Filamin A is a well-characterised cytoskeletal protein which regulates cell shape and migration by cross-linking with actin. Filamin A mutations cause a number of human developmental disorders, many of which exhibit skeletal dysplasia. However, the molecular mechanisms by which Filamin A affects skeletal development are unknown. The transcription factor Runx2 is a master regulator of osteoblast and chondrocyte differentiation. Data presented in this thesis show that Filamin A forms a complex with Runx2 in osteoblastic cell lines. Moreover, it is demonstrated that Filamin A is present in the nucleus in several cell lines, including those of osteoblastic origin. The data presented show that the Filamin A/Runx2 complex suppresses the expression of the gene encoding the matrix-degrading enzyme, matrix metalloproteinase-13 (MMP-13), which is an important osteoblastic differentiation marker. ChIP assays were employed to demonstrate that endogenously expressed Filamin A associates with the promoter of the MMP-13 gene. In addition, Filamin A is not only located in the nucleus but also in the nucleolus, an important nuclear compartment involved in ribosomal RNA (rRNA) transcription. Ribosomal DNA promoter-driven reporter assays, Filamin A-knockdown experiments and exogenous Filamin A transfections demonstrated that Filamin A and Runx2 can repress ribosomal gene expression activity. Importantly, Filamin A is recruited to the human ribosomal DNA promoter, suggesting its direct involvement in the regulation of rRNA transcription. These findings reveal a novel role of Filamin A in the direct regulation of ribosomal gene expression. Finally, by using microarray technology, changes in gene expression were identified when Filamin A was downregulated. Some of the differentially expressed genes were known orchestrators of bone development. The data presented in this thesis strengthen the link between Filamin A and bone development and provide a molecular rationale for how Filamin A, acting as a regulator of gene expression, might influence osteoblastic differentiation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19569号 / 医博第4076号 / 新制||医||1017(附属図書館) / 32605 / 京都大学大学院医学研究科医学専攻 / (主査)教授 江藤 浩之, 教授 武藤 学, 教授 中畑 龍俊 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In the United States, nearly half a million bone graft operations are performed annually to repair defects arising from birth defects, trauma, and disease, making bone the second most transplanted tissue. Autogenous bone is the current gold standard for bone grafts; however it is in limited supply and results in a second injury at the donor site. A promising alternative is a tissue engineered bone graft composed of a biomaterial scaffold, pharmaceutics, and osteoprogenitor cells. One source of osteoprogenitor cells is bone marrow stroma, which can be obtained from the patient - minimizing the risk of an immune response - directed in vitro to proliferate, and differentiate into a bone-like tissue. To date, tissue engineered bone grafts have not been clinically effective; thus, strategies must be developed to improve efficacy. I hypothesize that to facilitate tissue healing in a manner similar to autogenous bone tissue engineering bone must possess a mineralized collagen matrix to support tissue integration, and angiogenic factors to stimulate vascular infiltration, and osteogenic factors to direct normal bone remodeling. I propose that these factors can be synthesized by osteoprogenitor cells in vitro when cultured under the appropriate conditions. Previous work has demonstrated that perfusion culture of osteoprogenitor cells within 3D scaffolds stimulates phenotypic markers of osteoblastic differentiation, but those studies did not determine whether the effects were a consequence of shear stress or increased nutrient availability. Consequently, this work has involved studies in a planar geometry, where nutrient effects are negligible. Three studies that characterize the effect of fluid flow on osteoblastic differentiation of osteoprogenitor cells are presented here. The objective of the first study was to determine the effect of shear stress magnitude on cell density and osteocalcin deposition. In this study, radial flow chambers were used to generate a spatially dependent range of shear stresses (0.36 to 2.7 dynes/cm2) across single substrates, and immunofluorescent techniques were used to assay cell phenotype as a function of shear stress. The objective of the second study was to determine the effect of the duration of fluid flow on cell density and phenotypic markers of differentiation. Here, parallel plate flow chambers were used to generate a single shear stress at the cell surface, and entire cell layers were assayed for expression of osteoblastic genes. The objective of the third study was to compare continuous and intermittent fluid flow strategies. In this study, a microprocessor-controlled actuator was added to the flow loop to periodically halt flow, and markers of mechanosensation and osteoblastic differentiation were measured. These studies demonstrated that shear stresses of 0.36 to 2.7 dynes/cm2 stimulate late phenotypic markers of osteoblastic differentiation but not cell proliferation. In addition, this osteogenic effect is sensitive to duration of fluid flow but insensitive to the magnitude of shear stress. Further, intermittent fluid flow enhances cell retention, biochemical markers of mechanotransduction, and synthesis of the angiogenic factor vascular endothelial growth factor (VEGF). Thus, these studies suggest that intermittent fluid flow may be an attractive component of a biodynamic bioreactor for in vitro manufacture of clinically effective tissue engineered bone grafts. Future studies will further investigate intermittent fluid flow strategies and three-dimensional studies with scaffolds suitable for bone tissue engineering. / Ph. D.

