Dissertationen zum Thema „Decellularized matrix“

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.

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.

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.

Three-dimensional (3D) cancer models are overlooking the scientific landscape with the primary goal of bridging the gaps between two-dimensional (2D) cell cultures, animal models and clinical research. In this thesis, we describe an innovative tissue engineering approach applied to colorectal cancer (CRC) starting from decellularized human biopsies in order to generate an organotypic 3D bioactive model. This in vitro 3D system recapitulates the ultrastructural environment of native tissue as demonstrated by histology, immunohistochemistry, immunofluorescence and scanning electron microscopy analyses. Mass spectrometry of proteome and secretome confirmed a different stromal composition between decellularized healthy mucosa and CRC in terms of structural proteins (COL1A1, COL1A2, and COL3A1) and secreted proteins such as DEFA3. Importantly, we proved that our 3D acellular matrices retained their biological properties: using CAM assay, we observed a decreased angiogenic potential in decellularized CRC compared with healthy colon mucosa, caused by direct effect of DEFA3. In addition, we demonstrated that following a 5 days of recellularization with HT-29 cell line, the 3D tumor matrices induced an over-expression of IL-8, a DEFA3-mediated pathway and a mandatory chemokine in cancer growth and proliferation, compared with recellularized healthy mucosa and 2D conventional culture model. Given the biological activity maintained by the scaffolds after decellularization, we believe this approach is a powerful tool for future pre-clinical research and screenings.
I modelli tumorali tridimensionali (3D) si stanno affacciando sul panorama scientifico con l’obiettivo primario di superare le limitazioni di colture cellulari convenzionali (2D) e modelli animali negli approcci di ricerca clinica. In questa tesi di dottorato, si descrive un innovativo approccio di ingegneria tissutale applicata alla ricerca oncologica mediante il quale, partendo da una biopsia tissutale decellularizzata, si genera un modello organo-tipico 3D bioattivo. Questo modello 3D, ricapitola, in vitro, l’ambiente ultra-strutturale del tessuto nativo come dimostrato da indagini istologiche, immunoistochimiche, di immunofluorescenza e di microscopia elettronica a scansione. L’analisi del proteoma e del secretoma mediante spettrometria di massa ha confermato una differente composizione stromale tra la mucosa colica sana decellularizzata e quella della controparte tumorale (CRC) in termini di proteine strutturali (Collagene 1A1, Collagene 1A2, Collagene 3A1) e di proteine secrete, come la Defensina alfa 3. Abbiamo dimostrato che le nostre matrici 3D mantengono le loro proprietà biologiche dopo il processo di decellularizzazione: mediante la CAM, abbiamo osservato un decremento del potenziale angiogenico della matrice decellularizzata di CRC comparata con la mucosa colica sana, causata da un effetto diretto della Defensina alfa 3. Inoltre, abbiamo dimostrato che dopo 5 giorni di ricellularizzazione con cellule HT-29 (linea stabilizzata di cancro del colon), le matrici tumorali 3D (comparate con le rispettive mucose coliche sane ed il metodo di coltura 2D) hanno indotto una sovra-espressione di IL-8, una chemochina a valle del pathway della Defensina alfa 3, che gioca un ruolo molto importante nella crescita e proliferazione tumorale. In conclusione, avendo dimostrato la capacità dei delle nostre matrici acellulari 3D di mucosa colica sana e CRC di mimare gli stimoli ultra-strutturali e biologici dei rispettivi tessuti nativi, crediamo che questo approccio possa essere un efficace strumento per migliorare il livello delle ricerche precliniche e nei test di screening di farmaci.

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

Chronic obstructive pulmonary disease (COPD) including emphysema is a devastating condition, increasing in prevalence in the US and worldwide. There remains no cure for COPD, rather only symptomatic treatments. Due to unique challenges of the lung, translation of therapies for acute lung injury to target chronic lung diseases like COPD has not been successful. We have been investigating lung derived extracellular matrix (ECM) hydrogels as a novel approach for delivery of cellular therapies to the pulmonary system. During the course of this work we have developed and characterized a lug derived ECM hydrogel that exhibits “injectability,” allowing cells or dugs to be delivered in a liquid and encapsulated at body temperature. The hydrogel self assembles in <5 minutes and achieves mechanical stiffness similar to other soft tissue ECM hydrogels. The hydrogel can support 3D cell growth and encapsulated cell viability. Encapsulated hMSCs can also still be activated by simulated inflammatory environments. Naïve mouse macrophages exposed to the fully formed gel were not significantly induced to express markers for pro or anti-inflammatory polarized phenotypes, but increased expression for several secreted inflammatory mediators was observed. We also investigated a novel approach for preparing and solubilizing the isolated ECM proteins, using digestion time as a variable for controlling hydrogel density (interconnectivity), mechanical stiffness, component protein size distribution, and cell behavior on fully formed gels. The potential future impact for the presented research includes optimization for future animal studies, expansion to additional applications, and the development of new derivative materials.

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.

Articular cartilage is a highly organized tissue with cellular and matrix properties that vary with depth zones. Regenerating this zonal organization has proven difficult in tissue-engineered cartilage to treat damaged cartilage. In this thesis, we evaluated the effects of culture environments that mimic aspects of the native cartilage environment on chondrocyte subpopulations. We found that decellularized cartilage matrix can improve zonal tissue-engineered cartilage. Also, chondrocytes respond to signals from bone cells and compressive stimulation in a zone-dependent manner. These results highlight the importance of a zone-specific environment to improve tissue-engineered cartilage in vitro.

This thesis is a comparative study of cartilage tissue regeneration by the tissue’s resident cells with or without adding the tissue matrix’s particles, in the lab and followed by implantation in mouse. Incorporation of the particles with the cells appears to be a viable strategy to increase the cartilage-like matrix content in the short-term, and the particles appear to integrate into the regenerated tissue in the long-term.

