Orientador: Eduardo Hochuli Vieira / Banca: Osvaldo Magro Filho / Banca: Idelmo Rangel Garcia Júnior / Banca: Michel Reis Messora / Banca: Thallita Pereira Queiroz / Resumo: OBJETIVOS: A proposta deste estudo foi avaliar a expressão de proteínas que participam da fase de osteoindução (VEGF, BMP-2 e CBFA1) durante o processo de regeneração óssea de defeitos criados em calvária de ratos e preenchidos com enxerto autógeno em bloco. MATERIAIS E MÉTODOS: Para o presente estudo foram utilizados 10 ratos adultos machos (Rattus norvegicus albinus, Wistar) que receberam dois defeitos ósseos de 5 mm cada, em calvária. Os defeitos ósseos constituiram dois grupos experimentais (n=10): Grupo controle (CONT) (defeitos preenchidos com o próprio coágulo); Grupo enxerto (ENX) (defeitos preenchidos com osso autógeno removido do defeito contralateral). Os animais foram submetidos a eutanásia nos períodos de 7 e 30 dias pós-operatórios. RESULTADOS: A análise quantitativa demonstrou formação óssea significativamente maior no Grupo ENX, no entanto, a presença das proteínas estudadas foi significativamente maior no Grupo CONT em ambos os períodos de observação. CONCLUSÃO: O enxerto ósseo autógeno cortical em bloco não expressou de forma significativa as proteínas osteoindutoras estudadas durante o processo de reparo / Abstract: AIMS: The proposal of this study was to evaluate the expression of proteins that act in osteoinduction (VEGF, BMP-2, CBFA1) phase during the bone defects regeneration created in rat calvaria and filled with autogenous bone graft in block. METHODS: For the present study, 10 adult male rats (Rattus norvegicus albinus, Wistar) had two 5mm- bone defects in calvaria. Bone defects constituted two experimental groups: CONTROL Group (defects filled with blood clot); GRAFT Group (defects filled with bone graft). Animals were sacrificed at 7 and 30 days post operative. RESULTS: Quantitative analysis showed significantly higher bone formation in Graft Group, however, the presence of studied proteins was significantly higher in Control Group in both observation periods. CONCLUSION: Autogenous cortical bone graft in block did not express the studied osteoinductive proteins during bone repair / Doutor

The cardiovascular system is an essential component of mammalian biology. It is a complex network of various tissues and structures with unique functions. The function of the cardiovascular system is to supply nutrients including oxygen to the various cells, tissues and organs within the body, and remove waste products from them. Given the importance of this role, it is not surprising that there are countless regulatory mechanisms at the molecular, cellular and tissue levels that are required to support this functional system. Perturbations in parts of this system are likely to lead to abnormalities, and thus give rise to cardiovascular-related diseases. Despite the currently expanding list of genes reported to be involved in a variety of cardiovascular-related diseases, including calcific aortic valve disease (CAVD), the functions and associated pathways of these factors in both normal and pathological physiology have yet to be fully understood, such as at the transcriptomic level. In this thesis, a genome-wide transcriptomic atlas of the healthy mammalian cardiovascular system was generated using the sheep as a large animal model. This atlas was generated using RNA-seq, with the aim of further understanding normal gene expression patterns in the context of the known physiology of healthy mammalian tissues. Through this work, I identified novel gene networks and detailed functional clustering of co-expressed genes with region-specific expression and specialised cardiovascular roles. One interesting cluster was highly expressed in the cardiac valves, and shared genes found in physiological bone development, such as bone morphogenetic protein 4 (BMP4), collagen type I alpha 2 (COL1A2), Sry homeobox 8 (SOX8) and bone gamma-carboxyglutamate protein (BGLAP), some of which have been implicated in vascular calcification. Further to this work, I studied the expression profiles of these key cardiovascular genes during development in the sheep from foetal to adult stages. In addition, I investigated the gene expression patterns of various key vascular calcification genes. These studies showed differential expression of genes in the different cardiovascular tissues, demonstrating transcriptional differences between these different tissues known to have different functions. CAVD involves progressive valve leaflet thickening and severe calcification, resulting in impaired leaflet motion. The in vitro calcification of primary rat, human, porcine and bovine aortic valve interstitial cells (VICs) is commonly employed to examine the mechanisms of CAVD. However, to date, no published studies have utilised cell lines to investigate this process Thus, in this project, I generated and evaluated the calcification potential of an immortalised cell line derived from sheep aortic VICs (SAVICs). This novel large animal in vitro model of CAVD was demonstrated to calcify under high calcium and phosphate conditions. Changes in the expression of key calcification genes during VIC calcification was also