Philipp Hoffmann Untersuchung zur Fixierung von Knorpelgewebe mittels laserinduzierter Koagulation Aus der Chirurgischen Tierklinik der Veterinärmedizinische Fakultät der Universität Leipzig, angefertigt im Forschungszentrum für Medizintechnik und Biotechnologie GmbH, Bad Langensalza Eingereicht im Januar 2012 97 Seiten, 59 Abbildungen, 9 Tabellen, 318 Literaturangaben 10 Seiten Anhang Schlüsselwörter: Laser, Löten, Knorpelgewebe, Zugfestigkeit, thermische Schäden Gelenkerkrankungen zählen zu den häufigsten Ursachen von Bewegungseinschränkungen in der Human- und Veterinärmedizin. Neben der konservativen Therapie gibt es zahlreiche chirurgische Therapieansätze, unter denen die verschiedenen Verfahren der autologen Chondrozytenimplantation (ACI) vermehrt in den Fokus gerückt sind. Als unbefriedigend stellt sich aktuell die Fixierung der Implantate bzw. Transplantate dar. Ziel der vorliegenden Arbeit war es, zunächst in vitro, unter Nutzung von Gelenkknorpelgewebe aus Kadavermaterial (Schwein, Rind), ein Verfahren einzuarbeiten, mit dem es möglich ist, durch laserinduzierte Koagulation eines Lötmittels eine Verbindung zwischen zwei Knorpelfragmenten bei einer möglichst geringen Gewebeschädigung herzustellen. Als Lötmittel war ein geeignetes Chromophoren-Protein-Gemisch (CPG) herzustellen, welches so auf die Wellenlänge des zur Verfügung stehenden Lasers angepasst wurde, dass die Herstellung von Lötverbindungen möglich war. Die mechanische Festigkeit der Lötverbindungen wurde in verschiedenen Studien zur Optimierung der Lötmittelzusammensetzung und der Lasereinstellungen durch die Bestimmung der Zugkraft geprüft. Ebenso wurden Untersuchungen zum Auftreten thermischer Schäden am Gewebe durch das lasergestützte Löten vorgenommen. Ausgehend von der Untersuchung der Absorptionseigenschaften verschiedener Chromophore und Proteine wurden verschiedene, auf die Wellenlänge des Lasers (810 nm Diodenlaser) abgestimmte, CPG unter Verwendung des Farbstoffes Indocyaningrün (ICG), welcher in dem in der Humanmedizin zugelassenen Diagnostikum ICG-Pulsion® (PULSION Medical Systems AG, München) enthalten ist, und bovinem Serumalbumin (BSA) hergestellt. Knorpelgewebe absorbiert die Strahlung des Diodenlasers (810 nm) kaum (μa ≈ 0 bis 0,02 cm-1). Das Lötmittel (ICG + BSA), dessen Absorptionsmaximum mit 790 nm nah an der Emissionswellenlänge des Lasers liegt, absorbiert hingegen in diesem Wellenlängenbereich gut. Dadurch kann eine direkte Schädigung des Knorpelgewebes durch die Absorption der Laserstrahlung vermieden werden. In den Studien wurden drei Lötmittel mit unterschiedlichen Anteilen an ICG (1 %, 0,25 % und 0,025 %) bei einem BSA-Gehalt von 60 % verwendet. Die Lötmittel mit 0,025 % und 0,25 % ICG wurden zur Prüfung der Zugfestigkeit der gelöteten Verbindung in Abhängigkeit von der Leistungsdichte und der Expositionszeit untersucht. Das Lötmittel mit 0,025 % ICG wurde in den Untersuchungen zur Abhängigkeit der Zugfestigkeit von der Tierspezies, der Entnahmestelle des Knorpelgewebes und der Lötmitteldicke genutzt. Einflüsse der Lagerung des Lötmittels und der Anzahl an Lötmittelpunkten auf die Zugfestigkeit wurden mit dem Lötmittel mit 0,25 % ICG untersucht. Zusätzlich war zu prüfen ob durch ein Knorpelgewebefragment hindurch das CPG zu koagulieren ist. Zur Untersuchung thermisch bedingter Schäden wurden zum einen Temperaturmessungen an der Oberfläche des Knorpelgewebes, im Bereich des Lötmittels und in verschiedenen Tiefen unterhalb des Lötmittels durchgeführt. Zum anderen erfolgten histologische Untersuchungen der Knorpelgewebeproben nach Laseranwendung. Es ist möglich, mittels laserinduzierter Koagulation eines CPG eine Verbindung von Knorpelgewebe vom Schwein und Rind herzustellen. Mit Steigerung der Leistungsdichte und Verlängerung der Expositionszeit kommt es zur Erhöhung der Zugfestigkeit. Die Zugfestigkeiten waren bei Koagulation des CPG durch das Knorpelfragment hindurch niedriger als die Zugfestigkeiten mit aufgelegtem Lötmittel. Unter Laseranwendung kommt es zu einem steilen Ansteigen der Temperatur im Lötmittel bis zum Erreichen einer Höchsttemperatur. Die Steilheit des Temperaturanstieges und die sich einstellenden Temperaturen nehmen mit Erhöhung des im Lötmittel enthaltenen ICG-Gehaltes und der am Laser eingestellten Leistung zu. Die Temperaturerhöhung ist jedoch weitgehend auf das Lötmittel und dessen Randbereiche begrenzt. Die histologischen Untersuchungen verdeutlichten, dass die Laserbestrahlung von Knorpelgewebe mittels Diodenlaser (810 nm) nur eine sehr geringe Schädigung verursacht. Unter Verwendung eines Lötmittels kommt es durch die vom Lötmittel absorbierte Energie zu Schäden am umliegenden Knorpelgewebe. Diese Schädigung ist auf Randbereiche des Lötmittels begrenzt und nimmt mit steigender Leistung und Expositionszeit zu. Bei einer Leistungsdichte von (5,09 W/cm2) konnte eine Verbindung zwischen zwei Knorpelfragmenten erzielt werden, die bei einer Zugkraft von 13,3 N/cm2 nachgibt