Stem cell therapies have shown promising capabilities in regaining the functionality of scar tissue following a myocardial infarction. Biological sutures composed of fibrin have been shown to more effectively deliver human mesenchymal stem cells (hMSCs) to the heart when compared to traditional cell delivery mechanisms. While the biological sutures do show promise, improvements can be made. To enhance the fibrin sutures, we propose to incorporate native cardiac extracellular matrix (ECM) into the fibrin microthreads to produce a more in vivo-like environment. This project investigated the effects that ECM incorporation has on fibrin microthread structure, mechanics, stem cell seeding, and pro-angiogenic potential. Single microthreads composed of fibrin or fibrin and ECM were subjected to uniaxial tensile testing. It was found that the microthreads consisting of both fibrin and ECM had significantly high elastic moduli than fibrin only microthreads. Cell seeding potential was evaluated by performing a 24-hour hMSC seeding experiment using sutures of the varying microthread types. A CyQuant cell proliferation assay was used to determine the number of cells seeded onto each suture type. The results determined that there was no statistical difference between the numbers of cells seeded on the types of sutures. To examine the pro-angiogenic potential the microthreads had, a 24-hour endothelial progenitor outgrowth cell (EPOC) outgrowth assay was used. Fibrin and 15% ECM-fibrin microthreads were placed within the scratch of an EPOC culture and evaluated every 6 hours for 24 hours. We found that the 15% ECM microthreads had significantly increased the EPOC outgrowth, approximately 16% more distance travelled than fibrin microthreads and 18% more than no microthreads. Our combined results suggest that ECM does not affect hMSC attachment to biological sutures but does increase the pro-angiogenic potential of the microthreads due to their increase in guiding EPOC outgrowth.

archives@tulane.edu / Hydrogels serve as three-dimensional scaffolds whose composition can be customized to allow the attachment and proliferation of several different cell types. Decellularized tissue-derived scaffolds are considered close replicates of the tissue microenvironment. Decellularized adipose tissue (DAT) hydrogel has proven to be a useful tool for tissue engineering applications in pre-clinical models. The first aim of the present study was to characterize the biochemical composition of DAT hydrogel. The DAT hydrogel was prepared by processing adipose tissue acquired from three female human donors, and subsequently quantitatively analyzed using liquid chromatography-mass spectrometry (LC-MS). The enriched and depleted proteins were determined in DAT hydrogel and further analyzed by gene ontology (GO) analysis. Extracellular matrix proteins were found to be enriched, while cellular proteins were depleted relative to native adipose tissue. Furthermore, GO analysis identified that the enriched proteins could affect various biological processes via the regulation of a range of cellular pathways. The second aim was focused on the analysis of the effect of adipose-derived stromal/stem cells (ASCs) and DAT hydrogel interaction on cell morphology, proliferation, differentiation, and hydrogel microstructure. The ASCs seeded in DAT hydrogel remained viable and displayed proliferation. The adipogenic and osteogenic differentiation of ASCs seeded in DAT hydrogel was confirmed by marker gene expression and histochemical staining. Moreover, ASC attachment and differentiation altered the fibril arrangement, which indicated remodeling of the DAT hydrogel. The third aim was to analyze the regenerative potential of DAT hydrogel in a critical-sized mouse femoral defect model. The DAT hydrogel alone, or its composites with ASCs, osteo-induced ASCs (OIASC), and hydroxyapatite were tested for the ability to mediate repair of the femoral defect. The data indicated that DAT hydrogel promoted bone regeneration alone, while the regeneration was enhanced in the presence of OIASCs and hydroxyapatite. In summary, the current findings confirm that DAT hydrogel is a cytocompatible and bio-active scaffold, with potential utility as an off-the-shelf product for tissue engineering applications. In future, the analysis of DAT hydrogel using a wider range of donors representing different body mass index, age, gender, and ethnicity will provide a more comprehensive characterization. / 0 / Omair A. Mohiuddin

