<p dir="ltr">Tissue engineering utilizes polymers, cells, and other bioactive factors to promote regeneration within damaged tissue. The main works in this thesis employ naturally derived polymers for use in tissue engineering and explores ways to recapitulate native environments <i>in vitro</i>.</p><p dir="ltr">Collagen (col) is the most prevalent protein in the body. Col type I, II, and III are all fibril-forming collagens that provide structure to tissues. All three types polymerize <i>in vitro</i> to form hydrogels, and these hydrogels have often been studied for use in tissue engineering. Other applications include <i>in vitro </i>tissue models for studies on drug diffusion and drug delivery. Blending collagen types is of particular interest as col I is easier to source and is therefore cheaper than other collagen types. However, to confer biological signals to tissues where col II or III are more abundant (e.g., cartilage or cardiac tissue, respectively), col II or III can be added to col I to form col I/II or col I/III gels, respectively. Additionally, adding multiple types of col to hydrogel models better recapitulates the native environment and can better capture effects on drug diffusion. In this work, compared to col I alone, col I/II hydrogels polymerize more slowly, form more fibril bundles, result in softer hydrogels, and impede transport of larger macromolecules. On the other hand, col I/III gels polymerize at a similar rate to col I, create heterogenous fibril structures, are oftentimes stiffer than col I, and also impede transport of larger macromolecules. Additionally, this work explored the effect of polymerization temperature on blended gel polymerization and properties.</p><p dir="ltr">The second work evaluates col I/II hydrogels for a specific application: cartilage tissue engineering for osteoarthritic applications. Col II is the primary protein found in cartilage. Other components include: glycosaminoglycans, such as hyaluronic acid (HA) and chondroitin sulfate, chondrocytes (cartilage cells), and other small signaling molecules. Building on prior work in the group, high molecular weight hyaluronic acid (HA) was added to col I/II hydrogels, and cartilage differentiation of mesenchymal stem cells (MSCs) was assessed under ideal laboratory conditions and under pro-inflammatory, osteoarthritic conditions (i.e., cytokine-supplemented media of oncostatin M (OSM) at 10 ng/mL and tumor necrosis factor-α (TNF-α) at 20 ng/mL). The addition of HA did not dramatically impact cartilage differentiation of MSCs, however, HA did mitigate the effect of inflammation via downregulation of a degradative enzyme. HA had little impact on inflammatory cytokine production of interleukin (IL)-6 or IL-8, both of which are upregulated during osteoarthritis. However, a linear model suggests that HA and IL-8 are strongly correlated. Thus, this system should be explored further with different HA concentrations or presentations (e.g., chemically modified).</p><p dir="ltr">The last primary chapter of this thesis provides depth to the pro-inflammatory, osteoarthritic model used in the previous chapter. Different pro-inflammatory environments are studied using cytokines found in OA. MSC pellets (used in literature as controls to confirm chondrogenic potential of MSCs) were used to evaluate these inflammatory environments since MSCs are commonly used in tissue engineering. Six treatments were studied: negative control (without the chondrogenic growth factor TGF-β3), positive control (with the chondrogenic growth factor TGF-β3), and four cytokine treatments all with TGF-β3. First, IL-1β at 10 ng/mL was utilized as a comparison to literature. The other three cytokine groups used TNF-α at 20 ng/mL and OSM at 10 ng/mL individually or combined to form the main experimental group, OSM+TNF-α. All cytokine treatment groups limited cartilage production, but OSM decreased production to a statistically lesser extent than other cytokine groups. This trend was similar to observations made via immunostaining of cartilage matrix and gene expression analysis of aggrecan. Furthermore, OSM+TNF-α statistically lowered aggrecan gene expression. In terms of degradation, when compared to all other groups, OSM dramatically increased the protein expression of the degradative enzyme matrix metalloproteinase-13 (MMP-13). Evaluation of inflammatory markers (IL-6 and IL-8) revealed no signal for OSM-treated pellets. TNF-α yielded some signal after 1 week in culture but no signal after two weeks. IL-1β and OSM+TNF-α both resulted in sustained IL-6 and IL-8 expression, however, IL-1β exhibited large variance. Thus, each cytokine contributes to various pathways that are present in OA. Since the combination of OSM and TNF-α appeared to lower cartilage gene expression and resulted in sustained and reproducible IL-6 and IL-8 production, it may serve as a better model of OA than a single cytokine such as IL-1β.</p>