La restauration de la continuité digestive suite à l'ablation d'une portion de l'œsophage consiste à la réalisation chirurgicale d’une anastomose œsogastrique intra-thoracique. Néanmoins des complications post-opératoires sont décrites telles que des atteintes pulmonaires, des fistules, des sténoses, des nécroses de plastie, et un reflux gastro-œsophagien. Le développement d'un substitut issu de l'ingénierie tissulaire dérivé de la matrice œsophagienne biologique décellularisée (MBD) est prometteur dans la perspective d’améliorer la prise en charge du traitement chirurgical pour le remplacement de l’œsophage. L'objectif principal de cette étude était d'optimiser la conception d’une MBD de porcs et de caractériser ses propriétés biologiques et mécaniques. Le second objectif était de cellulariser la MBD au moyen de cellules immuno-privilégiées, facilement disponibles : les cellules stromales mésenchymateuses humaines issues de la gelée de Wharton (CSM-GW).La décellularisation de l'œsophage était réalisée selon un protocole basé sur la perfusion dynamique de solutions chimiques et enzymatiques de la lumière de l’organe. L’analyse histologique et la quantification de l’ADN résiduel de la MBD permettaient de déterminer l’efficacité du protocole de décellularisation. L'ultrastructure de la MBD était analysée par des marquages immunohistochimiques (IHC), et la composition du contenu protéique de la matrice extracellulaire (MEC) était décrite par spectrométrie de masse. Les tests de cytotoxicité in-vitro de la MBD étaient réalisés conformément à la norme ISO 10993-5. L’évaluation de la force de rétention à la suture, la résistance à la traction et la pression à l’éclatement de la MBD consistait à décrire le comportement mécanique du substitut en regard de son utilisation clinique.Les CSM-GW utilisées pour la cellularisation de la MBD étaient extraites à partir de cordons ombilicaux humains et leur profilage par cytométrie en flux permettait de confirmer la pureté de la population cellulaire. La réponse immunitaire des CSM-GW était quantifiée après une co-culture avec des cellules mononucléées du sang périphérique (PBMC). Le phénotypage des PBMC permettait d’évaluer l’expression des marqueurs immunitaires au contact des MSC-GW, et l’étude du sécrétome, par une méthode immuno-enzymatique (ELISA), quantifiait le relargage des cytokines. La stratégie de cellularisation de la MBD proposée reposait sur le développement de feuillets cellulaires de MSC-GW. La validation du protocole de fabrication des feuillets consistait en la caractérisation du phénotype cellulaire par IHC et l’étude mécanique des feuillets permettait de mesurer leur résistance à la perforation.L’absence de contenu cellulaire et la quantification de l’ADN résiduel de la MBD confirmaient l’efficacité de la décellularisation selon les critères de validation en vigueur. L’ultrastructure et les composants biologiques de la MEC étaient préservés et l'analyse protéomique de la MEC mettait en évidence une complexité protéique. Le traitement de décellularisation n’induisait pas de toxicité de la MBD et le comportement mécanique de la MBD était adapté à son utilisation en tant que substitut œsophagien.La culture des CSM-GW sous forme de feuillets favorisait la cellularisation de la MBD. Une fois ensemencés, les feuillets avaient conservé leur phénotype cellulaire et leur caractéristiques immuno-privilégiées. Un remodelage tissulaire in-vitro était visible ainsi que la formation d’une nouvelle MEC produite par les CSM-GW.Les caractérisations de la MBD obtenue offraient une complexité biologique et un comportement mécanique favorable à son utilisation en tant que substitut œsophagien. La MBD était cellularisable avec des feuillets cellulaires de CSM-GW, pouvant favoriser ainsi l'intégration et le remodelage des tissus
Upon removal of a portion of the esophagus, the restoration of the digestive continuity involves the surgical creation of an intrathoracic esophagogastric anastomosis. However, postoperative complications such as lung impairments, fistulas, strictures, graft necrosis, and gastroesophageal reflux are reported. The enhancement of surgical procedures for esophageal replacement has made promising progress by the development of a substitute through tissue engineering that utilizes a decellularized biological esophageal matrix (DEM). The primary objective of this study was to optimize the design of porcine DEM and characterize its biological and mechanical properties. The secondary objective was to cellularize DEM using readily available immune-privileged human mesenchymal stromal cells derived from Wharton's jelly (hMSCs-WJ).Esophageal decellularization was performed according to a protocol based on the dynamic perfusion of chemical and enzymatic solutions through the organ lumen. Histological analysis and residual DNA quantification of the DEM were conducted to determine the efficiency of the decellularization protocol. The ultrastructure of the DEM was analyzed using immunohistochemical (IHC) labeling, and the composition of the extracellular matrix (ECM) protein content was described by mass spectrometry. In-vitro cytotoxicity tests of DEM were conducted following ISO 10993-5 standards. The evaluation of suture retention strength, tensile strength, and bursting pressure of DEM aimed to describe the mechanical behavior of the substitute for clinical use.hMSCs-WJ used for DEM cellularization were extracted from human umbilical cords, and their flow cytometry profiling confirmed the purity of the cell population. The immune response of hMSCs-WJ was quantified after co-culture with peripheral blood mononuclear cells (PBMCs). PBMCs phenotyping assessed the expression of immune markers in contact with hMSCs-WJ, while enzyme-linked immunosorbent assay (ELISA) quantified cytokine release. The proposed DEM cellularization strategy involved the development of cell sheets from hMSCs-WJ. The validation of the cell sheet production protocol involved the characterization of the cellular phenotype by IHC analysis, and the mechanical study of the sheets measured their resistance to perforation.The absence of cellular content and residual DNA quantification in DEM confirmed the efficacy of decellularization according to current validation criteria. The ultrastructure and biological components of the ECM were preserved, and proteomic analysis highlighted protein complexity. Decellularization treatment did not induce DEM toxicity, and the mechanical behavior of DEM was suitable for its use as an esophageal substitute.Culturing hMSCs-WJ as cell sheets promoted the cellularization of the DEM. Once seeded, the sheets retained their cellular phenotype and immune-privileged characteristics. In-vitro tissue remodeling was visible, along with the formation of a new ECM produced by hMSCs-WJ.Characterization of the obtained DEM offered biological complexity and favorable mechanical behavior for its use as an esophageal substitute. DEM was cellularizable with hMSCs-WJ cell sheets, potentially promoting tissue integration and remodeling