observed, including increased mRNA expression of bone markers Runt-related transcription factor 2 (RUNX2) and sodium-dependent phosphate transporter 1 (PiT1), and a concomitant decrease in matrix Gla protein (MGP) mRNA expression. In addition, the role of extracellular nucleotides and their receptors (P2 receptors), which have been previously shown to be important in bone and vascular calcification, were investigated using SAVICs in vitro. This study has shown that extracellular nucleotides, particularly adenosine 5’-triphosphate (ATP) and uridine 5’-triphosphate (UTP) and other agonists of P2 receptors, reduced VIC calcification in vitro. Moreover, the cutting-edge gene-editing technology, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9), was successfully applied to generate large animal models of cardiovascular-related diseases. In this project, I applied the CRISPR/Cas9 technology to edit ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and fibrillin 1 (FBN1) to generate two models of vascular calcification and Marfan Syndrome (MFS), respectively. In the ENPP1-edited animals, soft tissue calcification has been observed in the biallelic mutant and homozygous pigs. In this project, I have developed a range of novel in vitro and in vivo tools to advance the study of cardiovascular disease. These studies demonstrate that large animal models are highly valuable in the field of cardiovascular biology. The in vivo and in vitro experimental models described should facilitate detailed analysis of cardiovascular molecular biology and ultimately lead to therapies which will minimise the morbidity and mortality currently arising from cardiovascular pathology.

Devido as limitações relacionadas ao emprego do enxerto autólogo, o uso dos biomateriais poliméricos naturais tornou-se uma opção viável em terapias regenerativas do tecido ósseo. O objetivo desse trabalho é avaliar de forma qualitativa e quantitiva a contribuição da membrana de elastina utilizada isoladamente ou em associação a hidroxiapatita e proteína morfogenética óssea, no reparo de defeitos ósseos no crânio de ratos. Foram utilizados 49 ratos (Rattus norvegicus, Wistar), machos, com peso aproximado de 330 gramas e 4 meses de idade. Os animais foram submetidos ao procedimento cirúrgico para a criação do defeito ósseo no osso parietal esquerdo e divididos em 7 grupos com 7 animais cada. Os grupos foram implantados com os seguintes bioamateriais: grupo 1 controle (G1-C) sem implante, grupo 2 (G2-E24h) membrana de elastina 24 h, grupo 3 (G3-E24h/HA) membrana de elastina 24 h com hidroxiapatita, grupo 4 (G4-E24h/BMP) membrana de elastina 24 h com proteína morfogenética óssea, grupo 5 (G5-E96h) membrana de elastina 96 h, grupo 6 (G6-E96h/HA) membrana de elastina 96 h com hidroxiapatita, grupo 7 (G7-E96h/BMP) membrana de elastina 96 h com proteína morfogenética óssea. Após a morte indolor induzida em 6 semanas, as calotas cranianas foram retiradas para análise macroscópica, radiográfica, histológica e morfométrica. As análises macroscópicas, radiográficas e histológicas demonstraram a biocompatibilidade dos biomateriais utilizados. As médias e desvios-padrão do volume percentual relativo de osso neoformado nos defeitos cranianos dos grupos G1 a G7 foram 7,87±2,53; 24,01±0,55; 9,59±1,27; 31,31±6,37; 19,77±2,62; 7,31±2,43; 43,25±3,72, respectivamente. Os biomateriais mostraram-se biocompatíveis e o grupo 7 (G7-E96h/BMP) resultou na maior neoformação óssea. / Due to the limitations related to the use of autologous grafts, the use of natural polymeric biomaterials has become a viable option in regenerative therapies of bone tissue. The objective of this dissertation is to evaluate in qualitative and quantitative way the contribution of the elastin matrice used alone or in combination with hydroxyapatite and bone morphogenetic protein in the repair of bone defects in the skull of rats. Were use 49 Mices (Rattus norvegicus, Wistar), weighting approximately 330 grams and 4 months of age, were used. The animals were submitted to the surgical procedure to create the bone defect in the left parietal bone and divided into 7 groups with 7 animals each. The groups were implanted with the following biomaterials: group 1 control (G1-C) without biomaterial, group 2 (G2-E24h) 24 h elastin membrane, group 3 (G3-E24h/HA) 24 h elastin membrane with hydroxyapatite, Group 4 (G4-E24h/BMP) elastin membrane 24 h with bone morphogenetic protein, group 5 (G5-E96h) elastin membrane 96 h, group 6 (G6- E96h/HA) elastin membrane 96 h with hydroxyapatite, group 7 (G7-E96h/BMP) 96 h elastin membrane with bone morphogenetic protein. After painless death induced at 6 weeks, the skull caps were removed for macroscopic, radiographic, histological and morphometric analysis. Macroscopic, radiographic and histological analysis demonstrated the biocompatibility of the biomaterials used. The mean and standard deviations of the relative percentage volume of newly formed bone in the cranial defects of the G1 to G7 groups were 7,87±2,53; 24,01±0,55; 9,59±1,27; 31,31±6,37; 19,77±2,62; 7,31±2,43; 43,25±3,72, respectively. The implanted biomaterials were shown to be biocompatible and the group 7 (G7-E96h/BMP) resulted with greater bone neoformation.