Bone is the second most transplanted tissue after blood and the need for bone graft materials continues to rise at an average annual growth rate of over 18%. An engineered bone substitute consisting of a bone-like extracellular matrix deposited on the internal pores of a resorbable biomaterial scaffold is postulated to stimulate normal bone remodeling when implanted in vivo. Part one of this engineering strategy, the deposition of bone-like extracellular matrix, can be achieved by the directed differentiation of progenitor cells such as bone marrow stromal cells (BMSCs). Part two of the engineering strategy, the biomaterial scaffold, can be fabricated with the appropriate mechanical properties using a synthetic polymer system with tunable properties like polyurethanes. Finally, BMSCs seeded within the biomaterial scaffold can be cultured in a perfusion flow bioreactor to stimulate osteoblastic differentiation and the deposition of bioactive factors. Using the three-part engineering strategy described, I hypothesize that the extracellular matrix produced by BMSCs can be modulated by two stimuli: the stiffness of the scaffold and perfusion flow. First, I propose that culturing BMSCs on polyurethane scaffolds with increasing stiffness will increase markers of osteoblastic differentiation. Secondly, I suggest that mechanically stimulating BMSCs with novel perfusion strategies will also increase markers of osteoblastic differentiation. In aim 1, a family of segmented degradable poly(esterurethane urea)s (PEUURs) were synthesized. The modulus of the PEUUR materials was systematically increased from 0.18 to 0.80 MPa by systematically increasing the molecular weight of the poly(ε-caprolactone) (PCL) soft segment from 1425 to 2700 Da. BMSCs were cultured on both rigid polymer films and on porous foam scaffolds to dissociate the effect of variation in polymer chemistry from the effect of scaffold modulus on cell phenotype. These studies demonstrated changes in osteoblastic differentiation as measured by prostaglandin E2 production, alkaline phosphatase activity (ALP) activity, and osteopontin gene expression. However, the increased levels of these phenotypic markers on the PCL 2700 material could not be attributed to scaffold chemistry or modulus. Instead, the differences may be related to polymer crystallinity or surface topography. In aim 2, novel dynamic perfusion strategies were used to investigate the influence of frequency on osteoblastic differentiation. BMSCs were seeded on porous foam scaffolds and exposed to both steady perfusion and pulsatile perfusion at 0.017, 0.050, and 0.083 Hz frequencies. The data presented here demonstrated that while some markers of osteoblastic phenotype such as ALP activity are enhanced by 0.05 Hz pulsatile flow over continuous flow, they are insensitive to frequency at low frequencies. Therefore, future studies will continue to investigate the effect of a larger range of frequencies. Additionally, fluid flow has also been shown to stimulate the deposition of bioactive factors such as BMP-2 and VEGF-A, and these growth factors are known to significantly enhance healing in bone defect models. Therefore, we plan to investigate the effect of dynamic flow strategies on the deposition of these bioactive factors. We propose that an engineered bone graft material containing a bone-like extracellular matrix and producing these growth factors will show more rapid formation of bone when implanted in vivo. / Ph. D.