Periodontitis is a highly prevalent chronic inflammatory disease affecting more than 60% of the population, which leads to destruction of the tooth-supporting tissues. The periodontium is composed of both hard (bone and cementum) and soft tissues (periodontal ligament and gingiva), requiring a tissue-engineering approach to allow a precisely coordinated and compartmentalised healing response for subsequent structural and functional regeneration. The present study investigated the functionalisation of highly porous scaffolds with decellularised cell-laid extracellular matrix for periodontal regeneration. This novel technique allowed the combination of a three-dimensional scaffold providing mechanical support with a native ECM providing tissue specific biological activity. The decellularisation of such constructs allows the maintenance of an intact ECM structure and composition while removing the immunogenic cellular component, thus generating an acellular implant. By combining a bone-like ECM-decorated scaffold (bone compartment) with periodontal ligament cell-sheets (PDLcs), the aim was to achieve specific bone and periodontal ligament regeneration. The first part of this study (Chapter 2) focused on the optimisation of cell seeding on highly porous scaffolds. Indeed, cell seeding on such structures is challenging, resulting in both poor and heterogeneous cellular attachment, impeding in vitro characterisation of the constructs and hence their clinical translation. Several parameters affecting the quality of cell seeding were investigated, and we successfully identified pre-incubation of the scaffolds in FBS as a reproducible and repeatable protocol, which significantly improved cell seeding efficiency and subsequent scaffold maturation. The second part of the study (Chapter 3) investigated the effect of culture time on ECM deposition and its composition. To this end, human osteoblasts were seeded on 250 μm pore size polycaprolactone melt electrowritten scaffolds and cultured in osteogenic medium for 1, 2 or 4 weeks, allowing cell proliferation, differentiation and ECM deposition. The constructs were subsequently decellularised, using an in-house optimised protocol for PDLcs decellularisation. Cellularised and decellularised constructs were then extensively characterised in vitro to assess cellular and extracellular composition. The decellularised constructs were recellularised with osteoblasts to study their biological activity in vitro. In vivo performance of the different groups for bone regeneration was assessed in vivo in a rodent calvarial defect model. The various culture periods demonstrated a significant difference in ECM morphology and quantity between 1, 2 and 4 weeks. At the early time points, the fibres were decorated with collagen which mineralised over time and gradually obstructed the pores of the PCL scaffold. Although longer culture times resulted in higher osteogenic activity of reseeded cells, the more mature matrix impeded in vivo bone regeneration. Scaffold porosity is crucial for host cell colonisation and vascularisation, which are indispensable for tissue regeneration. The decoration of the 250 μm pore size construct in the previous study altered its porosity and subsequent regeneration. In the third study (Chapter 4) scaffolds with different pore sizes (250, 500 and 750 μm) were cultured for 1, 2 and 4 weeks. The scaffolds with 750 μm pore sizes did not exhibit appropriate mechanical properties and were not further characterised. 250 and 500 μm scaffolds cultured for 1, 2 and 4 weeks were decellularised, characterised and recellularised with osteoblasts or macrophages. All decellularised constructs were implanted in a rodent calvarial defect and evaluated for bone regeneration. Although 500 μm pores enabled maintenance of the porosity even after 4 weeks of in vitro maturation, both pore sizes performed similarly in vivo. Again, shorter in vitro maturation was more beneficial for bone regeneration and more mature ECM impaired bone regeneration as observed 6 weeks post-implantation. In the last part of this study (Chapter 5), the best performing bone compartment (250 μm pore scaffold maturated for 1 week) was combined with a PDLcs prior to decellularisation, in order to fabricate a biphasic scaffold for periodontal regeneration. Cell removal and ECM preservation were confirmed in vitro before implanting in a periodontal defect. Freshly decellularised constructs were compared before and after freeze drying and long-term storage. Freeze drying allows stabilisation of biological components, potentially increasing products stability, shelf life and therefore clinical translation. Although our biphasic construct did not induce bone regeneration in vivo, fresh and freeze dried constructs displayed a higher potential in periodontal regeneration. Both groups displayed enhanced cementum formation and periodontal attachment, and prevented the formation of ankylosis, as opposed to the control groups. In conclusion, the ECM-decorated melt electrowritten scaffolds were shown to support bone and periodontal regeneration. Optimisation of the cell culture time was shown to be essential for efficient in vivo regeneration. Longer maturation time did not automatically increase scaffold performance, and indeed the more mature matrix appeared to inhibit in vivo bone regeneration. The combination of our optimised bone compartment with a mature periodontal ligament cell-sheet before decellularisation successfully generated a construct capable of promoting compartmentalised periodontal regeneration.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medicine & Dentistry
Griffith Health
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Skeletal muscle is an essential tissue for several vital functions. It displays an intrinsic regenerative ability in case of injury, thanks to the activation of satellite cells (SCs), the adult skeletal muscle stem cells. In presence of large defects, the renewing capacities of skeletal muscle are compromised. In such situations regenerative medicine may be a promising solution. This project is focused on a neonatal pathology known as congenital diaphragmatic hernia (CDH), in which the diaphragm fails to close during gestation. CDH is a severe anomaly with an incidence of 1 on 2,500-3,000 new-borns and high mortality rate. Currently, the most frequently used material for the surgical CDH repair is polytetrafluoroethylene (Gore-Tex[R]), but its application can lead to several drawbacks, as hernia recurrence and chest deformation. Great interest has been shown in alternative solutions based on tissue engineering approaches. In this regard, the use of decellularised extracellular matrix (ECM) revealed to be encouraging. When transplanted in vivo it can integrate with the native tissue, recruit host stem cells and influence their behaviour towards a regenerative process. The aim of this work is to characterise a novel tissue engineering approach based on the use of diaphragm decellularised ECM (dECM) as an alternative solution to the current CDH clinical options. The final purpose is to close the defect on the diaphragm and to induce its regeneration and functional recovery. In vivo, we created the first surgical CDH mouse model and we repaired the defect on the diaphragm using mouse dECM and expanded-polytetrafluoroethylene (ePTFE) as control. The transplantation of dECM patches did not cause any rejection effect nor hernia recurrence, differently from ePTFE treated mice. Moreover, ePTFE patches induced a foreign body reaction that was absent when dECM patches were used. We further considered three essential aspects of tissue regeneration: new muscle tissue formation, angiogenesis and re-innervation. In all the cases the biologic patch demonstrated to be better compared to ePTFE. The prolonged activation of muscle regeneration together with the angiogenic and re-innervation processes induced by dECM translated into an overall amelioration of diaphragmatic function compared to ePTFE-treated animals. Despite the positive clinical outcome, dECM patches did not activate complete regeneration of the defect. For this reason, we set up a tissue engineering technique to re-create in vitro diaphragmatic muscle tissues recellularising mouse diaphragm dECM and human MPCs cells. The aim was to obtain skeletal muscle-like substitutes for CDH capable to boost myofibers generation and further improve tissue functionality. We demonstrated that human MPCs not only were able to engraft the scaffold and repopulate the dECM in all its thickness, but most importantly, they differentiated giving rise to metabolic active myotubes. Moreover, a subpopulation of cells maintained SCs features, showing the ability to respond to in vitro injury. Given the positive outcomes obtained using dECM, the next step to get closer to clinic would be to use larger animal models. Moreover, the recellularisation could be improved by using mechanical stimulation, perfusion systems and by adding other cell types as endothelial and neural cells, in order to obtain a more complete in vitro construct for pre-clinical and clinical applications. Finally, the two parts of this project could be joined by closing the defect on the diaphragm using recellularised ECM, with the aim to favour tissue regeneration and reduce the drawbacks related to the use of current synthetic patches.
Il muscolo scheletrico ha un’intrinseca capacità rigenerativa grazie all’attività svolta dalle cellule satelliti. In presenza di danni estesi però tali capacità rigenerative possono essere compromesse. In queste situazioni un approccio di medicina rigenerativa può costituire una soluzione promettente. Questo progetto è focalizzato sull’ernia diaframmatica congenita, patologia neonatale caratterizzata da un’incompleta formazione del diaframma, con incidenza di 1 su 2,500-3,000 neonati e un alto tasso di mortalità. Attualmente, il materiale più usato per il riparo dell’ernia è il politetrafluoroetilene (Gore-Tex[R]), tuttavia il suo utilizzo può causare effetti collaterali, come la ricorrenza dell’ernia e malformazioni della cassa toracica. Grande interesse è stato rivolto a soluzioni di ingegneria tissutale, come l’uso di matrici extracellulari decellularizzate. Quando trapiantate in vivo esse riescono ad integrarsi in maniera fisiologica con il tessuto nativo e reclutano cellule staminali, modulando il loro comportamento verso un processo rigenerativo. Lo scopo di questo progetto è caratterizzare un approccio di ingegneria tissutale basato sull’uso di matrici decellularizzate come soluzione alternativa all’attuale metodo per il riparo l’ernia. L’obiettivo è chiudere il difetto sul diaframma ed indurne la rigenerazione. In vivo, abbiamo creato il primo modello murino di ernia diaframmatica e abbiamo riparato il difetto usando una matrice decellularizzata. Il politetrafluoroetilene espanso (ePTFE) è stato usato come controllo. Il trapianto di matrici decellularizzate non ha causato rigetto o ricorrenza dell’ernia, a differenza degli animali trattati con ePTFE. Inoltre, ePTFE ha indotto una reazione da corpo estraneo che era completamente assente negli animali trattati con la matrice biologica. Ci siamo poi concentrati su tre aspetti fondamentali della rigenerazione: la formazione di nuovo tessuto muscolare, angiogenesi e re-innervazione. In tutti i casi la matrice biologica ha dimostrato di essere migliore di quella sintetica. La prolungata attivazione della rigenerazione muscolare insieme ai processi angiogenici e di re-innervazione indotti dalla matrice extracellulare si sono tradotti in un generale miglioramento delle funzioni diaframmatiche rispetto a quanto ottenuto negli animali con ePTFE. Nonostante i risultati positivi, la matrice extracellulare non era in grado di indurre una completa rigenerazione del difetto. Perciò abbiamo messo a punto una tecnica di ingegneria tissutale per ricreare in vitro tessuti diaframmatici ricellularizzando matrici decellularizzate con precursori muscolari umani. Lo scopo era di ottenere dei possibili costrutti paragonabili al muscolo scheletrico da usare per il riapro dell’ernia in modo da stimolare maggiormente la generazione di nuove miofibre e migliorare la funzionalità tissutale. I precursori muscolari umani erano in grado di attecchire sulla matrice decellularizzata, di ripopolarla in tutto il suo spessore e di differenziare dando origine a miotubi attivi metabolicamente. Inoltre, una sottopopolazione di cellule manteneva le caratteristiche tipiche delle cellule satelliti, dimostrando di saper rispondere in vitro ad un danno. Visti i risultati positivi ottenuti usando la matrice decellularizzata, il passaggio successivo per avvicinarsi alla cinica è rappresentato dall’utilizzo di modelli animali più grandi. Inoltre, la ricellularizzazione potrebbe essere migliorata grazie a stimolazione meccanica, a sistemi di perfusione e all’aggiunta di altri tipi cellulari (cellule endoteliali e neurali) con lo scopo di ottenere un costrutto più completo per possibili applicazioni pre-cliniche e cliniche. Infine, le due parti di questo progetto potrebbero essere unite in futuro riparando il difetto sul diaframma usando matrici biologiche ricellularizzate al fine di favorire la rigenerazione e ridurre gli svantaggi legati all’uso delle matrici sintetiche.