Nas doenças que provocam perdas ósseas, destaca-se a utilização de enxerto ósseo autólogo como tratamento padrão-ouro. Entretanto, devido a comorbidades e limitação quantitativa, apresentam-se como alternativa o uso de biomateriais de elastina e hidroxiapatita, bem como a associação com a proteína morfogenética óssea. O objetivo desta pesquisa foi avaliar de forma qualitativa e quantitativa a contribuição da membrana de elastina utilizada isoladamente ou em combinação com a hidroxiapatita e a proteína morfogenética óssea no reparo de defeitos ósseos provocados experimentalmente no fêmur de ratos. Foram utilizados 77 ratos (Rattus norvegicus, Wistar), machos, com peso aproximado de 330 gramas e 4 meses de idade. Os animais foram submetidos ao procedimento cirúrgico para criação do defeito ósseo na superfície anterior da região supracondiliana do fêmur direito e separados em 7 grupos com 11 animais cada. Os grupos foram divididos da seguinte forma: Grupo controle (G1-C), ratos com defeito crítico induzido no osso femoral, sem preenchimento com implante. Grupo 2 (G2-E24/50), ratos com defeito crítico induzido no osso femoral preenchido com membrana de elastina hidrolisada durante 24h a 50°C; Grupo 3 (G3-E24/50/HA), defeito preenchido com membrana de elastina hidrolisada durante 24h a 50°C e hidroxiapatita. Grupo 4 (G4 E24/50/BMP), defeito preenchido com membrana de elastina hidrolisada durante 24h a 50°C e BMP. Grupo 5 (G5-E96/37), defeito preenchido com membrana de elastina hidrolisada durante 96 h a 37°C. Grupo 6 (G6-E96/37/HA), defeito preenchido com membrana de elastina hidrolisada durante 96 h a 37°C e hidroxiapatita. Grupo 7 (G7-E96/37/BMP), defeito preenchido com membrana de elastina hidrolisada durante 96 h a 37°C e BMP. Após a morte indolor induzida após 6 semanas, as peças anatômicas femorais foram retiradas para análise macroscópica, radiográfica, histológica, morfométrica e biomecânica. As médias e desvios-padrão do volume percentual relativo de osso neoformado no defeito femoral foram respectivamente 36,39±3,86 (G1); 66,40±3,69 (G2); 55,12±2,23 (G3); 58,46±1,79 (G4); 51,36±3,15 (G5); 71,28±4,26 (G6); 45,54±3,98 (G7). Os achados demonstraram biocompatibilidade, biodegradabilidade e osteorregeneração maior que o grupo controle nos biomateriais estudados. Os grupos com maior volume ósseo neoformado foram G2-E24/50 e G6-E96/37/HA. / In diseases that cause bone loss, the use of autologous bone graft is still a goldstandard treatment. However, due to comorbidities and quantitative limitation, the use of biomaterials of elastin, hydroxyapatite as well as the association with the bone morphogenetic protein are presented as alternatives. The objective of this research is to evaluate in a qualitative and quantitative way the contribution of the elastin membrane used alone or in combination with the hydroxyapatite and the bone morphogenetic protein in bone defects experimentally created in femur of rats. A total of 77 male rats (Rattus norvegicus, Wistar) weighing approximately 330 grams and 4 months of age were used. The animals were submitted to the surgical procedure to create the bone defect on the anterior surface of the supracondylar region of the right femur and were separated into 7 groups with 11 animals each. The groups were divided as follows: Control group (G1-C), rats with critical defect induced in the femoral bone without implant filling. Group 2 (G2-E24/50), defect filled with elastin membrane prepared for 24 h at 50°C. Group 3 (G3-E24/50/HA), defect filled with elastin membrane prepared for 24 h at 50°C and hydroxyapatite. Group 4 (G4-E24/50/BMP), defect filled with elastin membrane prepared for 24 h at 50°C and BMP. Group 5 (G5- E96/37), defect filled with elastin membrane prepared for 96 h at 37°C. Group 6 (G6- E96/37/HA), defect filled with elastin membrane prepared for 96 h at 37°C and hydroxyapatite. Group 7 (G7-E96/37/BMP) defect filled with elastin membrane