Mesenchymal stem cells (MSCs) have great potential to improve clinical outcomes for many inflammatory and degenerative diseases through delivery of exogenous MSCs via injection or cell-laden scaffolds and through mobilization and migration of endogenous MSCs to injury sites. MSC fate and function is determined by microenvironmental cues, specifically dimensionality, topography, and cell-cell interactions. MSC responses of migration and differentiation are the focus of this dissertation. Cell migration occurs in several physiological and pathological processes; migration mode and cell signaling are determined by the environment and type of confinement in three-dimensional (3D) models. Tendon injury is a common musculoskeletal disorder that occurs through cumulative damage to the extracellular matrix (ECM). Studies combining nanofibrous scaffolds and MSCs to determine an optimal topographical environment have promoted tenogenic differentiation under various conditions. We investigated cellular response of MSCs on specifically designed nanofiber matrices fabricated using a novel spinneret-based tunable engineered parameters production method (STEP). We designed suspended and aligned nanofiber scaffolds to study cellular morphology, tendon marker gene expression, and matrix deposition as determinants for tendon differentiation. The delivery and maintenance of MSCs at sites of inflammation or injury are major challenges in stem cell therapies. Enhancing stem cell homing could improve their therapeutic effects. Homing is a process that involves cell migration through the vasculature to target organs. This process is defined in leukocyte transendothelial migration (TEM); however, far less is known about MSC homing. We investigated two population subsets of MSCs in a Transwell system mimicking the vasculature; migrated cells that initiated transmigration on the endothelium and nonmigrated cells in the apical chamber that failed to transmigrate. Gene and protein expression changes were observed between these subsets and evidence suggests that multiple signaling pathways regulate TEM. The results of these experiments have demonstrated that microenvironmental cues are critical to understanding the cellular and molecular mechanisms of MSC response, specifically in homing and differentiation. This knowledge has identified scaffold parameters required to stimulate tenogenesis and signaling pathways controlling MSC homing. These findings will allow us to target key regulatory molecules and cell signaling pathways involved in MSC response towards development of regenerative therapies. / Ph. D. / Stem cell therapy is one way to improve tissue injury and inflammatory conditions, but to optimize such therapy, we need to study how the environment around cells influence turning them into the injured tissue and how to control their movement to these sites in order for mesenchymal stem cells (MSCs) to exert their therapeutic functions. MSCs move through and detect their environment through the material around them, including organization of the fibers they attach to and neighboring cells. Cell migration is an important cell behavior that occurs in normal and diseased processes. MSCs have great potential to improve clinical outcomes for many inflammatory and degenerative diseases whether through delivery of exogenous MSCs or through mobilization and migration of endogenous MSCs to injury sites. Tendon damage can occur slowly over time and optimal treatment for normal function after injury remains unknown. Equine MSCs were harvested from bone marrow and subjected to scaffolds of different fiber orientation to study whether cells develop characteristics of tendon cells. Cellular responses were similar between scaffolds of aligned fiber orientation. Manipulation of equine bone marrow MSCs through the use of specifically designed nanofiber scaffolds aid in understanding the mechanisms by which the cells respond and function in tendon development, injury, and repair. Inflammation is a necessary process after tissue injury; however, it must progress in a controlled manner and be resolved before it leads to tissue damage and dysfunction. MSCs function in regulating the effects of inflammation and immune cells; however, getting them to these sites and keeping them there remains challenging. MSCs adhere to and migrate through capillaries towards these sites, known as stem cell homing. Human bone marrow MSCs were loaded onto human synovial microvascular endothelial cells to study migration towards an inflammatory stimulus. This stimulus acted on the endothelial cells to produce another stimulus that attracted MSCs to the endothelial cells. These actions resulted in complete MSC migration through the endothelial cells and activated intracellular signals that can be used to increase the number of MSCs that reach the inflammatory sites and stimulate tissue-healing effects.

Partial or complete tears of the anterior cruciate ligament (ACL) can greatly afflict quality of life and often require surgical reconstruction with autograft or allograft tissue to restore native knee biomechanical function. However, limitations exist with these treatments that include donor site pain and weakness found with autografts, and longer "ligamentization" and integration times due to the devitalization of allograft tissue. Alternatively, a tissue engineering approach has been proposed for the fabrication of patient-specific grafts that can more rapidly and completely heal after ACL reconstruction. Electrospun micro-fiber networks have been widely utilized as biomaterial scaffolds to support the growth and differentiation of mesenchymal stem cells toward many tissue lineages including ligament. However, these micro-fiber networks do not possess suitable sizes and shapes for a ligament application and cannot support cell infiltration. The objective of this work was to develop techniques to 1) rapidly cellularize micro-fiber networks, 2) assemble micro-fiber networks into cylindrical composites, 3) provide cues to mesenchymal stem cells (MSCs) to guide their differentiation toward a ligament phenotype. The cellularization of micro-fiber networks was performed utilizing a co-electrospinning/electrospraying technique. Cells deposited within a cell culture medium solution remained where they were deposited and did not proliferate. The inclusion of space-filling hydrogel network such as collagen was necessary to reduce the density of the micro-fiber network to facilitate spreading. However, it became apparent that the incorporation of significant collagen phase was necessary for long-term MSC survival within the micro-fiber network. Next, two approaches were developed to fabricate large cylindrical, composites. The first approach utilized a co-electrospinning/electrospraying technique to generate micro-fiber/collagen composites that were subsequently rolled into cylinders. These cylindrical composites exhibited greater diameters and water weight percentages as collagen content increased. However, the high micro-fiber content of these composites was inhibitory to cell survival. In the second approach, thin layers (~5-10 fibers) of aligned electrospun PEUR fibers were encapsulated within a collagen gel and subsequently rolled the composites into cylinders. These sparse-fiber composites were nearly 98% by weight water and confocal imaging revealed the presence of sparse fiber layers (~5 fibers thick) separated by approximately 200 μm thick collagen layers. We hypothesize that the proliferation and migration of MSCs within these micro-fiber/collagen composites may not be restricted by the presence of a dense, non-manipulatable electrospun fiber network present in traditionally rolled fiber composites. Simple model platforms were then developed to study the influence of sparse micro-fibers on MSCs differentiation within a collagen hydrogel. MSCs in the presence of the softest (5.6 MPa) micro-fibers elongated and oriented to the underlying network and exhibited greater expression of scleraxis, and α-smooth muscle actin compared to the stiffest (31 MPa) fibers. Additionally, preliminary results revealed that the incorporation of fibroblast growth factor-2 and growth and differentiation factor-5 onto micro-fibers through chemical conjugation enhanced expression of the ligamentous markers collagen I, scleraxis, and tenomodulin. In conclusion, micro-fiber/collagen composite materials must possess sufficient space to support the infiltration and differentiation of MSCs. The strategies described in this document could be combined to fabricate large, micro-fiber/collagen composites that can support cell infiltration and provide relevant cues to guide the formation of an engineered ligament tissue. / Ph. D.

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

Yes / Mesenchymal stem cells (MSCs) are multipotent adult stem cells with great potential in regenerative medicine. One method for stimulating proliferation and differentiation of MSCs is via electrical stimulation (ES). A valuable approach for evaluating the response of MSCs to ES is to assess changes in gene expression, relative to one or more reference genes. In a survey of 25 publications that used ES on cells, 70% selected GAPDH as the reference gene. We conducted a study to assess the suitability of six potential reference genes on an immortalized human MSC line following direct current ES at seeding densities of 5000 and 10,000 cells/cm2 . We employed three methods to validate the most stable reference genes from qRT-PCR data. Our findings show that GAPDH and ACTB exhibit reduced stability when seeded at 5000 cell/cm2 . In contrast, we found that the most stable genes across both plating densities and stimulation regimes were PPIA and YWHAZ. Thus, in ES gene expression studies in MSCs, we support the use of PPIA and YWHAZ as an optimal reference gene pair, and discourage the use of ACTB and GAPDH at lower seeding densities. However, it is strongly recommended that similar verification studies are carried out based on cell type and different ES conditions.

