In pathological states such as osteoarthritis, the complex metabolic balance of cartilage is disrupted, leading to a loss in the integrity and biomechanical function of cartilage. Osteoarthritis affects more than 20 million Americans, costing the United States economy over $60 billion yearly. Risk factors for osteoarthritis include age, excessive joint loading, and joint injury. Tissue engineering offers a potential solution for the replacement of diseased and/or damaged cartilage. Unfortunately, plentiful donor cell populations are difficult to assemble, as chondrocytes have a well characterized lack of expansion potential. Mesenchymal progenitor cells offer an alternative with a high expansion potential capable of supplying large quantities of cells. Using an immature bovine model, the chondrogenic differentiation of articular chondrocytes and bone marrow stromal cells was found to be scaffold, media and mechanical stimulation dependent. TGF-beta signaling participated in the response of articular chondrocytes to dynamic compressive loading, as well as enhanced the chondrogenesis of bovine BMSCs, through interactions between loading and TGF-beta/Smad signaling. Also, dynamic loading altered gene expression, matrix synthesis rates and intracellular phosphorylation for bovine BMSCs. However the response of the cells to dynamic loading depends on both media supplementation and the duration of unloaded culture. These studies establish signaling through the TGF-beta pathway as a mechanotransduction pathway for chondrocytes and chondroprogenitors in 3D culture.

Current cartilage repair methods are ineffective in restoring the mechanical and biological functions of native hyaline cartilage. Therefore, using the paracrine actions of stem cell therapies to stimulate endogenous cartilage regeneration has gained momentum. Adipose stem cells (ASCs) are an attractive option for this endeavor because of their accessibility, chondrogenic potential, and secretion of factors that promote connective tissue repair. In order to use the factors secreted by ASCs to stimulate cartilage regeneration, the signaling pathways that affect postnatal cartilage development and morphology need to be understood. Next, approaches need to be developed to tailor the secretory profile of ASCs to promote cartilage regeneration. Finally, delivery methods that localize ASCs within a defect site while facilitating paracrine factor secretion need to be optimized. The overall objective of this thesis was to develop an ASC therapy that could be effectively delivered in cartilage defects and stimulate regeneration via its paracrine actions. The general hypothesis was that the secretory profile of ASCs can be tailored to enhance cartilage regeneration and be effectively delivered to regenerate cartilage in vivo. The overall approach used the growth plate as an initial model to study changes in postnatal cartilage morphology and the molecular mechanisms that regulate it, different media treatments and microencapsulation to tailor growth factor production, and alginate microbeads to deliver ASCs in vivo to repair cartilage focal defects.

Mesenchymale Stammzellen bieten eine interessante Option in der regenerativen Medizin, da sie praktisch unlimitiert verfügbar sind. Um das Verhalten von humanen MSC zu studieren, werden Untersuchungen momentan an immundefizienten Mäusen durchgeführt, deren Verwendung kostenintensiv und aufwendig ist. Fra-gestellung war, ob durch Immunisolation (Alginat, Dialyseschlauch, Diffusionskammer) die Knorpel erhaltenden -, bzw. bildenden Eigenschaften von MSC-Konstrukten ebenso gut in immunkompetenten Mäusen untersucht werden können. Gleichzeitig sollte geprüft werden, ob die mit einer Immunabschirmung einhergehende Reduktion der Zellversorgung und damit die Annäherung an die Gelenksituation ihre Mineralisierung vermindern kann und ob Mauszellen für eine Veränderung der vordifferenzierten Knorpelpellets verantwortlich sind. Hierzu wurden hBMSC chondrogen differenziert. Die Zellpellets wurden mit Alginat, dem Dialyseschlauch oder der Diffusionskammer verkapselt und parallel zu unver-kapselten Kontrollpellets subkutan in immundefiziente SCID-Mäuse sowie in immunkompetente BDF1-Mäuse implantiert. Die Explantate wurden mit Alzianblau-, Alizarinrot-, Kollagen Typ II-Färbungen, sowie einer ALU in-situ Hybridisierung mar-kiert und mittels Histologiescore doppelt blind bewertet (MannWhitneyU). Überra-schenderweise zeigten die unverkapselten Kontrollen in den BDF1-Mäusen weder Zeichen von Inflammation noch von Destruktion und 4/5 der Pellets waren auf Kol-lagen Typ-II und Alzianblau positiv. Gleichzeitig war der Grad der Mineralisierung in den BDF1-Mäusen gegenüber SCID-Mäusen reduziert (p = 0,03). Durch Alginat wurde die Mineralisierung in den BDF1 Mäusen (0/8) völlig verhindert, während in den SCID-Mäusen noch 7/8 der Pellets Kalzifizierung zeigten (p = 0,001). Die Verkapselung mit Alginat verglichen mit der Kontrolle führte in beiden Mausstämmen zu höheren Scores für Kollagen Typ II (SCID: p = 0,013, BDF1: p = 0,042) und zeigte gleichzeitig eine Reduktion der Mineralisierung (SCID: p = 0,018, BDF1: p = 0,031). In SCID-Mäusen war außerdem der Alzianblau-Wert gegenüber den Kontrollen erhöht (p = 0,003). Die Diffusionskammer erwies sich als ungeeignet, da die Pellets ihre knorpeligen Eigenschaften verloren. Durch die Verwendung des Dialyseschlauches konnte lediglich in der SCID-Maus eine Erhöhung der Kollagen Typ II (p = 0,03) und eine Reduktion der Kalzifizierung (p = 0,004) erreicht werden. Sowohl im Alginatbead in der BDF1-Maus (1/3 Spendern), als auch im Dialyseschlauch mit Kollagenmembran (2/3 Spendern) konnte eine erfolgreiche in vivo Chondrogenese durchgeführt werden. Zur Untersuchung der in vivo Stabilität knorpeliger MSC-basierter Konstrukte stellt die BDF1-Maus eine attraktive, kostengünstige Alternative mit einer gegenüber der SCID-Maus verringerten Mineralisierungsrate dar. Die in vitro gebildete knorpelige Extrazellulärmatrix erzeugt dabei bereits eine Immunisolation, welche die Transplantatdestruktion verhindert. Ob ein intaktes lymphozytäres System die Knorpelstabilität gegenüber defizienten Immunsystemen begünstigt, muss durch die Untersuchung weiterer Ansätze belegt werden. Im Gegensatz zur Diffusionskammer bietet Alginat das richtige Maß an Versorgungsreduktion, um die Stabilisierung des Knorpelphänotyps der Konstrukte zu ermöglichen.

Co-delivery of the embedded growth factor-loaded microspheres and adult stem cells in a hydrogel matrix was studied for its potential as a cell-based therapeutic strategy for cartilage regeneration in partial thickness chondral defects. A photopolymerizable N-methacrylate glycol chitosan (MGC) was employed to form an in situ gel that was embedded with two formulations of growth factor-loaded microspheres and human adipose-derived stem cells (ASC). The polymeric microspheres were used as a delivery vehicle for the controlled release of growth factors to stimulate differentiation of the ASC towards the chondrocyte lineage. The microspheres were made of amphiphilic low molecular weight (Mn < 10,000 Da) poly(1,3-trimethylene carbonate-co--caprolactone)-b-poly(ethylene glycol)-b-poly(1,3-trimethylene carbonate-co--caprolactone) (P(TMC-CL)2-PEG)). This triblock copolymer is solid below 100C, but liquid with a low degree of crystallinity at physiological temperature and degrades slowly, and so acidic degradation products do not accumulate locally. Bone morphogenetic protein-6 (BMP-6) and transforming growth factor-3 (TGF-3) were delivered at 5 ng/day with initial bursts of 14.3 and 23.6%, respectively. Both growth factors were highly bioactive when released, retaining greater than 95% bioactivity for 33 days as measured by cell-based assays. To improve ASC viability within the MGC vehicle, an RGD-containing ligand was grafted to the MGC backbone. Prior to chondrogenic induction within the MGC gel, ASC viability was assessed and greater than 90% of ASC were viable in the gel grafted with cell-adhesive RGD peptides as compared to that in non-RGD grafted gels. For ASC chondrogenesis induced by the sustained release of BMP-6 and TGF-3 in MGC gels, the ASC cellularity and glycosaminosglycan production were similar for 28 days. The ratio of collagen type II to I per cell (normalized to deoxyribonucleic acid content) in the microsphere delivery group was significantly higher than that of non-induced ASC or with soluble growth factor administration in the culture media, and increased with time. Thus, the co-delivery of growth factor-loaded microspheres and ASC in MGC gels successfully induced ASC chondrogenesis and is a promising strategy for cartilage repair. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2011-06-07 19:32:50.94