Chronic respiratory diseases affects many people worldwide with little known about the mechanisms diving the pathology, making it difficult to find a cure. Improving the understanding of smooth muscle and extracellular matrix (ECM) interaction is key to developing a remedy to this leading cause of death. With currently no relevant or controllable in vivo or in vitro model to investigate diseased and normal interactions of small airway components, the development of a physiologically relevant in vitro model with comparable cell attachment, signaling, and organization is necessary to develop new treatments for airway disease. The goal of this study is to create a mechanically, biologically and structurally relevant in vitro model of small airway smooth muscle tissue. Synthetic Poly-L-Lactic Acid (PLLA) and decellularized pig lung ECM (DPLECM) were electrospun to form nanofibrous mats that can closely mimic natural bronchial tissue. The addition of DPLECM significantly changed the PLLA scaffold mechanically, biologically, and physically to bring it closer to the characteristics of the human lung. DPLECM scaffolds exhibited a significant decrease in the elastic modulus compared with PLLA alone. Histological staining and SDS-PAGE showed that after scaffold fabrication, essential proteins or protein fragments in natural ECM are still present after processing. Human bronchial smooth muscle cells (HBSMCs) seeded onto PLECM scaffolds formed multiple layers of cells compared to scaffolds composed solely of PLLA. Phenotype of smooth muscle is better maintained when DPLECM is incorporated into the scaffold shown by enhanced contractile protein expression and increased collagen production for normal smooth muscle remodeling of the scaffold. In summary, this research demonstrates that a PLLA/DPLECM composite electrospun mat is a promising tool to produce an in vitro model with the potential to uncover unknown characteristics of bronchiole smooth muscle behavior in diseased or normal states.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22302号 / 医博第4543号 / 新制||医||1040(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 川口 義弥, 教授 妹尾 浩, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20995号 / 医博第4341号 / 新制||医||1027(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 武藤 学, 教授 川口 義弥, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Heart failure, predominately caused by myocardial infarction (MI), is the leading cause of death in the United States. Currently the only treatment for heart failure is cardiac transplantation, but studies show that progenitor cell, biomaterial, or combined therapies have improved cardiac function post-MI. The endogenous environment of CPCs is drastically different from commonly used culture conditions. Further the endogenous environment changes with age and disease state. We evaluated the behavior of CPCs cultured on a naturally-derived, cardiac extracellular matrix (cECM) as compared to the standard culture coating collagen I, that also mimics fibrotic tissue. In this study, CPCs cultured on cECM had improved cell numbers and cardiomyogenic maturation. However, the microenvironmental cues responsible for stimulating CPC activation are largely unknown. During development, aging and disease the myocardium changes in matrix composition and stiffness exposing endogenous cells to a wide variety of stimuli. In a combinatorial study, we evaluated the effect of cyclic strain and extracellular matrix composition on CPC behavior. The response of CPCs to signals from the microenvironment is complex, with more matrix-dependency observed at lower strains. Alignment, cell division and paracrine signaling are extracellular matrix and strain dependent. Extracellular matrix conditions affect CPC maturation and calcium signaling. Mechanotransduction pathways, including focal adhesion kinase and extracellular signal-regulated kinase, are activated through adhesion and maintained under cyclic strain. Insights from this work will advance pragmatic cell therapy attempts to regenerate healthy myocardium post-MI.

Recreating organ-specific microenvironments of the extracellular matrix (ECM) in vitro has been an ongoing challenge in biofabrication. In this study, I present a biofunctional ECM-mimicking protein scaffold with tunable biochemical, mechanical and topographical properties. This scaffold, formed by microfibres, displays three favorable characteristics as a cell culture platform: high-loading of key ECM proteins, single-layered mesh membrane with controllable mesh size, and flexibility for supporting a range of cell culture configurations. Decellularized extracellular matrix (dECM) powder was used to fabricate this protein scaffold, as a close replicate of the chemical composition of physiological ECM. The highest dECM concentration in the solidified protein scaffold was 50 wt%, with gelatin consisting the rest. In practice, a high density of dECM-laden nano- to microfibres was directly patterned on a variety of substrates to form a single layer of mesh membrane, using the low-voltage electrospinning patterning (LEP) method. The smallest fibre diameter was measured at 450 nm, the smallest mesh size of the membrane was below 1 μm, and the thickness of the membrane was estimated to be less than 2 μm. This fabrication method demonstrated a good preservation of the key ECM proteins and growth factors, including collagen IV, laminin, fibronectin, VEGF and b-FGF. The integrated fibrous mesh exhibited robust mechanical properties, with tunable fibril Young’s modulus for over two orders of magnitude in the physiological range (depending on the dECM concentration). Combining this mesh membrane with 3D printing, a cell culture device was constructed. Co-culture of human glomerulus endothelial cells and podocytes was performed on this device, to simulate the blood-to-urine interface in vitro. Good cell attachment and viability were demonstrated, and specific cell differentiation and fibronectin secretion were observed. This dECM-laden protein scaffold sees the potential to be incorporated into a glomerulus-on-chip model, to further improve the physiological relevance of in vitro pathological models.