Poor skeletal health results from aging and metabolic diseases such as obesity and diabetes and involves impaired homeostatic balance between marrow osteogenesis and adipogenesis. Tissue engineering provides researchers with the ability to generate improved, highly controlled and tailorable in vitro model systems to better understand mechanisms of homeostasis, disease, and healing and regeneration. Model systems that allow assembly of modules of MSCs, osteoblasts, and adipocytes in a number of configurations to engage in signaling crosstalk offer the potential to study integrative physiological aspects and complex interactions in the face of changes in local and systemic microenvironments. Thus, the overall goal of this dissertation was to examine integrative physiological aspects between MSCs, osteoblasts, and adipocytes that exist within the marrow microenvironment. To investigate the effects of intercellular signaling in different microenvironmental contexts, methods were developed to photolithographically pattern and assemble cell-laden PEG-based hydrogels with high spatial fidelity and tissue-scale thickness for long-term 3D co-culture of multiple cell types. This platform was applied to study effects of crosstalk between MSCs, osteoblasts and adipocytes on markers of differentiation in each cell type. Additionally, responses of MSCs to systemic perturbations in glucose concentration were modulated by mono-, co-, and tri-culture with these cell types in a model of diabetes-induced skeletal disease. Together, these studies provided valuable insight into unique and differential effects of intercellular signaling within the niche environment of MSCs and their terminally differentiated progeny during homeostatic and pathological states, and offer opportunities further study of integrative physiological interactions between mesenchymal lineage cells.

Les lymphocytes Th17 forment une population de cellules T CD4+ pro-inflammatoires, impliqués non seulement dans l'élimination d'agents pathogènes, mais ayant aussi un rôle délétère dans l'induction de maladies inflammatoires chroniques. Ils expriment spécifiquement le récepteur de chimiokines CCR6, qui a pour ligand le CCL20 mais aussi les β-defensine-1, 2 et 3, peptides ayant une activité antimicrobienne. Les cellules souches mésenchymateuses (CSMs) représentent une population cellulaire hétérogène exerçant diverses propriétés immunomodulatrices.Les résultats obtenus dans ce travail de thèse montrent que l'environnement inflammatoire contribue à augmenter l'adhésion des lymphocytes Th17 aux CSMs, et qu'elle est régulée par l'interaction du CCR6 avec ses ligands ; que les CSMs exercent, en partie via la sécrétion de PGE2, des effets anti-inflammatoires en faisant acquérir un phénotype régulateur aux lymphocytes Th17 différenciés, soulignant ainsi la plasticité de ces derniers.De plus, nous avons montré que les lymphocytes Th17 activés par l'antigène produisent du CCL20 et induisent, via la production de l'IL-17 et de l'IL-22, la sécrétion d'hBD-2, mais pas celle des hBD-1 et 3, par des kératinocytes épidermiques humains et de la peau reconstituée; que le CCL20, ainsi que la hBD-2, induisent l'arrêt de ces cellules sur l'endothélium enflammé in vitro en conditions de cisaillement. Finalement, l'activation spécifique d'antigène des lymphocytes Th17 entraîne une perte de l'expression de CCR6, ce qui provoque ainsi un état transitoire de non réponse à une nouvelle stimulation de ces cellules avec les ligands de CCR6, permettant leur migration ultérieure hors du tissu enflammé. / Th17 cells form a population CD4+ T cells with strong pro-inflammatory properties that are not only involved in the clearance of pathogens, but also play a deleterious role of in the pathogenesis of inflammatory disease. Th17 cells specifically express CCR6, a chemokine receptor that binds to its unique chemokine ligand, CCL20, as well as to human β-defensin (hBD)-1, 2 and 3, peptides with anti-microbial activity. Mesenchymal stem cells (MSC) represent a heterogenous population that exert broad immunomodulatory effects.The results from the studies carried out during this thesis show that the inflammatory environment contributes to increased adhesion of Th17 cells to MSCs, which is mediated via the interaction of CCR6 with its ligands, and that MSCs exert, in part via the secretion of PGE2, anti-inflammatory effects through the induction of a T regulatory cell phenotype in fully differentiated tissue-infiltrating Th17 cells, thereby underscoring the plasticity of the latter cells.Furthermore, the results show that antigen-activated Th17 cells produce CCL20 and induce, via the production of both IL-17 and IL-22, the secretion of hBD-2, but not 1 and 3, by normal human epidermal keratinocytes and reconstituted skin, and that CCL20, as well as hBD-2, induce arrest of these cells onto inflamed endothelium in vitro under conditions of shear stress. Finally, antigen-specific activation of Th17 cells also causes a loss of CCR6 expression from their cell surface and thus results in a transitory state of non-responsiveness to further stimulation of these cells with CCR6 ligands, which is likely to permit their subsequent migration out of inflamed tissue.