und bei der die Schädigungen des Knorpelgewebes minimal sind. Die vorliegenden Untersuchungen haben gezeigt, dass es möglich ist, Knorpelfragmente mittels laserinduzierter Koagulation eines CPGs miteinander zu fixieren. / Philipp Hoffmann Investigation for the fixation of articular cartilage tissue using laser-induced coagulation From the Large Animal Clinic for Surgery, Faculty of Veterinary Medicine, University of Leipzig, prepared at Research Centre of Medical Technology and Biotechnology GmbH, Bad Langensalza Submitted in January 2012 97 Pages, 59 figures, 9 tables, 318 references, 10 pages appendices Keywords: laser, soldering, cartilage tissue, tensile strength, thermal damage Joint diseases are among the most common causes of restricted movement of patients in the human and veterinary medicine. In addition to the conservative therapy, there are numerous surgical therapies, under which the various methods of autologous chondrocyteimplantation, have moved increasingly into the focus of scientific and clinical interest. As problematic and unsatisfactory is currently the fixation of the implants. The aim of this study was, first in vitro, taking advantage of articular cartilage tissue from cadaver material (pig, cattle) to incorporate a process by which it is possible to produce by laser-induced coagulation of solder a connection between two cartilage fragments with the smallest possible tissue damage. As solder was a suitable chromophore-protein-mixture (CPG) to establish which it was adapted to the wavelength of the laser is available, that the production of solder joints was possible. The mechanical strength of solder joints has been examined in several studies to optimize the laser settings and the solder ingredients by determining the tensile strength. Likewise, studies on the occurrence of thermal damage to the tissues were made by the laser-assisted soldering. Based on the study of the absorption properties of various chromophores and proteins the wavelength of the laser (810 nm diode laser) was tuned, and different CPG using the dye indocyanine green (ICG), which is within the acceptable in human medicine ICG-Pulsion ® (Pulsion Medical Systems AG, Munich) is included, and bovine serum albumin (BSA) were prepared. Articular cartilage tissue absorbs the radiation of the diode laser (810 nm) hardly (uA ≈ 0 to 0.02 cm–1). The solder (ICG + BSA), whose absorption maximum at 790 nm is close to the emission wavelength of the laser is absorbed. This can be avoided direct damage to the cartilage tissue through the absorption of laser radiation. In the studies, three solders were used with different proportions of ICG (1 %, 0.25 % and 0.025 %) at a content of 60 % BSA. The solder with 0.025 % and 0.25 % ICG were studied to test the tensile strength of the soldered connection as a function of power density and exposure time. The solder containing 0.025 % ICG was used in the investigations of the dependence of tensile strength of the animal species, the donor site of the cartilage and the solder thickness. Influences of storage the solder and the number of solder dots on the tensile strength were investigated with the solder with 0.25 % ICG. In addition it was to examine if it is possible to coagulate the CPG through an articular cartilage fragment. To investigate thermally induced damage to temperature measurements were performed on the surface of the cartilage tissue in the area of the solder and at various depths below the solder. Secondly, histological examinations were made of the articular cartilage after laser application. It is possible to produce by laser-induced coagulation of a CPG an articular cartilage bonding of pig and cattle. With increasing power density and lengthening the exposure time leads to the increase in tensile strength. The tensile strengths were measured with coagulation of the CPG passed through the cartilage fragment is lower than the tensile strengths with applied solder. Under laser application leads to a steep rise in temperature in the solder to reach a maximum temperature. The rate of temperature rise increases with increasing the solder contained in ICG content and on the laser power set. The temperature rise is limited largely to the solder and its peripheral areas. The histological examinations showed that the laser irradiation of cartilage tissue using diode laser (810 nm) only a very little damage caused. Using a solder it comes through the energy absorbed by the solder and damage to the surrounding articular cartilage tissue. This damage is limited to border areas and the flux increases with increasing power and exposure time. At a power density of (5.09 W/cm2) was a connection between two cartilage fragments are obtained, which yields at a tensile force of 13.3 N/cm2 and where the damage to the cartilage tissue is minimal. The present studies have shown that it is possible cartilage fragments by laser-inducedcoagulation of a CPG to fix each other.