The regenerative potential of adult stem cell populations within the human body bears great promises for their use in regenerative medicine. The bone marrow (BM) harbours two different types of adult stem cells, haematopoietic stem and progneitor cells (HSPCs) and multipotent mesenchymal stromal cells (MSCs), which are tightly regulated in their distinct anatomically defined niches by multiple cues such as cytokines, cell-cell contacts, the extracellular matrix (ECM) and the physical microenvironment. The ex vivo expansion of these cells for applications in regenerative therapies is of great interest and several biomaterial approaches attempt to mimic the natural BM niche and its components to control stem cell maintenance and differentiation. However, as of now the complexity of such stem cell niches is hard to recapitulate. Towards this goal, this work was focussing on the ECM environment of BM stem cells and was set out to engineer improved in vitro culture systems. MSC themselves are one of the most important cell types within the BM that secrete and construct ECM-networks and thereby shape the microenvironment of the residing cells. The potential of primary human BM-MSC to secrete ECM in vitro has been exploited to generate niche-like ECM surrogates in a robust and versatile format. Application of decellularisation regimes allowed the fabrication of complex matrices which demonstrated suprastructural, compositional and physicochemical properties compareable to those of the native BM-ECM environment. Reliable stability and reproduciblity was achieved by a dedicated procedure of maleic anhydride co-polymer-mediated covalent binding of fibronectin and subsequent anchorage of cell-secreted ECM molecules. As a result of the high reproducibility, a complete proteomic register of ECM molecules was obtained in combination with determining the complex fibrillar and soft gel-like characteristics of MSC-derived matrices. Based on the established BM niche-like substrate, the impact of extracellular matrices on MSC and HSPC ex vivo behavior has been explored. Both cell types demonstrated strong adhesion to ECM substrates and depicted a changed cellular morphology upon contact with native ECM structures compared to standard culture substrates or simple ECM protein coatings, indicating an intense interplay between the cell and the microenvironment. MSC that re-grew into their own matrices have shown advantageous proliferation and cytokine secretion levels as well as enhanced differentiation intensity (upon differentiation induction) compared to MSC that were cultured on less complex substrates. Similarly, HSPC were also instructed for enhanced expansion on MSC-derived matrices without exhaustion of stem cell-marker expressing progenitor cells. The efficiency of these matrices was related to their ability to mimic the native composite suprastructure, ligand nano-topography, molecular composition and physical properties of natural BM ECM environments. The data obtained within this thesis set the ground for a more rational design of artificial stem cell niches with defined and distinct properties, offering exciting options for the in-depth analysis and understanding of stem cell regulation by exogenous cues.

Human tissue-derived hydrogels are highly biocompatible and inherently possess cell-instructive cues that are important for repairing tissue, yet control over the physicochemical properties is severely limited. This thesis describes the creation of a photocrosslinkable hydrogel derived from human placental tissue. It outlines the procedure of removing cellular material, as well as the solubilisation and chemical functionalisation of extracellular matrix components to create hydrogels which irreversibly crosslink upon exposure to blue light. The findings demonstrate the formation of stable hydrogels with highly tuneable physicochemical properties which support cellular viability and growth, suggesting their suitability for tissue engineering applications.

碩士
國立臺灣大學
生物產業機電工程學研究所
104
In order to overcome the shortage of organ donation and injury of patients after partial hepatectomy (PH) treatment, we aimed at developing a liver film for hepato-cytes regeneration through liver tissue engineering. In this study, a HGF/heparin-immobilized decellularized liver matrix film (HGF/heparin complex coated on DLM film) was developed for hepatocytes regeneration in liver injury. In the result, (1) the amounts of immobilized heparin on DLM film was 30 μg/cm2 when the initial heparin concentrations were 1 mg/mL. (2) The relative cell viability and albu-min synthesis of the hepatocytes on HGF/heparin-immobilized DLM film was 20-30 % and 20 % superior than on normal dish at 3 days of culture, respectively. (3) The lactate dehydrogenase activity of the D-galactosamine-induced injury of hepatocytes on hepa-rin-immobilized DLM film cultures was 10 milliunits/mL, which was 50 % lower than that in the D-galactosamine-induced injury of hepatocytes on normal dish cultures, and albumin synthesis can recover to the same level as non-toxic hepatocytes cultured on normal dish. The HGF/heparin-immobilized DLM film showed highly potential in maintaining hepatocyte culture and also in repairing injured hepatocytes from D-galactosamine. It is believed that this HGF/heparin-immobilized DLM film has promising potential for hepatocyte transplantation, and could be applied for future use in liver tissue engineering.