Os eventos celulares e extracelulares que ocorrem durante o processo de osseointegração do titânio (Ti) são influenciados pelas propriedades físicas e químicas de sua superfície. Modificações bioquímicas de topografias complexas de Ti permitem o desenvolvimento de novas superfícies de implantes funcionalizadas com moléculas bioativas, visando a promover a osteogênese de contato e a osseointegração. O objetivo do presente estudo foi avaliar os efeitos, sobre a osteogênese in vitro, da funcionalização de microtopografia de Ti com concentrações distintas de peptídeo sintético análogo a uma seqüência de amino-ácidos do colágeno tipo I, relacionada a adesão e diferenciação celulares. Células osteogênicas primárias derivadas de calvárias de ratos foram plaqueadas sobre superfícies de Ti: 1) usinada e lixada (Usinado); 2) com microtopografia (Plus); 3) Plus com recobrimento de hidroxiapatita (Plus+HA); 4) Plus+HA, com baixa concentração de P-15 (P-15 low); 5) Plus+HA, com alta concentração de P-15 (P-15 high). Por períodos de até 21 dias, foram avaliados: morfologia celular e estágios de adesão e espraiamento celulares; viabilidade celular, proporção de células no ciclo celular e número total de células; imunolocalização de proteínas da matriz extracelular não-colágena; expressão de marcadores do fenótipo osteoblástico por reação em cadeia da polimerase em tempo real (Real-time PCR); atividade de fosfatase alcalina (ALP); proporção de células em apoptose e formação de matriz mineralizada. Avaliaram-se, também, os aspectos topográficos das superfícies, por microscopia eletrônica de varredura (MEV) de alta resolução, e o molhamento de superfície, pelo método da gota séssil. As superfícies Plus modificadas apresentavam camada superficial constituída por agregados de material acicular, os quais eram menos evidentes em P-15 high. Todas as superfícies eram hidrofóbicas, sendo que a funcionalização com P-15 proporcionava tendência à hidrofilicidade em equilíbrio. Após 4 h, observou-se que as superfícies com microtopografia apresentavam menor proporção de células nos estágios 3 e 4 de espraiamento quando comparadas com o Usinado (p<0,05). A viabilidade celular por MTT demonstrou valores maiores para as superfícies Plus modificadas aos 3 dias (p<0,05). Em 1 dia, acúmulos extracelulares de osteopontina (OPN) foram evidentes apenas sobre Plus+HA, P-15 low e P-15 high, com maior extensão em 3 dias. Em 7 dias, áreas imunomarcadas para sialoproteína óssea (BSP) eram menos extensas sobre Plus e Plus+HA. Para os grupos com microtopografia, foram observados valores de RNAm: menores para RUNX2 em 7 dias em comparação ao Usinado; para fosfatase alcalina (ALP), maiores em 10 se comparados a 7 dias; menores para BSP e maiores para OPN em 7 e 10 dias, quando comparados ao Usinado. Em 10 dias, observou-se redução significante (p<0,05) na atividade de ALP nas superfícies com microtopografia em comparação ao Usinado. Aos 14 dias, formações nodulares típicas de matriz mineralizada, marcadas para BSP em sua periferia, foram observadas apenas nos grupos Usinado e Plus. No entanto, a quantificação do vermelho de Alizarina (ARS) revelou valores maiores para as culturas sobre superfícies Plus modificadas em 14 e 21 dias (p<0,05). Concluiu-se que a microtopografia de Ti Plus, nanoestruturada com HA para funcionalização de P-15, altera o processo de aquisição do fenótipo osteogênico in vitro, resultando no aumento da formação de matriz calcificada, em padrão predominantemente diferente ao de típicas formações nodulares observadas sobre superfícies planas na microescala. / Surface functionalization of metallic surfaces with bioactive molecules has been developed aiming to promote specific cellular response at biomaterial-tissue interface. The present study evaluated the effects of surface functionalization of a microstructured titanium (Ti) surface with a synthetic peptide (P-15) analogue of the cell-binding domain of collagen I on key parameters of the progression of the osteogenic phenotype in vitro. Calvaria-derived osteogenic cells were plated on Ti disks: i) Machined; ii) with microtopography (Plus); iii) Plus with hydroxyapatite coating (Plus+HA); iv) Plus+HA with a low concentration of P-15 (P-15 low); v) Plus+HA with a high concentration of P-15 (P-15 high). High resolution SEM analysis showed that Plus exhibited a complex microtopography. In addition, a superficial layer of nano-sized needle-shaped HA was noticed for all modified Plus surfaces, although less apparent for P-15 high. Whereas all surfaces were hydrophobic at time