<p><p> Articular cartilage is a smooth connective tissue that covers the ends of bones and protects joints from wear. Cartilage has a poor healing capacity, and the lack of treatment options motivates the development of tissue engineering strategies. The widespread cartilage degeneration associated with osteoarthritis (OA) is dramatically accelerated by joint injury, but the defined initiating event presents a therapeutic window for preventive treatments. In vitro model systems allow investigation of OA risk factors and screening of potential therapeutics. This dissertation develops stem-cell based strategies to 1) treat cartilage injury and OA using tissue-engineered cartilage, 2) prevent the development of OA by delivering stem cells to the joint after injury, and 3) study cartilage by establishing systems to model genetic and environmental contributors to OA.</p><p> Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are promising human adult cell sources for cartilage tissue engineering, but require distinct chondrogenic conditions. As compared to ASCs, MSCs demonstrated enhanced chondrogenesis in both alginate beads and cartilage-derived matrix scaffolds. </p><p> We hypothesized that MSC therapy would prevent post-traumatic arthritis (PTA) by altering the balance of inflammation and regeneration. Highly purified MSCs (CD45-TER119-PDGFR&#945;+Sca-1+) rapidly expanded under hypoxic conditions. Unexpectedly, MSCs from control C57BL/6 (B6) mice proliferated and differentiated more than MSCs from MRL/MpJ (MRL) "superhealer" mice. We injected B6 or MRL MSCs into mouse knees immediately after fracture, and MSCs of either strain were sufficient to prevent PTA. </p><p> Genetically reprogramming adult cells into induced pluripotent stem cells (iPSCs) generates large numbers of patient-matched cells with chondrogenic potential for therapy and cartilage modeling. We produced murine iPSC-derived cartilage constructs with a multi-phase approach involving micromass culture with bone morphogenetic protein-4, flow cytometry cell sorting of chondrocyte-like cells, monolayer expansion, and pellet culture with transforming growth factor-beta 3. Successful differentiation was confirmed by increased chondrogenic gene expression, robust synthesis of glycosaminoglycans and type II collagen, and the repair of an in vitro cartilage defect. </p><p> The diverse applications pursued in this research illustrate the power of stem cells to deepen the understanding of cartilage and guide the development of therapies to prevent and treat cartilage injury and OA.</p> / Dissertation

Cartilage tissue provides compressive resistance in diarthrodial joints, and has been shown to be regulated by mechanical signals, in particular with regard to production of extracellular matrix proteins. However, less is understood about how chondrocytes in regions not solely purposed to provide compressive resistance may also be affected by mechanical forces. The growth plate is a small layer of cartilage that functions to facilitate longitudinal growth of the long bones from in utero through post-adolescent development. The growth plate maintains distinct regions of chondrocytes at carefully regulated stages of endochondral ossification that are in part characterized by their morphology and differential responsiveness to vitamin D metabolites. Understanding if mechanical cues could be harnessed to accelerate or delay the process of endochondral ossification might be beneficial for optimizing tissue engineering of cartilage or osteochondral interfaces. This study focused on three aims to provide a basis for future work in this area: 1) Develop a cell line culture model useful for studying growth plate chondrocytes, 2) Determine the response of primary growth plate chondrocytes and the cell line model to fluid shear stress, and 3) determine if expression of integrin beta 1 is important for the observed responses to shear stress. The findings of this study suggest that inorganic phosphate can promote differentiation in coordination with the 24,25(OH)2D3 metabolite of vitamin D, and that fluid shear stress generally inhibits differentiation and proliferation of growth plate chondrocytes in part through an integrin beta 1 mediated pathway.