碩士
國立臺灣大學
生物產業機電工程學研究所
106
More and more scholars believe that the pathological changes of cirrhosis are a series of gradual clinical stages rather than a single disease. In the beginning, chronic hepatitis can lead to hepatocyte necrosis, and then if it is not controlled in time, it will evolve into liver fibrosis, which eventually causes the liver to become hard and form liver cirrhosis. However, such a cirrhosis process is irreversible, and the current treatment can only stop or slow down the damage of the liver. In addition, cirrhosis has long been one of the common causes of death in the adult population of the world, so the treatment of cirrhosis has been studied in the field of biomedicine. The previous study shows that the decellularized liver matrix-film can promote the recovery of D-galactosamine-induced injured hepatocytes. Therefore, in this study, we proposed a novel hypothesis that the injection of liver extracellular matrix into the fibrotic liver via the hepatic portal vein, may be an effective treatment for patients with cirrhosis. First, we developed a three-dimensional porous biomaterial by mixing liver extracellular matrix (LECM) and gelatin-hydroxyphenylpropionic acid (Glt-HPA). Then we cultured injured hepatocytes (Treated by GaIN, CHCl3, CCl4, repesctively) in the above-mentioned biomaterials with LECM-containing medium, the viability and functionality of injured hepatocytes was determined thereafter. In the result: (1) After 3 days culture of primary rat hepatocytes in Glt-HPA:LECM = 4:6, the secretion of albumin was about 50% and 20% higher than that in Glt-HPA:LECM = 5:5 and Glt-HPA (without LECM), separately; (2) After 5 days culture, the albumin secretion of the D-galactosamin-induced injury of hepatocytes in Glt-HPA-LECM (4:6) with the LECM-containing medium cultures was 2 times superior to that in Glt-HPA without the LECM-containing medium cultures (Negative). Also, the lactate dehydrogenase (LDH) activity of the GaIN-induced injury of hepatocytes in Glt-HPA-LECM (4:6) with the LECM-containing medium cultures was reduced to be similar to the non-injured hepatocytes in Glt-HPA with normal medium cultures (Blank). (3) After 5 days culture, the albumin secretion of the CHCl3-induced injury of hepatocytes in Glt-HPA-LECM (4:6) with the LECM-containing medium cultures was 1 time superior to Negative condition. Also, the LDH activity of the GaIN-induced injury of hepatocytes in Glt-HPA-LECM (4:6) with the LECM-containing medium cultures was reduced to be lower than Blank. (4) After 5 days culture, the albumin secretion of the CCl4-induced injury of hepatocytes in Glt-HPA-LECM (4:6) with the LECM-containing medium cultures was 12 % superior to Negative condition. Also, the LDH activity of the GaIN-induced injury of hepatocytes in Glt-HPA-LECM (4:6) with the LECM-containing medium cultures was reduced to be similar to Blank. In summary, Glt-HPA-LECM as a three-dimensional porous substrate showed high potential in increasing cell viability and albumin secretion of the primary hepatocyte. Furthermore, the LECM-containing medium did have the effect of restoring the activity of damaged hepatocytes and delaying the toxicity. Such results are believed to be of great significance for the future treatment of clinical cirrhosis and liver tissue engineering.

碩士
國立臺灣大學
醫學工程學研究所
101
The soft tissue defects caused by congenital malformation, trauma, tumor removal and other various reasons affect the patient''s psychology and interpersonal relationship, so it has been gaining popularity for Plastic and Reconstructive Surgery. Due to advances in medicine, the emphasis on the requirements of the quality of medical care and physical appearance is increasing, and it also increases the demand for medical cosmetic. However, current challenge of adipose tissue engineering failed to maintain volume of adipose after transplantation, so it is important to find the soft fillers that have both functionality and aesthetics. In the study, we used decellularized porcine adipose matrix and silk fibroin as composite materials for scaffolds. The loss of the ECM content mixed in the scaffolds was slowed down by cross-linking. Eighty percentage collagen and thirty percentage GAG contents were retained after removing most cells and lipids from porcine adipose tissue. These bioactive contents have been proved to induce cell differentiation, and hydrolyzed silk fibroin also has the ability to promote cell proliferation. In addition, both of these materials have high biocompatibility, and they can increase the overall mechanical properties after blending. In vitro experiments, we observed that 3T3-L1 and adipose stem cells attached to the composite scaffolds successfully, and their GAG content increased 30 % to 45 % after culture several days, and there was remarkable difference between the composite scaffolds and the silk fibroin scaffolds. In vivo experiments, we observed that adipose matrix-silk fibroin composite microspheres were more effective to promote adipose stem cells proliferation and differentiation than silk fibroin microspheres. There are great potentials for the application of these hybrid materials in dermal fillers and soft tissue regeneration.

It would be a great advantage in reconstructive surgery to have an off-the-shelf biomaterial to promote regeneration and volume augmentation following soft tissue damage. With this long-term objective, human adipose tissue (fat) is an abundant and accessible source of extracellular matrix (ECM) for bioscaffold fabrication. The main goal of the current research project was to optimize the established 5-day detergent-free decellularization protocol developed by the Flynn group, by shortening it to a maximum of 3 days, while achieving comparable results in terms of cell and lipid extraction with preservation of the ECM. The effectiveness of the optimized protocol was assessed by examination of the decellularized adipose tissue (DAT) and its characteristic biological properties, including in vitro bioactivity assays with human adipose-derived stem cells (ASCs) to measure adipogenic potential, as well as in vivo testing of scaffold biocompatibility. In the optimized approach, the addition of mechanical processing steps including repeated pressing and centrifugation were shown to enhance cell extraction. Fibrous ultrastructure was observed under scanning electron microscopy (SEM) for the original and optimized protocols. The preservation of collagen fibres was assessed with picro-sirius red staining and confirmed by high hydroxyproline content. Enhanced preservation of glycosaminoglycans (GAGs) was determined for the optimized protocol. Residual DNA content was higher in the DAT scaffolds processed with the optimized protocol, including larger DNA fragments that were not typically observed in the samples treated with the original protocol, which incorporated additional enzymatic treatment stages with DNase, RNase and lipase. However, no residual nuclei were visualized through DAPI staining for both protocols. Enhanced removal of DNA was achieved with electron beam (e-beam) sterilization. E-beam sterilization caused some changes in the fine fibrous structure of the ECM, but did not negatively affect the adipo-conductive potential in vitro. In comparison to the original protocol, DAT produced via the optimized protocol exhibited similar adipo-conductive properties in vitro. The in vivo biocompatibility study over a 16 week period using an immunocompetent Wistar rat model showed promising results. DAT implants produced with the original and optimized protocols promoted adipogenesis and angiogenesis, gradually being remodelled to resemble mature adipose tissue.
Thesis (Master, Chemical Engineering) -- Queen's University, 2014-01-30 12:25:22.044

碩士
國立陽明大學
生物醫學工程學系
105
Decellularized blood vessels provide better biocompatibility than synthetic scaffolds. Nevertheless, decellularization tends to disrupt ultrastructure and the components of ECM, which in turn affects the mechanical properties and the ability of cell proliferation and migration in the scaffolds, especially for treatments by chemical agents. This study investigates the effects of ultrasound, perfusion and different medium for decellularization processes on the preservation of ECM components, DNA removal, and the viability of seeded cells on the decellularized scaffolds. Many kinds of scaffolds with various decellularization levels were tested for cell seeding to confirm whether the scaffolds are suitable for cell growth. The quantification of ECM components and DNA are measured by biochemical assay. The ultrastructure of the scaffold were observed by SEM and by histological staining. The growth of cells are examined by the reduction level of alamarblue and Live/Dead staining. 　　The results show that ultrasound treatment together with medium perfusion can promote decellularization efficiency. Using a hydrophilic solution of Pluronic C as perfusion medium, more than 85 % of the nucleus can be removed within 3 days, compared to 50% of nucleus removal rate of use water as a perfusion medium.The decellularized matrix treated by pluronic C solution was also less damaged. For cell seeding tests on flat scaffolds, it is found that the scaffold treated by pluronic solutions show best results for cell proliferation compared to scaffolds which are either with insufficient DNA removal rate or with the ECM structure too severely damaged scaffolds treated by Pluronic B and C is with a lot of nucleus removed meanwhile with some matrix retained. This might explain its superior performance observed in the cell seeding tests. This is possibly due to the fact that using a Pluronic solution as decellularization medium can effecting remove DNA while keep the composition and ultrastructure of the ECM properly preserved.