zero, biofunctionalization showed a tendency to hydrophilicity at equilibrium. At 4 hours, Plus and modified Plus surfaces exhibited a lower proportion of spread osteogenic cells. At day 3, cells were less spread on the microtopographies, showing long cytoplasmic extensions. Epifluorescence revealed a large extracellular OPN accumulation for modified Plus surfaces. Although at day 3 cell viability was higher for modified Plus surfaces, at day 7 no major differences were detected among groups. Real time PCR showed for Plus and modified Plus surfaces: i) lower levels for RUNX2 at day 7 and for BSP at days 7 and 10, and higher OPN levels at days 7 and 10 compared with Machined; ii) higher ALP levels at day 10 compared with day 7. At day 10, Plus and modified Plus surfaces showed lower ALP activity compared with Machined. At days 14 and 21, higher proportions of Alizarin red stained areas were detected for cultures grown on modified Plus surfaces. The modification of Ti Plus surface by means of HA coating and functionalization with peptide P-15 alters the osteogenic potential of osteoblastic cell cultures, leading to an enhancement in mineralized matrix formation.

Os eventos celulares e extracelulares que ocorrem durante o processo de osseointegração do titânio (Ti) são influenciados pelas propriedades físicas e químicas de sua superfície. Modificações bioquímicas de topografias complexas de Ti permitem o desenvolvimento de novas superfícies de implantes funcionalizadas com moléculas bioativas, visando a promover a osteogênese de contato e a osseointegração. O objetivo do presente estudo foi avaliar os efeitos, sobre a osteogênese in vitro, da funcionalização de microtopografia de Ti com concentrações distintas de peptídeo sintético análogo a uma seqüência de amino-ácidos do colágeno tipo I, relacionada a adesão e diferenciação celulares. Células osteogênicas primárias derivadas de calvárias de ratos foram plaqueadas sobre superfícies de Ti: 1) usinada e lixada (Usinado); 2) com microtopografia (Plus); 3) Plus com recobrimento de hidroxiapatita (Plus+HA); 4) Plus+HA, com baixa concentração de P-15 (P-15 low); 5) Plus+HA, com alta concentração de P-15 (P-15 high). Por períodos de até 21 dias, foram avaliados: morfologia celular e estágios de adesão e espraiamento celulares; viabilidade celular, proporção de células no ciclo celular e número total de células; imunolocalização de proteínas da matriz extracelular não-colágena; expressão de marcadores do fenótipo osteoblástico por reação em cadeia da polimerase em tempo real (Real-time PCR); atividade de fosfatase alcalina (ALP); proporção de células em apoptose e formação de matriz mineralizada. Avaliaram-se, também, os aspectos topográficos das superfícies, por microscopia eletrônica de varredura (MEV) de alta resolução, e o molhamento de superfície, pelo método da gota séssil. As superfícies Plus modificadas apresentavam camada superficial constituída por agregados de material acicular, os quais eram menos evidentes em P-15 high. Todas as superfícies eram hidrofóbicas, sendo que a funcionalização com P-15 proporcionava tendência à hidrofilicidade em equilíbrio. Após 4 h, observou-se que as superfícies com microtopografia apresentavam menor proporção de células nos estágios 3 e 4 de espraiamento quando comparadas com o Usinado (p<0,05). A viabilidade celular por MTT demonstrou valores maiores para as superfícies Plus modificadas aos 3 dias (p<0,05). Em 1 dia, acúmulos extracelulares de osteopontina (OPN) foram evidentes apenas sobre Plus+HA, P-15 low e P-15 high, com maior extensão em 3 dias. Em 7 dias, áreas imunomarcadas para sialoproteína óssea (BSP) eram menos extensas sobre Plus e Plus+HA. Para os grupos com microtopografia, foram observados valores de RNAm: menores para RUNX2 em 7 dias em comparação ao Usinado; para fosfatase alcalina (ALP), maiores em 10 se comparados a 7 dias; menores para BSP e maiores para OPN em 7 e 10 dias, quando comparados ao Usinado. Em 10 dias, observou-se redução significante (p<0,05) na atividade de ALP nas superfícies com microtopografia em comparação ao Usinado. Aos 14 dias, formações nodulares típicas de matriz mineralizada, marcadas para BSP em sua periferia, foram observadas apenas nos grupos Usinado e Plus. No entanto, a quantificação do vermelho de Alizarina (ARS) revelou valores maiores para as culturas sobre superfícies Plus modificadas em 14 e 21 dias (p<0,05). Concluiu-se que a microtopografia de Ti Plus, nanoestruturada com HA para funcionalização de P-15, altera o processo de aquisição do fenótipo osteogênico in