As cartilage tissue has limited repair capacities, tissue engineering has emerged as a promising alternative for cartilage repair. The scaffold is a primary component of the tissue engineering design, yet little information exists regarding the effects of polymer and scaffold properties on tissue growth. In this study, we have developed a novel scaffold, PLG microspheres, for use in cartilage tissue engineering, which has the capacity for alterations in polymer and scaffold. We examined the effects of molecular weight, hydrophobic capping, delivery of Mg(OH)2, microsphere size, and controlled release of IGF-I. Our findings demonstrated that polymer parameters distinctively affect tissue and matrix output. Specifically, micro spheres with high molecular weight polymer produced tissue with high GAG content and tissue mass in vivo and in vitro, while micro spheres with capped polymer induced steady tissue and matrix accumulation, but may have precluded cell attachment. Release of buffer to the growing cartilage had negative effects on tissue formation in vivo and in vitro. Additionally, increasing microsphere diameter generated more samples with center of necrotic tissue. The presence of microspheres induced greater cartilage mass and matrix content than cartilage from cells alone. Delivery of IGF-I induced a dose-dependent effect on matrix and tissue production in vivo, with the highest effective load of IGF-I (0.3%) generating the most matrix and tissue accumulation. In contrast, the in vitro IGF-I dose-dependent effect induced on matrix and tissue production peaked at a dose of 0.02% IGF-I, with higher doses generating less tissue and matrix. Taken together, changes in polymer or scaffold composition and release of growth factor can be optimized to form cartilage with enhanced tissue parameters. Moreover, these results demonstrate a novel scaffold with potential to support cartilage regeneration and provide simultaneous drug delivery.

Made available in DSpace on 2014-06-11T19:24:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-19Bitstream added on 2014-06-13T20:52:31Z : No. of bitstreams: 1 moroz_a_me_botfm.pdf: 608341 bytes, checksum: 715f3ed5332daf7487462c8fa4111838 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Levando-se em consideração avanços tecnológicos na área médica e o impacto dos programas de saúde que determinaram curvas de longevidade cada vez maiores e taxas de natalidade cada vez menores, o novo desafio do gestor público são as conseqüências: o envelhecimento e a vida como uma “doença crônica”. Entre os principais desafios está a abordagem das doenças crônico-degenerativas que determinam o aumento de lesões cartilaginosas articulares. A débil capacidade de regeneração e as limitações das alternativas de tratamento fazem as técnicas derivadas da biotecnologia, como o transplante autólogo de condrócitos (TAC) e o uso de células-tronco, o foco das investigações. O TAC requer coleta de material cartilaginoso de área sadia, podendo causar nova lesão; no entanto, pode-se evitar este perigo com o uso de células-tronco. As células-tronco mesenquimais, adultas, podem se diferenciar em condrócitos mediante o uso de meio de cultura específico em consonância a um arcabouço 3D, mas muitos problemas como evitar a calcificação e estimular a condrogênese em meio favorável constituem o desafio para os pesquisadores na atualidade. 