Le muscle strié squelettique possède de grandes capacités de régénération grâce à ses cellules souches, les cellules satellites. Après une lésion, le processus de régénération musculaire qui se met en place est finement régulé dans le temps et l’espace par le microenvironnement, constitué de cellules avoisinantes mais également par des éléments de la matrice extracellulaire (MEC). Cette dernière se compose de molécules structurales comme les collagènes et de composants possédant un rôle trophique comme les glycosaminoglycanes (GAGs). La MEC musculaire est peu étudiée à cause d’une organisation tridimensionnelle complexe rendant son exploration difficile. Lors d’une lésion avec perte de substance musculaire, la régénération est altérée, associée à une fibrose et une inflammation chronique. Ce type de lésion est fréquemment rencontré en traumatologie mais survient également chez le blessé de guerre. Malgré un traitement optimal, une invalidité fonctionnelle persiste chez ces patients. L’utilisation d’un biomatériau décellularisé, constitué de MEC pourrait fournir ce support physique et trophique faisant défaut dans ce type de lésion. Dans ce travail, nous avons entrepris l'établissement d'une MEC d’origine musculaire et nous avons établi un protocole de décellularisation permettant d’obtenir un biomatériau conservant l’architecture spécifique de la MEC musculaire avec une élimination de la majorité des antigènes cellulaires afin d'éviter une réponse immunitaire délétère après implantation. Néanmoins, le protocole retenu ne permet de conserver certaines molécules trophiques d’intérêt comme les GAGs. Les « ReGeneRaTing Agent®» (RGTA®) sont des mimétiques fonctionnels de ces GAGs, utilisés en clinique pour améliorer la cicatrisation cutanée et cornéenne. Ces mimétiques conservent une capacité de liaison aux facteurs de croissance avec une résistance aux dégradations enzymatiques. Nous avons évalué l’utilisation de ces molécules au cours de la réparation musculaire, dans un modèle in vivo chez le rongeur. Nous avons réalisé une analyse histologique précoce (8e jour de régénération) mettant en évidence une augmentation du nombre de noyaux par myofibre en faveur d’une augmentation de la fusion, validée également in vitro sur des progéniteurs musculaires. Nous avons également observé une augmentation du nombre de vaisseaux, suggérant une amélioration de l’angiogenèse. Le nombre de gouttelettes lipidiques, marqueur d’une mauvaise régénération, était en diminution. L’exploration histologique plus tardive (28e jour de régénération) n’a retrouvé que l’augmentation du nombre de vaisseaux en faveur d’un effet durable sur l’angiogenèse. Ces RGTA® peuvent être couplés aux biomatériaux et sont particulièrement résistants dans un environnement inflammatoire pouvant être rencontré dans les lésions avec perte de substance musculaire. Des chimiokines et des facteurs de croissance pourront également être ajoutés au biomatériau matriciel afin de favoriser la migration des différents progéniteurs nécessaires à une néoformation musculaire. L’efficacité thérapeutique de ces biomatériaux optimisés nécessitera d’être évaluée dans un modèle in vivo de perte de substance / Skeletal muscle exhibits high capacity for regeneration after an injury that relies on resident stem cells. Muscle regeneration is tightly regulated by both the immune response and other resident cells, as well as by cues from the local extracellular matrix (ECM), contributing to a coordinated repair process. Muscle ECM is a network of structural macromolecules with a large majority of collagens and trophic molecules such as glycosaminoglycans (GAGs). In the skeletal muscle tissue, ECM was overlooked due to its complex organization making investigations difficult. Muscle regenerative ability can be overtaken in large muscle wasting, such as in volumetric muscle loss (VML), leading to fibrosis formation and chronic inflammation. This type of injury predominantly occurs in traumatology and in war-wounded patients, with functional disability despite an optimal treatment. The use of biomaterials could provide the biochemical and physical cues that are missing in this pathologic repair. In this work we have focused on obtaining a biomaterial composed of skeletal muscle ECM. We have tested several decellularization protocols both to preserve the three-dimensional architecture of the muscle ECM and to completely remove cell components in order to avoid a deleterious immune response after implantation. However, the protocol did not allow the preservation of trophic molecules such as GAGs, in the scaffold.“ReGenerating Agents” (RGTA®) are functionally analogous of GAGs with a crucial property to resist enzymatic degradation. They function to restore a proper microenvironment for tissue healing with already a clinical application in skin and corneal repair. We have explored the effects of RGTA® in muscle regeneration using an in vivo model in mouse. At early time of regeneration (day 8), we performed histologic analysis. We showed that regenerating myofibers contained more nuclei in the treated animals, in favor of an increase of progenitor fusion, which has been validated in vitro in myogenic cultures. The number of capillaries was higher in favor of a better angiogenesis. Lipid droplets, a marker of impaired regeneration, were reduced by RGTA® administration. At later time of regeneration (day 28), capillary number was still improved in favor of a durable effect of RGTA® on angiogenesis. RGTA® could be incorporated into biomaterials and are particularly resistant in an inflammatory environment, such as that occurring after a VML injury. Chemokines and growth factors could also be added in ECM-based scaffolds to promote the migration of progenitors that are essential for myofiber neoformation. Therapeutic efficacy of these optimized biomaterials will require to be evaluated in an in vivo model of VML

No description available.