vitro, resultando no aumento da formação de matriz calcificada, em padrão predominantemente diferente ao de típicas formações nodulares observadas sobre superfícies planas na microescala. / Surface functionalization of metallic surfaces with bioactive molecules has been developed aiming to promote specific cellular response at biomaterial-tissue interface. The present study evaluated the effects of surface functionalization of a microstructured titanium (Ti) surface with a synthetic peptide (P-15) analogue of the cell-binding domain of collagen I on key parameters of the progression of the osteogenic phenotype in vitro. Calvaria-derived osteogenic cells were plated on Ti disks: i) Machined; ii) with microtopography (Plus); iii) Plus with hydroxyapatite coating (Plus+HA); iv) Plus+HA with a low concentration of P-15 (P-15 low); v) Plus+HA with a high concentration of P-15 (P-15 high). High resolution SEM analysis showed that Plus exhibited a complex microtopography. In addition, a superficial layer of nano-sized needle-shaped HA was noticed for all modified Plus surfaces, although less apparent for P-15 high. Whereas all surfaces were hydrophobic at time zero, biofunctionalization showed a tendency to hydrophilicity at equilibrium. At 4 hours, Plus and modified Plus surfaces exhibited a lower proportion of spread osteogenic cells. At day 3, cells were less spread on the microtopographies, showing long cytoplasmic extensions. Epifluorescence revealed a large extracellular OPN accumulation for modified Plus surfaces. Although at day 3 cell viability was higher for modified Plus surfaces, at day 7 no major differences were detected among groups. Real time PCR showed for Plus and modified Plus surfaces: i) lower levels for RUNX2 at day 7 and for BSP at days 7 and 10, and higher OPN levels at days 7 and 10 compared with Machined; ii) higher ALP levels at day 10 compared with day 7. At day 10, Plus and modified Plus surfaces showed lower ALP activity compared with Machined. At days 14 and 21, higher proportions of Alizarin red stained areas were detected for cultures grown on modified Plus surfaces. The modification of Ti Plus surface by means of HA coating and functionalization with peptide P-15 alters the osteogenic potential of osteoblastic cell cultures, leading to an enhancement in mineralized matrix formation.

Orientador: Luana Marotta Reis de Vasconcellos / Resumo: Os cimentos de silicatos de cálcio (CaSiO3) podem ser utilizados em tratamentos de reparo ósseo, tanto para aplicações médicas quanto odontológicas. A fim de atender às necessidades da engenharia de tecidos e aprimorar o leque de opções foram produzidos três cimentos de silicatos de cálcio utilizando α-wollastonita como precursora, juntamente com soluções ativadoras com potencial hidrogeniônico neutro, com três diferentes cátions, Na+, K+ e NH4+. A topografia superficial dos cimentos foi analisada por microscopia eletrônica de varredura com canhão de emissão por campo (MEV-FEG). Estudos in vitro, in vivo, relacionados a capacidade de interação com microrganismos e testes biomecânicos foram realizados para avaliar a influência de diferentes soluções ativadoras de fosfato e carbonato nos cimentos produzidos. Nos testes in vitro foram utilizadas células mesenquimais provenientes de fêmures de ratos. Após períodos pré-determinados foram realizados os testes de viabilidade celular, conteúdo de proteína total (PT), atividade de fosfatase alcalina (ALP) e formação de nódulos de mineralização. Para análise da interação microbiana a formação dos biofilmes monotípicos de S. aureus, P. aeruginosa e C. albicans foi mensurada. Posteriormente, os cimentos obtidos, a base de silicato de cálcio, foram submetidos ao estudo in vivo utilizando 20 ratos Wistars que passaram por procedimento cirúrgico para confecção de um defeito crítico de 3,0 mm nas tíbias direita e esquerda. Após a eutanásia, ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Calcium silicate cements (CaSiO3) can be used in bone repair treatments, both for medical and dental applications. In order to meet the needs of tissue engineering and improve the range of options, three calcium silicate cements were produced using αwollastonite as a precursor, with an activating solution of neutral potential hydrogen ionic, made of three different cations Na+, K+ and NH4 +. A superficial topography of the cements was analyzed by Field Emission Gun Scanning Electron Microscopy (SEMFEG). In vitro, in vivo studies related to the ability to interact with microorganisms and biomechanical tests were carried out