prepared for 96 h at 37°C and BMP. After painless death induced after 6 weeks, the femoral anatomical pieces were removed for macroscopic, radiographic, histological, morphometric and biomechanical analysis. The mean and standard deviations of the relative percentage volume of newly formed bone in the femoral defect were respectively 36,39±3,86 (G1); 66,40±3,69 (G2); 55,12±2,23 (G3); 58,46±1,79 (G4); 51,36±3,15 (G5); 71,28±4,26 (G6); 45,54±3,98 (G7). The findings demonstrated biocompatibility, biodegradability and osseous regeneration greater than the control group in the studied biomaterials. The groups with the highest newly formed bone volume were G2-E24/50 and G6-E96/37/HA.

Methods for preparing an array of potential small molecule inhibitors of Bone Morphogenetic Protein Receptor 1b (BMPR1b) are described. Target molecules were prepared from two general classes: (1) N9-aryl-N6-ureidoadenines, and (2) dicarbamyl iodoacetamides. Recent data from the Peterson lab indicated that both classes might bind to BMPR1b and thus inhibit this key receptor. Docking studies performed using Sureflex Dock suggested the N9-aryl-N6-ureidoadenines would bind to the active site of BMPR1b. In addition antiproliferative activities of dicarbamyl iodoacetamides previously synthesized in the Peterson lab pointed to this moiety as an attractive target for structure activity relationship (SAR) development. Compounds were prepared in good to excellent yields and 40 derivatives were screened for antiproliferative activity. Of the N9-aryl-N6-ureidoadenine derivatives, N9-phenyl-N6-N-phenylureaadenine was most potent and exhibited selective activity against HeLa cells (IC50 = 11± 1 uM). Dicarbamyl iodoacetamide derivatives had similar activities compared to the previously reported compound (JRS-150).

The objective of this thesis is to design nanoparticle (NP)-based drug delivery systems suitable for treatment of bone diseases. Two types of nanocarriers, (1) polymer coated bovine serum albumin (BSA) NPs and (2) lipid based NPs (micelles and liposomes) were investigated. The BSA NPs were prepared by a coacervation method and stabilized with a polymer coating approach. For bone-specific delivery of bone morphogenetic protein-2 (BMP-2), a copolymer polyethyleneimine-graft-poly(ethylene glycol) conjugated with 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (PEI-PEG-thiolBP) was synthesized and used for coating the BSA NPs. The particle size and ζ-potential of the NPs could be effectively modulated by the processing parameters. All the NPs showed no or low cytotoxicity (except for a high concentration of PEI), and the NP encapsulated BMP-2 displayed full retention of its bioactivity. By encapsulating 125I-labeled BMP-2, the polymer-coated NPs were assessed for hydroxyapatite (HA) affinity; all NP-encapsulated BMP-2 showed significant affinity to HA as compared with free BMP-2 in vitro, and the PEI-PEG-thiolBP coated NPs improved the in vivo retention of BMP-2 compared with uncoated NPs. However, the biodistribution of NPs after intravenous injection in a rat model indicated no beneficial effects of thiolBP-coated NPs for bone targeting. Alternatively, micelles and liposomes were prepared with a conjugate of distearoylphosphoethanolamine-polyethyleneglycol with thiolBP (DSPE-PEG-thiolBP) to create mineral-binding nanocarriers. The thiolBP-decorated liposomes also displayed a stronger binding affinity to HA and a collagen/HA (Col/HA) scaffold and gave increased retention in the scaffold in a subcutaneous implant model in rats. Taking advantage of the high HA affinity of the BP-liposomes, a sustainable release system was developed by sequestering the liposomal drugs in the Col/HA scaffolds. Three different model drugs, carboxyfluorescein, doxorubicin and lysozyme, were used to evaluate the drug release profiles from the liposome-loaded scaffolds, and all showed a slowing effect of the BP on the release of the liposome-encapsulated drugs from the Col/HA scaffolds. This liposome-scaffold combination will provide a platform for the application of various therapeutic agents for bone regeneration. In conclusion, the BP-modified NPs showed strong mineral-binding affinity. Although the systemic bone targeting was limited by physiological barriers, these NPs are promising in local delivery and controlled release of bioactive molecules for treatment of bone diseases. / Chemical Engineering