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.

L'utilisation grandissante des cellules souches mésenchymateuses (CSM) en ingénierie tissulaire augmente la nécessité d'améliorer leur expansion. Ces travaux ont concerné l'étude d'un procédé performant d'expansion de CSM porcines en mode agité. Tout d'abord, un milieu de culture a été adapté aux CSM porcines multipotentes. Puis, différents modes d'expansion en conditions agitées ont été évalués avec les cellules fixées sur des microporteurs. La culture sur le microporteur Cytodex 1 a permis d'atteindre une vitesse spécifique de croissance de 0,54 j-1, supérieure à celle observée en flacon statique (0,31 j-1), avec les mêmes conditions de culture. En parallèle, une méthode de comptage innovante a été proposée pour le dénombrement automatique des cellules cultivées sur Cytodex 1, sans passer par une étape de trypsination. Enfin, les conditions opératoires du procédé d'expansion ont été étudiées. En comparaison d'une culture de CSM sur Cytodex 1 sans agitation, une agrégation des cellules et une baisse apparente de la concentration cellulaire ont été observées à 25 et 75 rpm. Par ailleurs, l'ajout de microporteurs au cours d'une culture de 300 h, réalisée dans un système de culture agité à 25 rpm et dans un volume de 200 mL, a permis de prolonger la prolifération cellulaire en évitant l'agrégation tout en maintenant la multipotence des CSM. Une concentration cellulaire de 3 x 105 cellules/mL a été obtenue, au lieu de 1,2 x 105 cellules/mL en flacons statiques avec les mêmes conditions de culture. Un procédé performant d'expansion de CSM porcines en conditions agitées a ainsi pu être proposé / The extensive use of mesenchymal stem cells (MSC) in tissue engineering increases the necessity to improve the expansion performance. This work aimed at studying an efficient expansion process for porcine MSC in agitated mode. First, a culture medium was adapted to the multipotent porcine MSC. Then, various expansion modes and agitation conditions were evaluated with the cells fixed on microcarriers. Cultures on the Cytodex 1 microcarrier enabled to reach a specific growth rate of 0.54 d-1, which was higher than the one observed in static T-flasks (0.31 d-1), with the same culture conditions. In parallel, an innovative counting method was proposed for the automatic enumeration of cells cultivated on Cytodex 1, without passing by a trypsination step. Finally, the operating conditions of the expansion process were studied. Compared to a culture of MSC on non-agitated Cytodex 1 microcarriers, cell aggregation occurred and an apparent decrease in the cell concentration was observed at an agitation rate of 25 and 75 rpm. Moreover, the addition of microcarriers during a 300 h culture, performed in an agitated culture at 25 rpm and in a volume of 200 mL enabled to prolong the cell proliferation without any aggregation, while maintaining the multipotency of the cells. A cell concentration of 3 x 105 cells/mL was obtained, instead of the 1.2 x 105 cells/mL in static flasks with the same culture conditions. An efficient expansion process for porcine MSC under agitated conditions has therefore been proposed

La régénération osseuse est un processus impliquant de nombreux types cellulaires comme les ostéoblastes, les chondrocytes ou les cellules stromales mésenchymateuses (CSM). Les CSM possèdent des capacités de différenciation suggérant leur implication dans ce processus de réparation. La régénération osseuse est le fruit de la coordination complexe de l'activité de nombreux facteurs de croissance. Parmi eux, l'« Heparin affin regulatory peptide » (HARP) est fortement exprimé dans le callus durant la régénération mais son rôle n'est pas clairement établi. Le but de ce travail de thèse a été (1) d'évaluer les effets de HARP sur les propriétés de migration, de prolifération et de différenciation des CSM in vitro ; (2) évaluer la capacité de HARP à induire une formation osseuse ou une régénération osseuse in vivo.Nos résultats démontrent que HARP est chémoattractant pour les CSM et potentialise leur prolifération. De plus, nous montrons pour la première fois que le traitement de CSM par HARP durant leur chondroinduction conduit à une différenciation chondrocytaire de type hypertrophique. Ce type cellulaire est primordial dans les derniers stades de la formation osseuse endochondrale qui se met en place durant la croissance osseuse, mais également durant la réparation. L'implantation de biomatériaux associés à HARP dans un défaut osseux de condyle fémoral a