to evaluate the influence of different phosphate and carbonate activating solutions in the cements produced. In in vitro tests, mesenchymal cells from rat femurs were used. After predetermined periods, cell viability tests, total protein content (PT), alkaline phosphatase activity (ALP) and formation of mineralization nodules were performed. For the analysis of microbial interaction, the formation of monotypic biofilms from S. aureus, P. aeruginosa and C. albicans was measured. Subsequently, the obtained cements, based on calcium silicate, were subjected to an in vivo study using 20 Wistars rats that underwent a surgical procedure to make a 3.0 mm critical defect in the right and left tibiae. After euthanasia, the pieces were submitted to histological, histomorphometric analysis and biomechanical flexion test of three points. The data obtained were statisti... (Complete abstract click electronic access below) / Mestre

Bone is the second most transplanted tissue in the body, with approximately 2.2 million bone graft procedures performed annually worldwide. Currently, autogenous bone is the gold standard for bone grafting due to its ability to achieve functional healing; however, it is limited in supply and results in secondary injury at the donor site. Tissue engineering has emerged as a promising means for the development of new bone graft substitutes in order to overcome the limitations of the current grafts. In this research project, the specific approach for bone tissue engineering involves seeding osteoprogenitor cells within a biomaterial scaffold then culturing this construct in a biodynamic bioreactor. The bioreactor imparts osteoinductive mechanical stimuli on the cells to stimulate the synthesis of an extracellular matrix rich in osteogenic and angiogenic factors that are envisioned to guide bone healing in vivo. Fluid flow, which exerts a hydrodynamic shear stress on adherent cells, has been identified as one of the strongest stimuli on bone cell behavior. It has been shown to enhance the deposition of osteoblastic matrix proteins in vitro, and is particularly important for the delivery of oxygen and nutrients to cells within large scaffolds suitable for bone tissue regeneration. In particular, dynamic flow profiles have been shown to be more efficient at initiating mechanotransductive signaling and enhancing gene expression of osteoblastic cells in vitro relative to steady flow. However, the molecular signaling mechanisms by which bone cells convert hydrodynamic shear stress into biochemical signals and express osteoblastic matrix proteins are not fully understood. Therefore, the overall goal of this research project was to determine the effect of dynamic fluid flow on mechanotransductive signaling and expression of bioactive factors and bone matrix proteins. In the first study, an intermittent flow regimen, in which 5 min rest periods were inserted during fluid flow, was examined. Results showed that signaling molecules, mitogen activated protein kinases (MAPKs) and prostaglandin E2, were modulated with the flow regimen, but that expression of bone matrix proteins, collagen 1α1, osteopontin, bone sialoprotein (BSP), and osteocalcin (OC), were similar under continuous and intermittent flow. Thus, this study suggested that variation of the flow regimen modulates mechanotranductive signaling. In the second study, four flow conditions were examined: continuous flow, 0.074 Hz, 0.044 Hz, and 0.015 Hz pulsatile flow. This study demonstrated that pulsatile flow enhances expression of BSP and OC over steady flow. Similarly, bone morphogenetic protein (BMP)-2 and -7 were enhanced with pulsatile flow, while BMP-4 was suppressed with all flow conditions, suggesting that the mechanism by which fluid flow enhances bone matrix proteins may involve the induction of BMP-2 and -7, but not BMP-4. In the third study, the molecular mechanism by which fluid flow simulates expression of BMPs was examined. Results from this study suggest that this mechanism may involve activation of MAPKs, but BMP-2, -4, and -7 are regulated through multiple different signaling pathways. Overall, the results from this research demonstrate that dynamic flow modulates mechanotransductive signaling and expression of osteoblastic matrix proteins by osteoblast cells. In particular, BMPs, important for formation in vivo, were shown to be induced by fluid flow. Therefore, this work may be beneficial in understanding and developing 3D perfusion culture systems for the creation of a clinically effective engineering bone tissue. / Ph. D.