The osteogenic effects of bone morphogenetic protein-2 (BMP-2) on human mesenchymal stem cells (MSCs) are less profound than expected as compared with rodent cells, and supraphysiological dose of BMP-2 is required to achieve desired clinical outcome. The mechanism for this phenomenon is unclear. In this study, we examined the effects of macrophages and noggin suppression on the BMP-2-induced osteogenesis of human bone marrow MSCs in vitro. Our data show that macrophage conditioned medium significantly decreased the migration capacity, metabolic activity and BMP-2-induced osteogenesis of MSCs. In addition, knocking down noggin by small interfering RNA (siRNA) also significantly decreased BMP-2-induced osteogenesis and proliferation of MSCs. In summary, our studies demonstrated that macrophages and knocking down the expression of noggin decreased BMP-2-induced osteogenesis of human MSCs in vitro. In the future, manipulation on macrophage activation and noggin expression may allow us to achieve higher BMP-2-induced osteogenesis that leads to better bone healing. / Experimental Surgery

Glioblastoma multiforme (GBM), the most common and malignant brain tumor, is characterized by high molecular and cellular heterogeneity within and among tumors. Parameters such as invasive growth, infiltration of immune cells and endothelial proliferation contribute in a systemic manner to maintain the malignancy. Studies in this thesis show that the expression of Sox2 is correlated with Sox21 in human gliomas. We demonstrate that an upregulation of Sox21 induces loss of proliferation, apoptosis and differentiation in glioma cells in vitro and in vivo and seems to correlate with decreased Sox2 expression. Induced expression of Sox21 in vivo significantly reduces the tumor size and increase the survival extensively, suggesting that Sox21 can act as a tumor suppressor Our studies indicate that the balance of Sox21-Sox2 in glioma cells is decisive of either a proliferative or a non-proliferative state. Several TGFß family members have an important role in glioma development. TGFß promotes proliferation and tumorigenicity whereas BMPs mostly inhibit proliferation. We demonstrate that BMP7 can induce the transcription factor Snail in glioma cells and that this reduces the tumorigenicity with a concomitant increase in invasiveness. Thus, we have identified a mechanism to the double-edged sword of proliferation versus invasiveness in GBM, the latter contributing to relapse in patients. Experimental gliomas were induced with the Sleeping Beauty (SB) model in mice with different immunological status of their T cells. The tumors that developed were either GBMs or highly diffuse in their growth, reminiscent of gliomatosis cerebri (GC). GC is a highly uncommon form of glioma characterized by extensive infiltrative growth in large parts of the brain. It is an orphan disease and today there is practically a total lack of relevant experimental models. The SB system would constitute a novel experimental model to study the mechanisms behind the development of diffusely growing tumors like GC. The presence or absence of T cells did not affect tumor development. The work in this thesis demonstrates that the proliferative and the invasive capacities of glioma cells can be dissociated and that the SB model constitutes an excellent model to study the highly proliferative cells in GBMs versus the highly invasive cells in diffuse tumors like .GC.