碩士
國立陽明大學
生物醫學工程學系
104
Decellularized blood vessels provide better biocompatibility than synthetic scaffolds. Nevertheless, decellularization tends to disrupt ultrastructure and the components of ECM, which in turn affects the mechanical properties and the ability of cell proliferation and migration in the scaffolds, especially for treatments by chemical agents. This study investigates the effects of ultrasound and perfusion for decellularization processes on the preservation of ECM components, DNA removal, and the viability of seeded cells on the decellularized scaffolds. Three kinds of scaffolds with various decellularization levels were tested for cell seeding to confirm whether the scaffolds are suitable for cell growth. The quantification of ECM components and DNA are measured by biochemical assay. The growth of cells are examined by the reduction level of alamarblue and observed by SEM and Live/Dead staining. 　　The results show that ultrasound with perfusion can effectively promote decellularization efficiency. Using NaOH or PS-1 as perfusion medium, the nucleus can be removed for more than 70 % within 3 hours, which would take for at least 5 days by using regular chemical agents, such as Triton X-100. Together with some post processing, like treated by DNase/RNase, could remove more than 98% nucleus and with the ECM ultrastructure better preserved. For cell seeding tests on flat scaffolds, it is found that the TX-100 scaffold shows best results for cell attachment. Compared to scaffolds which are either with insufficient DNA removal rate or with the ECM structure too severely damaged, scaffolds treated by TX-100 is with 80% nucleus removed meanwhile with some matrix retained. This might explain its superior performance observed in the cell seeding tests. It is also found that the cell growth is poor for static cultured tubular cell seeding scaffolds. To improve cell growth, the mass transfer problem needed to be resolved.

Cardiovascular diseases (CVDs) are still the leading cause of death and disabilities globally. Among CVDs, ischemic heart disease (IHD) has remained the leading cause of death worldwide in the last 16 years. IHD is caused by a sudden blockage of blood flow through coronary arteries that prevents the supply of oxygen and nutrients to the region of myocardium fed by the affected vessels. This condition causes the necrosis of the myocardium that is followed by a reparative process that starts from the infarcted area, but then involves, at later stages, also the uninjured myocardium, causing progressive fibrosis that may lead eventually to heart failure. Unfortunately, there is no cure for IHD and therapy can at best control symptoms and prevent a second ischemic event. The induction of post-infarction cardiac regeneration by the means of three factors, cells, scaffold and signals, is currently the target of cardiac tissue engineering. However, the field is still at its infancy and all three factors are yet to be defined. Since the ECM is the naturally occurring scaffold loaded with uncountable biological and mechanical signals, we aimed at obtaining and characterizing a biological three-dimensional scaffold for cardiac repair and regeneration from the adult human skin. Our results provided evidence that the scaffold of decellularized human skin (d-HuSk) was acellular and had a preserved architecture, retained components of the ECM that are also typical of cardiac matrix and are critical for cardiac functions and mechanical properties of the ECM, like collagen, fibronectin, laminin, tenascin, elastin and GAGs. Additionally, growth factors stored in d-HuSk matrix were similar to those found in cardiac matrix and, as similar were the signals, similar were the effects of d-HuSk and cardiac matrix on human cardiac progenitor cells (hCPCs). Indeed, as emerged from cytocompatibility study, the environment offered by d-HuSk did not differ from the cardiac native one in supporting engraftment and survival of hCPCs. Furthermore, d-HuSk attracted hCPCs from the cardiac native matrix and sustained their differentiation and differentiation towards cardiac myocytes. Therefore, d-HuSk is a biological scaffold that is easily obtained and might be used as an autograft. It shares to a large extent the composition of the cardiac native matrix, exerts on hCPCs similar effects in vitro and is also capable of stimulating their mobilization and engraftment. Overall, d-HuSk fulfills the key requirements needed for a scaffold to warrant its use in tissue engineering and, then, holds great promise as substitute for cardiac environment. Additionally, consisting of ECM proteins and being a storage of growth factors, d-HuSk might alone provide two of the three pillars of tissue engineering, namely the scaffold and the signals, and might be exploited as stand-alone scaffold to boost cardiac regeneration by recruiting resident cardiac progenitor cells, or as a cellularized scaffold by preparing a cardiac engineered tissue in vitro with the cell population of choice.

In vivo, adipose tissue demonstrates only a limited capacity for self-repair, and the long-term treatment of subcutaneous defects remains an unresolved clinical problem. With the goal of regenerating healthy tissues, many tissue-engineering strategies have pointed to the potential of implementing three-dimensional (3-D), cell-seeded scaffolds for soft tissue augmentation and wound healing. In particular, microcarriers have shown promise as both cell expansion substrates and injectable cell-delivery vehicles for these applications. However, limited research has investigated the engineering of tissue-specific microcarriers, designed to closely mimic the native extracellular matrix (ECM) composition. In this work, methods were developed to fabricate microcarriers from decellularized adipose tissue (DAT) via non-cytotoxic protocols. Characterization by microscopy confirmed the efficacy of the fabrication protocols in producing stable beads, as well as the production of a microporous surface topography. The mean bead diameter was 934 ± 51 μm, while the porosity was measured to be 29 ± 4 % using liquid displacement. Stability and swelling behavior over 4 weeks indicated that the DAT-based microcarriers were effectively stabilized with the non-cytotoxic photochemical crosslinking agent rose bengal, with only low levels of protein release measured within a simulated physiological environment. In cell-based studies, the DAT-based microcarriers successfully supported the proliferation and adipogenic differentiation of human adipose-derived stem cells (hASCs) in a dynamic spinner flask system, with a more favorable response observed in terms of adhesion, proliferation, and adipogenesis on the DAT-based microcarriers relative to gelatin control beads. More specifically, dynamically-cultured hASCs on DAT-based microcarriers demonstrated greater lipid loading, as well as higher glycerol-3-phosphate dehydrogenase (GPDH) activity, a key enzyme involved in triacylglycerol biosynthesis, at 7 days and 14 days in culture in an inductive medium. Overall, the results indicated that the DAT-based microcarriers provided a uniquely supportive environment for adipogenesis. Established microcarrier sterility and injectability further support the broad potential of these tissue-specific microcarriers as a novel, adipogenic, clinically-translatable strategy for soft tissue engineering.
Thesis (Master, Chemical Engineering) -- Queen's University, 2010-12-01 14:28:14.628