Among the most common knee ligament injuries are those to the anterior cruciate ligament (ACL). Annually, approximately 350,000 people require surgical ACL reconstruction, accounting for more than $6 billion of health-care costs in the United States alone. An injured ACL loses its functions as it cannot heal with larger injuries and heals slowly with smaller ones. This may introduce complications, such as abnormal joint kinematics and deterioration, prior to complete rupture. Although the use of an autologous graft is the current gold standard for ACL reconstruction surgery, it is associated with donor site morbidity and a decrease in mechanical strength at the donor site. The use of allogenic grafts instead of autografts introduces the risk of disease transmission. Furthermore, integration of soft tissue grafts (e.g., hamstring tendon) to native bone is slow and risks graft pullout. To circumvent these limitations, tissue engineering seeks to fabricate suitable biomaterials that could replace the entire ACL, stimulate regeneration of the ligament tissue, and integrate with host bone tissue. Numerous efforts have led to the development of complex, multi-phased biomaterial scaffold designs that are intended to deliver an array of cell types and biological cues. Particularly, scaffolds that possess bone-regenerating biomaterials at the ends are envisioned to facilitate rapid integration with the femur and tibia. Electrospun fiber scaffolds continue to be regularly utilized for their high tensile strength, flexibility, and ability to bend. Nevertheless, fibrous scaffolds are inert and require the incorporation of trophic factors to guide tissue regeneration. Additionally, electrospun fibers are often densely packed, which can hinder cell infiltration and subsequent tissue formation. The objective of this work was to guide bone remodeling through the incorporation of trophic factors with 1) electrospun fiber scaffolds or 2) nanoparticles that could be combined with electrospun fiber scaffolds, and 3) to develop model three-dimensional fiber-hydrogel composites that support cell viability and proliferation. Two approaches were utilized to present the trophic factor bone morphogenic protein (BMP)-2 to stimulate bone formation. In the first approach, electrospun fibers were modified through the adsorption or covalent conjugation of BMP-2. These fibers exhibited increased BMP-2 concentrations with covalent conjugation over adsorption, and the incorporation of heparin into the fibers improved both adsorption and conjugation. Mesenchymal stem cells (MSCs) – that have the capacity to differentiate into osteoblastic cells – were able to attach and proliferate on all films yet appeared to do so to a greater extent on surfaces with higher heparin contents. Additionally, markers of osteoblastic differentiation were significantly higher on surfaces with covalently conjugated BMP-2 than on those with adsorbed BMP-2. In the second approach, a nanoparticle system was produced to control BMP-2 delivery and release. Importantly, this flexible system can be fabricated separately, and then combined with a scaffold for tissue regeneration. In this approach, BMP-2 was combined with chitosan nanoparticles through adsorption, encapsulation, or covalent conjugation. The particular BMP-2 incorporation technique had no significant effect on BMP-2 incorporation efficiencies, but affected particle size and BMP-2 release kinetics. Specifically, covalent conjugation method caused the aggregation of particles while adsorption method allowed the most sustainable release. MSCs cultured in the presence of the different particles survived and proliferated, but only particles with adsorbed BMP-2 stimulated osteoblastic differentiation. Finally, three-dimensional fiber-hydrogel composites of various models were fabricated to mimic the complexity of full-sized scaffolds for ACL regeneration, and to study cell infiltration, differentiation, and tissue formation. A collagen hydrogel phase was introduced to electrospun fiber scaffolds using different approaches. MSCs seeded within a thin collagen layer were able to proliferate, sense underlying substrate and spread according to fiber orientation, while those within thicker layers were not. Additionally, cells initially present in only the collagen phase infiltrated to the fiber phase. These results demonstrate that minor changes in fabrication steps to combine the two phases could significantly alter cell function during the formation of three-dimensional fiber-hydrogel composites for tissue regeneration. / Doctor