conduit à la formation de cartilage et d'os dans l'implant, reproduisant le mécanisme physiologique de formation osseuse endochondrale. Le biomatériau seul n'a été envahi que par du tissu fibreux.Durant les processus de réparation tissulaire, les glycosaminoglycannes (GAG), des chaînes polysaccharidiques sulfatées, composants majeurs de la matrice extracellulaire, participent à la modulation des effets des facteurs de croissance durant la réparation. Récemment, des mimétiques structuraux et fonctionnels des GAG ont été développés. Durant ma thèse, j'ai été associé au travail d'un doctorant de l'équipe de P.Albanese, qui a montré que des mimétiques de GAG induisent une différenciation ostéoblastique des CSM en l'absence de traitement ostéoinducteur. L'implantation sous-cutanée de biomatériaux covalemment associés aux mimétiques ont également été menées, et ont permis d'observer des potentialisations des processus de vascularisation de l'implant et de l'activité ostéoclastique. Ces resultats ont permis de valider l'interêt des GAG mimétiques dans le cadre des thérapies de régénération osseuse.Cette étude démontre pour la première fois les effets chondroinducteurs directs de HARP sur la production de molécules de la matrice cartilagineuse par les CSM in vitro, mais également sur la synthèse de tissu cartilagineux in vivo. Les effets de HARP observés sur la régénération osseuse confirment qu'il pourrait être un bon candidat en chirurgie orthopédique en permettant une régénération de type endochondrale typique de la réparation physiologique. De plus les nouvelles stratégies developpées dans le laboratoire sur la fonctionnalisation covalente de biomateriaux par des GAG mimétiques, meriteraient d'etre testées en association avec HARP, afin d'augmenter sa demi-vie et de controler son relarguage et ses activités biologiques in vivo. / Bone regeneration is a complicated process which involved many cellular types such as osteoblasts, chondrocytes and mesenchymal stromal cells (MSC). MSC can differentiate toward chondrocytes and osteoblasts, suggesting their implication in bone regeneration processes. Bone reparation involved a complex coordination of growth factors. Among them, heparin affin regulatory peptide (HARP) is found in callus during regeneration. However, its role is poorly understood. The aim of this thesis was (1) to evaluate HARP effects on proliferation, migration and differentiation of MSC in vitro, (2) to evaluate HARP ability to promote bone regeneration or bone formation.Our results demonstrate that HARP has chemoattractive and proliferative properties on human MSC. Moreover, we show for the first time that HARP commits human MSC toward hypertrophy during chondrogenesis. This is of great interest since hypertrophic chondrocytes are of primary importance in the late stage of endochondral bone formation. We further tested the association of HARP to scaffolds in a model of bone regeneration in femoral defect in rat. HARP associated scaffolds showed an invasion of cartilage and bony tissues, mimicking endochondral bone formation, whereas scaffold alone was just filled with fibrous tissue.During regenerative processes glycosaminoglycans, polysaccharides sulfated chains, are known as major components of the extracellular matrix and modulate the effects of growth factors during regenerative processes. Recently, structurally and functionally mimetics of GAG had been developed. During my PhD thesis, I was associated to the work of a doctoral student of P. Albanese who showed that GAG mimetics induce osteoblastic differentiation of MSC without any other osteoinductive treatment. The ectopic implantation of mimetic associated scaffolds didn't show effects on osteoformation but induced an enhancement of vascularization and of osteoclastic activity, both related to tissue remodeling. These results validate that GAG mimetics are of great interest in bone regenerative field.This study demonstrates for the first time the chondroinductive potential of HARP through its ability to induce cartilage specific matrix production by MSC in vitro but also by inducing cartilage tissue synthesis in vivo. The effects of HARP observed on bone regeneration, by inducing an endochondral bone formation similar to that observed in normal bone regeneration, confirm that HARP could be a good candidate in orthopedic surgery. Moreover, scaffold covalently linked with GAG mimetics should be tested in association with HARP. This strategy could increase the half life, control the release and potentiate HARP properties in vivo.