The Spemann organizer in amphibians gives rise to the anterior mesendoderm (AME) and is capable of inducing neural tissues. This inductive activity is thought to occur largely via the antagonism of Bone Morphogenetic Protein (BMP) signaling in the organizer. In the mouse, BMP antagonists Chordin and Noggin function redundantly in the AME and are required during forebrain maintenance. However, the timing of forebrain initiation and the function of BMP antagonism in forebrain initiation remained unclear prior to this study. In addition, the Transforming Growth Factor β (TGFβ) ligand Nodal patterns the forebrain via its function in the anterior primitive streak (APS), the precursor tissue of the AME. Whether BMP and Nodal signaling pathways interact has not been previously investigated. The goal of this dissertation was to investigate the cellular and molecular mechanisms involved in early mammalian forebrain establishment by embryonic and genetic manipulations. This study determined that forebrain initiation occurs during early gastrulation and requires signals from the AVE and AME. The AVE was identified as a source of active BMP antagonism in vivo, and the BMP antagonism supplied by exogenous tissues was capable to promote forebrain initiation and maintenance in the murine ectoderm. It is likely that BMP antagonism enhances forebrain gene expression via inhibiting posteriorization. This study further identified a possible crosstalk between BMP and Nodal signaling. Loss of Chordin or Noggin in combination with heterozygosity for Nodal or Smad3 results in holoprosencephaly. Molecular analyses suggest that the BMP-Nodal interaction occurs in the APS and/or the AME. Failure of this interaction results in an imbalance of BMP and Nodal signal levels that devastate APS and AME patterning during early forebrain establishment, ultimately leading to holoprosencephaly in mid-gestation. This interaction is likely to occur extracellularly, possibly by formation of a BMP-Nodal heteromeric complex. Furthermore, the spatiotemporal expression of phospho-Smad1/5/8, an effector of BMP signaling pathway, was characterized during early mouse embryogenesis. Distribution of phospho-Smad1/5/8 serves as a faithful readout of BMP signaling activity and helps to better understand how BMPs are involved in patterning early embryos. The implication of phospho-Smad1/5/8 expression in both wildtype and mutant embryos is also discussed. / Dissertation

<p>Articular cartilage is the thin, load-bearing connective tissue that lines the ends of long bones in diarthroidal joints, providing predominantly a mechanical function. Because cartilage is avascular and aneural, it has little capacity for self-repair if damaged. One repair strategy is through a functional tissue engineering approach using adipose-derived stem cells (ASCs). ASCs are an abundant progenitor cell source easily obtained through a minimally invasive liposuction procedure. When appropriately stimulated, ASCs have demonstrated significant potential for chondrogenic differentiation. Though studies have demonstrated the ability of ASCs to synthesize cartilage-specific macromolecules, a more thorough understanding of factors that modulate ASC chondrogenesis is required. Accordingly, the central aim of this dissertation was to study the chondrogenic response of ASCs to biochemical, biomechanical, and biomaterial factors.</p><p>We hypothesized that factors, other than TGF-beta and dexamethasone, would improve ASC chondrogenesis. BMP-6 emerged as a potent regulator of ASC chondrogenesis, particularly in early culture, as noted by significant upregulation of cartilage-specific extracellular matrix (ECM) genes and downregulation of cartilage hypertrophy markers.</p><p>Hypothesizing that biomechanical factors would accelerate the formation of cartilage-specific macromolecules, we designed and manufactured an instrument to apply dynamic deformational loading to ASC seeded constructs. Dynamic loading significantly inhibited ASC metabolism and downregulated cartilage-specific ECM genes. However, 21 days of dynamic loading induced the production of type II collagen, a principal component of articular cartilage.</p><p>We hypothesized that a biomaterial derived from cartilage would serve as a bioactive scaffold and induce chondrogenic differentiation. The novel, ECM-derived scaffold promoted the most robust differentiation of ASCs relative to both biochemical and biomechanical factors, particularly noted by a type II collagen-rich matrix after 28 days of culture. After 42 days of culture, biphasic mechanical testing revealed an aggregate modulus of 150 kPa, approaching that of native cartilage. These data suggest that the ECM-derived scaffold may retain important signaling molecules to drive differentiation or that ASC differentiation is dependent on proper cell anchorage.</p><p>In summary, we have shown that biochemical, biomechanical, and biomaterial factors have strong influences on the chondrogenic potential of ASCs. Optimization of these factors will ultimately be required to successfully engineer a functional tissue.</p> / Dissertation