1) produzir anticorpo monoclonal específico a CTMs de coelho para monitorá-las, 2) determinar o volume ideal de coleta de medula óssea para microencapsulação e condrogênese, 3) realizar a microencapsulação das CTMs em novos arcabouços: BIOGEL3D e BACTCELL3D, comparando seu desempenho com o modelo clássico em alginato. Foram utilizados 25 coelhos Nova Zelândia sendo divididos em diferentes grupos em função do volume de MO coletado: G1 = 6mL, G2 = 9mL, G3 = 12mL, G4 = 15mL e G5 = volume ideal de coleta determinado pelos indicadores dos outros grupos. O material coletado foi diluído 1:2 em RPMI 1640 com 3.000U de heparina sódica. Após a contagem celular, as amostras foram submetidas a separação em gradiente... / Technological advances in the medical area combined with the impact of health programs that enhance longevity, together with lower natality rates created new challenges to the public manager such as aging and life as a “chronic disease”. Among the major problems are the chronic degenerative diseases that increase articular lesions. The limited regeneration capabilities and the limitations of actual treatment alternatives made biotechnology derived techniques the focus of investigations. The autologous chondrocyte transplantation (ACT) requires a small biopsy of health cartilage, which can lead to a new lesion. However, the use of stem cells can avoid this possibility. Mesenchymal stem cells (MSCs) can differentiate into chondrocytes by using a specific culture medium together with a 3D scaffold, but some questions such as the risks of calcification remain as key factors to researchers. 1) to produce a monoclonal antibody that recognizes MSCs in order to characterize them, 2) to determine the optimal bone marrow collection volume for cell microencapsulation and chondrogenesis and 3) to microencapsulate MSCs in two novel scaffolds: BIOGEL3D and BACTCELL3D, comparing them with sodium alginate. 25 New Zealand rabbits were divided into 5 groups related to bone marrow collection volume: G1 = 6mL, G2 = 9mL, G3 = 12mL, G4 = 15mL and G5 = optimal volume determined by the study. The collected material was diluted in RPMI1640 medium 1:2, with 3000U sodium heparin. After cell count and viability assessment the samples were submitted to density gradient centrifugation in order to isolate the lymphomononuclear (LMN) fraction. These cells were seeded to obtain and expand the MSCs in DMEM Knockout® (InvitrogenTM) supplemented with antibiotic/antimycotic, Lglutamine, essential aminoacids, non essential aminoacids and fetal bovine serum (all from InvitrogenTM). The cells were cultivated in 5% CO2 ...(Complete abstract click electronic access below)

Orientador: Lúcia Elvira Alvares / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T20:53:58Z (GMT). No. of bitstreams: 1 Peterlini_DennerJefferson_M.pdf: 3626461 bytes, checksum: 754caba3e8964b3c91965dc6f4912b32 (MD5) Previous issue date: 2011 / Resumo: Os membros de vertebrados representam uma aquisição importante do grupo, os quais possibilitaram a expansão destes pela Biosfera. As bases moleculares do desenvolvimento dos membros estão sob intensa investigação, e o papel de diversos genes e moléculas de sinalização começam a ser bem compreendidos tanto no contexto do estabelecimento de seus eixos quanto da padronização dos tecidos e estruturas. A família dos genes Dapper (Dpr) tem sido associada a diversos processos da embriogênese de vertebrados, desde a coordenação de movimentos morfogenéticos durante a gastrulação à morfogênese de estruturas tão distintas quanto encéfalo, olhos e coração. No entanto, pouco se sabe sobre o papel destes genes no desenvolvimento dos membros, um sítio marcante de sua expressão durante a embriogênese de vertebrados. Resultados preliminares obtidos com emprego de hibridação in situ em embrião de galinha no nosso laboratório já haviam mostrado a expressão destes genes nos membros, e isto sugeriu que eles pudessem desempenhar um papel importante na ontogênese destas estruturas. assim, os padrões de expressão dos genes Dpr1 e Dpr2 entre os estádios HH24 e HH34 da ontogênese de aves foram caracterizados por meio de hibridação in situ neste trabalho. Também foram avaliadas a expressão dos marcadores moleculares MyoD para desenvolvimento de músculo esquelético, e Sox9 para desenvolvimento de cartilagem, bem como foi feita coloração dos membros com alcian blue, que evidência matriz extra-celular de tecido cartilaginoso. Os resultados obtidos revelaram que, no estádio HH24, a expressão de Dpr1 está presente no