Yes
Implementing the principles of tissue engineering within the clinical management of non-vital immature permanent teeth is of clinical interest. However, the ideal scaffold remains elusive. The aim of this work was to assess the feasibility of decellularising rat dental pulp tissue and evaluate the ability of such scaffold to support stem cell repopulation. Rat dental pulps were retrieved and divided into control and decellularised groups. The decellularisation protocol incorporated a low detergent concentration and hypotonic buffers. After decellularisation, the scaffolds were characterised histologically, immunohistochemistry and the residual DNA content quantified. Surface topography was also viewed under scanning electron microscopy. Biocompatibility was evaluated using cytotoxicity assays utilising L-929 cell line. Decellularised scaffolds were recellularised with human dental pulp stem cells up to 14 days in vitro. Cellular viability was assessed using LIVE/DEAD stain kit and the recellularised scaffolds were further assessed histologically and immunolabelled using makers for odontoblastic differentiation, cytoskeleton components and growth factors. Analysis of the decellularised scaffolds revealed an acellular matrix with histological preservation of structural components. Decellularised scaffolds were biocompatible and able to support stem cell survival following recellularisation. Immunolabelling of the recellularised scaffolds demonstrated positive cellular expression against the tested markers in culture. This study has demonstrated the feasibility of developing a biocompatible decellularised dental pulp scaffold, which is able to support dental pulp stem cell repopulation. Clinically, decellularised pulp tissue could possibly be a suitable scaffold for use within regenerative (reparative) endodontic techniques.

The regenerative potential of adult stem cell populations within the human body bears great promises for their use in regenerative medicine. The bone marrow (BM) harbours two different types of adult stem cells, haematopoietic stem and progneitor cells (HSPCs) and multipotent mesenchymal stromal cells (MSCs), which are tightly regulated in their distinct anatomically defined niches by multiple cues such as cytokines, cell-cell contacts, the extracellular matrix (ECM) and the physical microenvironment. The ex vivo expansion of these cells for applications in regenerative therapies is of great interest and several biomaterial approaches attempt to mimic the natural BM niche and its components to control stem cell maintenance and differentiation. However, as of now the complexity of such stem cell niches is hard to recapitulate. Towards this goal, this work was focussing on the ECM environment of BM stem cells and was set out to engineer improved in vitro culture systems. MSC themselves are one of the most important cell types within the BM that secrete and construct ECM-networks and thereby shape the microenvironment of the residing cells. The potential of primary human BM-MSC to secrete ECM in vitro has been exploited to generate niche-like ECM surrogates in a robust and versatile format. Application of decellularisation regimes allowed the fabrication of complex matrices which demonstrated suprastructural, compositional and physicochemical properties compareable to those of the native BM-ECM environment. Reliable stability and reproduciblity was achieved by a dedicated procedure of maleic anhydride co-polymer-mediated covalent binding of fibronectin and subsequent anchorage of cell-secreted ECM molecules. As a result of the high reproducibility, a complete proteomic register of ECM molecules was obtained in combination with determining the complex fibrillar and soft gel-like characteristics of MSC-derived matrices. Based on the established BM niche-like substrate, the impact of extracellular matrices on MSC and HSPC ex vivo behavior has been explored. Both cell types demonstrated strong adhesion to ECM substrates and depicted a changed cellular morphology upon contact with native ECM structures compared to standard culture substrates or simple ECM protein coatings, indicating an intense interplay between the cell and the microenvironment. MSC that re-grew into their own matrices have shown advantageous proliferation and cytokine secretion levels as well as enhanced differentiation intensity (upon differentiation induction) compared to MSC that were cultured on less complex substrates. Similarly, HSPC were also instructed for enhanced expansion on MSC-derived matrices without exhaustion of stem cell-marker expressing progenitor cells. The efficiency of these matrices was related to their ability to mimic the native composite suprastructure, ligand nano-topography, molecular composition and physical properties of natural BM ECM environments. The data obtained within this thesis set the ground for a more rational design of artificial stem cell niches with defined and distinct properties, offering exciting options for the in-depth analysis and understanding of stem cell regulation by exogenous cues.

Tese de mestrado em Biologia Evolutiva e do Desenvolvimento, Universidade de Lisboa, Faculdade de Ciências, 2021
A matriz extracelular é o componente não celular de todos os tecidos. Esta estrutura detém não só funções de suporte das células, mas também é um importante mediador de todos os processos biológicos necessários para o correto desenvolvimento dos organismos assim como na manutenção da homeostase. Cada tecido ou órgão apresenta uma matriz extracelular com uma composição distinta. Várias moléculas constituem esta matriz, e é a natureza modelar destas que permite a formação de estruturas com diferentes propriedades mecânicas e bioquímicas, permitindo uma grande diversidade funcional. O desenvolvimento de uma matriz extracelular específica para cada tecido resulta da interação entre células e microambiente, sendo um processo altamente dinâmico e em constante remodelação. A matriz extracelular pode ser dividida em duas principais categorias: a matriz intersticial e as membranas basais. A matriz intersticial é principalmente associada ao tecido conjuntivo e é constituída por proteínas como os colagénios intersticiais, a elastina, fibronectina e proteoglicanos. Estas moléculas atuam com substrato para as células, mas estão envolvidas também na regulação da adesão, migração, proliferação e diferenciação celular. As membranas basais localizam-se mais proximamente das células e devido a isso têm grande influência em como estas interpretam o seu meio. As membranas basais são constituídas essencialmente por colagénio tipo IV, lamininas, nidogénio e perlecan. À semelhança da matriz intersticial, regulam também processos celulares como a proliferação, diferenciação, migração, polarização e sobrevivência ou apoptose. As células recebem informação acerca do seu meio exterior através de recetores membranares. A matriz extracelular é um fator chave para o correto desenvolvimento de todos os tecidos. A miogénese do músculo esquelético é um exemplo de um processo altamente dependente de sinalização da matriz extracelular. O desenvolvimento do músculo inicia-se cedo durante a embriogénese dos vertebrados com a formação do sómitos. Estas estruturas crescem e desenvolvem-se originando o dermomiótomo. O dermomiótomo possui os percursores miogénicos que irão dar origem ao músculo esquelético. No estádio 8.5 o dermomiótomo “desepiteliza” e células musculares estaminais migram para o espaço abaixo dando origem ao miótomo, iniciando-se assim a miogénese do músculo esquelético. No miótomo desenvolvem-se os mioblastos que proliferam e fundem dando origem aos miotubos. Nesta etapa tanto a presença de fibronectina como de lamininas é crucial para o correto desenvolvimento destas estruturas. No miótomo, as células presentes sofrem diferentes processos reorganizacionais transformando-se em miofibras primárias. A miogénese primária ou embrionária ocorre entre o estádio 11.5 até ao 14.5, a partir do qual se inicia a miogénese secundária ou fetal que decorrer até ao nascimento. Esta fase de miogénese é caracterizada pelo aparecimento das miofibras. Durante a miogénese primária formam-se as miofibras primárias que estabelecem o padrão corporal do músculo esquelético. A miogénese secundária é caracterizada pelo aumento da massa muscular. Este crescimento pode ser dividido em duas fases: células musculares estaminais (Pax7-positivas) podem produzir novos mioblastos que fundem entre si (crescimento por hiperplasia) contribuindo para a formação de miofibras secundárias enquanto outras se diferenciam e fundem com as miofibras primárias levando ao seu crescimento (hipertrofia mediada por células). A ação conjunta destas duas fases leva a um crescimento tanto em número como em tamanho das fibras musculares. Para que este processo ocorra corretamente a presença de laminina-211 parece ser fulcral. A contribuição da matriz extracelular é muito importante para o desenvolvimento dos tecidos e devido a isso, quando esta se encontra perturbada, pode dar origem a diversas patologias. Quando a laminina-211 não está presente o desenvolvimento esquelético muscular é severamente afetado, dando origem à distrofia muscular congénita merosina-negativa (MDC1A), uma das distrofias musculares mais comuns na Europa. Esta condição é provocada por mutações no gene LAMA2, responsável pela codificação da cadeia α2 da laminina-221 e -221 levando à produção de uma proteína não funcional. Os portadores desta doença manifestam diversos sintomas como atrofia e hipotonia muscular, mas também o sistema nervoso parece ser afetado, entre outros sistemas de órgãos. Não existem ainda tratamentos eficazes para esta doença e a maioria do conhecimento existente é proveniente de informação pós-natal, não se conhecendo ainda o momento em que se origina esta condição nem os processos moleculares por detrás da mesma. Estudos anteriores no nosso laboratório revelaram que a origem desta doença poderá ser in utero durante a miogénese secundária, onde a ausência de laminina-211 parece levar a uma depleção precoce das células musculares estaminais, não permitindo que ocorra o crescimento das massas musculares. Este trabalho tem como objetivo acrescentar conhecimento acerca das dinâmicas celulares durante o desenvolvimento fetal desta doença e para isso foi utilizado o modelo de ratinho dyW/ dyW durante o estádio fetal 18.5. Inicialmente começou-se por caracterizar a composição da matriz extracelular de ratinhos normais e distróficos, recorrendo a imuno-histoquímica e western blot. Esta comparação parece demonstrar que a ausência de laminina-211 nos ratinhos distróficos poderá afetar também outras proteínas presentes na matriz extracelular. O colagénio I sofre um aumento de cerca de 3 vezes nos ratinhos mutantes. O aumento desta proteína está documentado em ratinhos após o nascimento e em associação a fibronectina, é responsável por processos inflamatórios e formação de tecido fibrótico (um dos principais sintomas da MDC1A), o que poderá indicar que este sintoma, apesar de não ser observado morfologicamente no feto, poderá iniciar-se ainda in utero. No entanto, contrariamente ao expectável, os nossos resultados parecem indicar que existe uma menor quantidade de fibronectina nos ratinhos mutantes. As lamininas parecem também sofrer uma diminuição em quantidade. Após a caracterização da matriz extracelular de ambos os genótipos, propusemo-nos criar um sistema que permitisse estudar a contribuição relativa das duas fontes de laminina-211 (células e matriz extracelular) para o normal desenvolvimento do músculo esquelético. Esta proteína já está presente no nicho das miofibras quando a nova onda de células musculares estaminais entra no programa miogénico. No entanto, estas células parecem também ser capazes de produzir laminina-211 e assim contribuir para a construção do seu nicho. O conhecimento da contribuição relativa de cada uma destas fontes para o normal desenvolvimento do músculo esquelético poderá permitir identificar e desta forma desenvolver terapias para determinados momentos fulcrais para este processo. A descelularização é uma técnica que permite a produção de matrizes extracelulares com uma composição semelhante à do tecido nativo sem a presença de células. Estas matrizes mantêm assim não só a composição química, mas também as suas propriedades mecânicas, o que permite uma maior aproximação ao in vivo, apresentando assim várias aplicações terapêuticas. Utilizando esta técnica testámos diferentes protocolos com objetivo de produzir uma matriz descelularizada que mantivesse uma composição semelhante à do músculo inteiro, mas sem células presentes. A utilização do detergente SDS a baixa concentração, em conjunto com outros compostos, permitiu manter na matriz extracelular a maioria das proteínas testadas (especialmente laminina-211) e apenas uma reduzida quantidade de conteúdo celular. No entanto, as matrizes descelularizadas parecem apresentar uma ligeira diminuição na quantidade de proteínas presentes após o protocolo de descelularização. Posteriormente estas matrizes descelularizadas foram cultivadas com células C2C12. Estas células são mioblastos pertencentes a uma linha celular imortalizada originada a partir de células satélite pós-lesão de ratinho, apresentando assim características de células musculares estaminais. A contagem do número de células, após 8 dias em ambos os genótipos, permitiu chegar à conclusão que estas matrizes eram capazes albergar e manter estas células. Os nossos resultados parecem mostrar uma tendência para a presença de um menor número de células nas matrizes descelularizadas mutantes, demonstrando que a ausência de laminina-211 poderá de alguma forma dificultar a adesão ou proliferação das células C2C12. Estas células são capazes de infiltrar nas matrizes e até de contrai-las, mudando-lhes a forma. As células parecem também adquirir propriedades características de células musculares diferenciadas (alinhamento e afunilamento dos núcleos) assim como também produzem e secretam diferentes proteínas da matriz extracelular como fibronectina, lamininas e especialmente laminina-211. Estes resultados podem indicar que células poderão ter a capacidade de recuperar um nicho incompleto como é o caso da MDC1A. Este trabalho permitiu estabelecer um sistema in vitro, que quando otimizado, poderá representar uma nova abordagem para a aquisição de conhecimento acerca das dinâmicas moleculares desta doença durante os estádios fetais, e no futuro, ajudar no desenvolvimento de novas terapias.
The extracellular matrix (ECM) plays a crucial role in myogenesis and when disrupted can originate various conditions. When laminin-211 is not present skeletal muscle development is severely impaired leading to Merosin-deficient congenital dystrophy type 1A (MDC1A). Previous works in our group shed some light on the possible origin of this condition occurring during in utero development, when secondary myogenesis is undergoing. The absence of laminin-211 seems to lead to an early depletion of the muscle stem cell pool impairing myogenesis. In this work we used embryonic day (E) 18.5 fetus of the dyW/dyW mice model and characterized the main ECM proteins in both wild-type and mutant mice. The comparison of both genotypes showed that the absence of laminin α2 may somehow perturb other proteins expression. After characterization, we aimed to produce a system that allowed to study the relative contribution of both sources of laminin-211 (ECM and cells) during skeletal muscle development. We decellularized fetal skeletal muscle, producing a decellularized matrix (dECM) with a similar composition to the native tissue (most importantly laminin-211) but depleted of cellular con-tent. A low concentration SDS treatment, among other component, was optimized to better fulfill this compromise. The dECMs of both genotypes were seeded with C2C12 myoblasts and the cell number and protein production were analyzed. Our results show a tendency to have fewer cells in the mutant dECMs, suggesting that the absence of laminin-211 may difficult C2C12 cells adhesion/proliferation. These cells were able to colonize and contract the dECMs and express different ECM proteins, including laminin-211, opening the possibility for cells to be able to recover a defective niche. This work results in the production of an in vitro model representing a possible novel approach to better understand the molecular dynamics of MDC1A and, in the future, the potential development of new therapies.