of Philosophy / The anterior cruciate ligament (ACL) is one of four ligaments that connect the thigh bone to the shin bone and stabilize the knee. Injuries to the ACL often occur during high impact sports, and ruptures can necessitate surgical intervention. ACL reconstruction surgery involves drilling tunnels through the ends of leg bones, deploying the tissue graft through the knee joint and bone tunnels, and anchoring it within the bone tunnels. The most common grafts are autografts that use tendons of the patient's own body or allografts that are obtained from cadavers. The complications associated with autografts include pain at the site of tissue harvest, while allografts risk disease transmission. Additionally, directly affixing a soft tissue graft (e.g., the hamstring tendon) to bone within the bone tunnel suffers from slow tissue integration and risk of pull-out. Tissue engineering is a field that seeks to develop devices to direct the regeneration of damaged tissues and organs. In the context of ACL repair, it seeks to achieve a biomaterial device with the properties of ACL, that can both guide the regeneration of ligament tissue and facilitate integration with bone tunnels, eliminating the need for autografts and allografts and their associated risks. Toward the development of an engineered ACL, this work focuses on improving graft-to-bone integration. In the first project, fibrous materials are surface-modified with bone morphogenetic protein (BMP)-2 (a bone-forming protein), and then tested for their ability to stimulate formation of a bone-like tissue in cell culture. In the second project, the deployment of BMP-2 either on the surface of or within nanoparticle delivery vehicles is evaluated as an alternative strategy to stimulate bone-like tissue formation. The third project explores the inclusion of a hydrogel phase to facilitate cell infiltration and bone-like tissue formation within fibrous materials. Together these studies provide insights into how the architecture of the engineered tissue and the deployment of bone-forming proteins can be used to enhance ACL regeneration.

Long-term exposure to pharmacological doses of glucocorticoids has been associated with the development of osteopenia and avascular necrosis. Bone loss may be partially attributed to a steroid-induced decrease in the osteoblastic differentiation of multipotent progenitor cells found in the bone marrow. In order to determine if there is a change in the osteogenic potential of the bone marrow stroma following glucocorticoid treatment, Sprague-Dawley rats were administered methylprednisolone for up to six weeks, then sacrificed at 0, 2, 4, or 6 weeks during treatment or 4 weeks after cessation of treatment. Femurs were collected and analyzed for evidence of steroid-induced osteopenia and bone marrow adipogenesis. Although glucocorticoid treatment did inhibit bone growth, differences in ultimate shear stress and mineral content were not detected. The volume of marrow fat increased with increasing duration of treatment, but returned to near control levels after cessation of treatment. Marrow stromal cells were isolated from tibias, cultured in the presence of osteogenic supplements, and analyzed for their capacity to differentiate into osteoblast-like cells in vitro. Glucocorticoid treatment diminished the absolute number of isolated stromal cells, but did not inhibit the relative levels of bone-like mineral deposition or osteocalcin expression and secretion. Although pharmacological glucocorticoid levels induce bone loss in vivo, physiologically equivalent concentrations have been shown to enhance the formation of bone-like tissue in vitro. However, glucocorticoids have also been reported to inhibit proliferation and type I collagen synthesis in marrow stromal cell cultures. In order to assess the effects of intermittent dexamethasone treatment on the progression of osteogenesis in rat marrow stromal cell culture, this synthetic glucocorticoid was removed from the culture medium after a variable period of initial supplementation. Cell layers were analyzed for total cell number, collagen synthesis, phenotypic marker expression, and matrix mineralization. Prolonged supplementation with dexamethasone decreased proliferation, but did not significantly affect collagen synthesis. Furthermore, increased treatment duration was found to increase bone sialoprotein expression and mineral deposition. The duration of glucocorticoid treatment may be a key factor for controlling the extent of differentiation in vitro. / Master of Science