mesênquima proximal e medial dos membros anterior e posterior, e ausente da região distal (autópode). Neste estádio, a expressão de Dpr2 é claramente associada à agregação das células mesenquimais em condensações pré-condrogênicas. No estádio HH25, transcritos de Dpr1 e Dpr2 foram localizados pela primeira vez no autópode, delimitando uma região com o formato dos moldes cartilaginosos dos dígitos em formação. No estádio HH28, o padrão de expressão de Dpr1 ainda acompanha o contorno dos dígitos, além de serem observados altos níveis de expressão nos precursores dos tarsos e carpos. Por sua vez, Dpr2 é expresso fortemente nos dígitos 1 e 5 dos membros anterior e posterior, bem como nos blastemas dos dígitos posteriores. Finalmente, no estádio HH34, transcritos Dpr1 e Dpr2 estão concentrados nas regiões das articulações dos membros em desenvolvimento, enquanto Dpr2 é expresso também em tendões e em anexos ectodérmicos em formação. Este estudo suporta fortemente a hipótese de que os genes Dpr1 e Dpr2 desempenham um papel no processo de condrogênese que antecede a formação dos ossos dos membros de aves, bem como no desenvolvimento de outras estruturas, como articulações, tendões e anexos cutâneos / Abstract: The acquisition of vertebrates limbs represents an important novelty for this group and allowed the expansion of vertebrates through the Biosphere. The molecular basis of limbs development are under intense investigation, and the role of several genes and signaling molecules begin to be understood both within the context of axis determination as well as in the patterning of tissues and structures. The Dapper (Dpr) gene family has been associated with different processes of vertebrates embryogenesis, from the coordination of morphogenetic movements during gastrulation to morphogenesis of structures as different as brain, eyes and heart. However, nothing is known about the role of these genes in limb development, am important domain of Dpr expression during the embryogenesis of vertebrates. Preliminary results of in situ hybridization in chicken embryo obtained in our laboratory had already shown the expression of these genes in limb, suggesting that they could play an important role in the ontogeny of these structures. Thus, in this study the Dpr1 and Dpr2 expression pattern was characterized by in situ hybridization between stages HH24 and HH34 of chicken development. We also determined the expression of the molecular markers MyoD (skeletal muscle) and Sox9 (cartilage) and stained limbs at the different stages with alcian blue, that labels the extracellular matrix of cartilage. The results revealed that, at stage HH24, Dpr1 expression is observed in the proximal and medial mesenchyme in the fore and hindlimb buds but avoids the autopod. At this stage, the expression of Dpr2 is clearly associated with mesenchymal condensations of pre-chondrogenic cells. At stage HH25, Dpr1 and Dpr2 transcripts were found for the first time in the autopod, delimiting a region with the shape of the cartilaginous templates of the developing digits. At stage HH28, Dpr1 is still expressed around the developing digits, and transcripts are found at high levels in the tarsi and carpi precursors. In turn, Dpr2 is expressed strongly in the first and fifth digits of the forelimbs and hindlimbs, as well as in the digit blastemas. Finally, at stage HH34, Dpr1 and Dpr2 transcripts are concentrated in the developing joints, while Dpr2 is also expressed in ectodermal tendons and developing skin appendages. This study strongly supports the hypothesis that Dpr1 and Dpr2 play a role in the process of chondrogenesis before the formation of the limb bones in birds as well as the development of other structures such as tendons and skin appendages / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13113号 / 論医博第2131号 / 新制||医||1022(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 安達 泰治, 教授 開 祐司 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM