after injury, the GB and GBL had better organized newly formed bone compared to the CG. At day 45 was possible to observe granulation tissue in the defects of the GBL. In the GB, the peak of immunoexpression of COX-2 occurred on the 15th day and in the GBL, on the 30th day. The GB and GBL showed positive immunoexpression of BMP-9 up to 45th day after surgery, while RANKL immunoexpression was higher in the GBL at day 30. However, 30 and 45 days after injury, the animals of GB and GBL showed statistically lower values of maximum load compared to the CG. Thus, the association of the scaffold Biosilicate® with laser irradiation has osteogenic activity during the bone repair, however, the scaffold Biosilicate® associated or not with the laser irradiation is not effective to improve mechanical properties of the bone callus. Finally, we concluded that LLLT (λ = 830 nm, 120J/cm2) and implantation of Biosilicate® scaffolds, associated or not, were effective to stimulate the bone consolidation by improving the development of newly formed bone and activating immunoexpression of proteins, growth and transcription factors related to different stages of bone healing in tibial defects in rats. However, these therapeutic modalities associated or not, were unable to improve mechanical properties of the bone callus. / Este trabalho teve como objetivo avaliar os efeitos da terapia laser de baixa intensidade (LLLT) (830nm, 120J/cm2, 100mW) e do implante de scaffolds de Biosilicato®, associados ou não, nos aspectos histológicos, propriedades biomecânicas do calo ósseo e na imunoexpressão de proteínas, fatores de crescimento e de transcrição relacionados a diferentes etapas do reparo ósseo, ao 15º, 30º e 45º dia após a cirurgia de defeitos ósseos induzidos em tíbias de ratos. Cento e vinte ratos machos da linhagem Wistar (3 meses de idade ± 280 gramas) foram submetidos a defeitos tibiais bilaterais e distribuídos aleatoriamente em 4 grupos experimentais com 30 animais cada. No primeiro estudo investigaram-se os efeitos do implante de scaffolds de Biosilicato® em defeitos ósseos de ratos, a partir de dois grupos experimentais: grupo defeito ósseo controle (GC) e grupo defeito ósseo tratado com scaffold de Biosilicato® (GB). O implante do scaffold foi realizado em seguida à cirurgia de defeito ósseo. A análise histológica revelou que os animais do GB apresentavam osso neoformado mais organizado ao 30º e 45º dia após a cirurgia. A imunoistoquímica demonstrou que o scaffold de Biosilicato® promoveu maior expressão de COX-2 nos dias 15 e 30 de após a cirurgia, imunoexpressão positiva de RUNX-2 em todos os períodos, maior expressão de RANKL no 15º dia e imunoexpressão positiva de BMP-9 no 45º dia. Porém, o scaffold de Biosilicato® não aumentou as propriedades mecânicas do calo ósseo. Assim, o implante de scaffold de Biosilicato® foi eficaz em estimular o reparo de defeitos tibiais, porém, não foi capaz de melhorar suas propriedades mecânicas. No segundo estudo, foram avaliadas as mudanças temporais-espaciais no processo de reparo ósseo em defeitos tratados com LLLT, a partir de dois grupos experimentais: GC e grupo defeito ósseo tratado com laser (GL). O tratamento com laser iniciou-se imediatamente após a cirurgia dos defeitos ósseos e realizaram-se 8, 15 ou 23 sessões, com um intervalo de 48h entre elas. As análises histológica e morfométrica revelaram que o GL apresentou melhor organização tecidual aos 15º e 30º dias após a cirurgia, e maior área de osso neoformado no 15º dia. A imunoistoquímica mostrou que a LLLT promoveu maior expressão de COX-2 no 15º dia, imunoexpressão positiva de RUNX-2 em todos os períodos avaliados, maior imunoexpressão de BMP-9 no 30º dia e maior imunorreatividade do RANKL no 15º dia. Porém, a LLLT não aumentou as propriedades biomecânicas do calo ósseo. Assim, a LLLT melhorou o processo de consolidação óssea, mas não foi capaz de melhorar suas propriedades biomecânicas. O terceiro estudo investigou os efeitos da associação da LLLT com implantes de scaffolds de Biosilicato® na consolidação óssea, a partir de três grupos experimentais: GC, GB e grupo scaffold de Biosilicato® irradiado com laser (GBL). O implante do scaffold foi realizado em seguida à cirurgia de defeito ósseo. O tratamento com laser iniciou-se imediatamente após a cirurgia e foram realizadas 8, 15 ou 23 sessões, com um intervalo de 48h entre elas. Ao 15º dia pós-lesão a análise histológica revelou tecido de granulação e osso neoformado justapostos à superfície dos scaffolds no GB e GBL. Trinta dias após a lesão, o GB e GBL apresentavam osso neoformado mais organizado em comparação ao GC. Ao 45º dia, foi possível observar tecido de granulação nos defeitos do GBL. No GB, o pico de imunoexpressão da COX-2 ocorreu no 15º dia e no GBL, no 30º dia. Os GB e GBL apresentaram imunoexpressão positiva da BMP-9 até o 45º dia após a cirurgia, enquanto que para o RANKL, a imunoexpressão foi maior no GBL no 30º dia. No entanto, 30 e 45 dias após a lesão, os animais dos GB e GBL apresentaram valores estatísticamente menores de carga máxima em comparação ao GC. Assim, a associação do scaffold de Biosilicato® com o laser exerce atividade osteogênica durante o reparo ósseo, no entanto, o scaffold de Biosilicato® associado ou não a irradiação laser não é eficaz em melhorar as propriedades mecânicas do calo ósseo. Finalmente, podemos concluir que a LLLT e o implante de scaffolds de Biosilicato®, associados ou não, foram eficazes em estimular a consolidação óssea, melhorando o desenvolvimento de osso neoformado e ativando a imunoexpressão de proteínas, fatores de crescimento e de transcrição relacionados a diferentes etapas do reparo ósseo em defeitos tibiais em ratos. No entanto, estas modalidades terapêuticas, associadas ou não, não foram capazes de melhorar as propriedades mecânicas do calo ósseo em ensaio de flexão na posição de tração.