Manual segmentation of cartilage tissue in micro CT images of mouse embryos is a very time consuming process and significantly increases the time required for the research of mammal facial structure development. This problem might be solved by using a fully-automatic segmentation algorithm. In this diploma thesis a fully-automatic segmentation method is proposed using a convolutional neural network trained on manually segmented data. The architecture of the proposed convolutional network is based on the U-Net architecture with it's encoding part substituted for the encoding part of the VGG16 classification convolutional neural network pretrained on the ImageNet database of labeled images. The proposed network achieves Dice coefficient 0.8731 ± 0.0326 in comparison to manually segmented images.

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Cartilage regeneration is one of the most widely studied areas in tissue-engineering. Despite significant progress, most efforts to date have only focused on generating homogenous tissues whose bulk properties are similar to articular cartilage. However, anatomically and functionally, articular cartilage consists of four spatially distinct regions: the superficial, transitional, deep, and calcified zones. Each zone is characterized by unique extra-cellular matrix (ECM) compositions, mechanical properties, and cellular organization. The ECM is primarily composed of type II collagen and glycosaminoglycans (GAGs), whose relative concentrations vary between zones and therefore lead to distinctive mechanical properties. One of the major unsolved challenges in engineering cartilage has been the inability to regenerate tissue that mimics the zonal architecture of articular cartilage. Recent studies have attempted to imitate this spatial organization using zone-specific chondrocytes isolated from donor animal cartilage. Directed differentiation of a single stem population into zonally organized native-like articular cartilage has not yet been reported. This dissertation reports that hydrogels, incorporating both synthetic and natural polymers as well as cell-induced degradability, are suitable for generating zone-specific chondrogenic phenotypes from a single MSC population. Specifically, cues provided from the unique combinations of chondroitin sulfate (CS), hyaluronic acid (HA), and MMP-sensitive peptide (MMP-pep) within a PEG-based hydrogel, direct the chondrogenic differentiation of MSCs. The findings of this dissertation demonstrate the capability of creating native-like and mechanically relevant articular cartilage consisting of zone specific layers. This ability provides a new direction in cartilage tissue engineering and could be invaluable for cartilage repair if incorporated with current minimally invasive surgical techniques. / text

Cartilage tissue engineering has been emerging as a promising therapeutic approach, where engineered constructs or scaffolds are used as temporary supports to promote regeneration of functional cartilage tissue. Hybrid constructs fabricated from cells, hydrogels, and solid polymeric materials show the most potential for their enhanced biological and mechanical properties. However, fabrication of customized hybrid constructs with impregnated cells is still in its infancy and many issues related to their structural integrity and the cell functions need to be addressed by research. Meanwhile, it is noticed that nowadays monitoring the success of tissue engineered constructs must rely on animal models, which have to be sacrificed for subsequent examination based on histological techniques. This becomes a critical issue as tissue engineering advances from animal to human studies, thus raising a great need for non-invasive assessments of engineered constructs in situ. To address the aforementioned issues, this research is aimed to (1) develop novel fabrication processes to fabricate hybrid constructs incorporating living cells (hereafter referred as “construct biofabrication”) for cartilage tissue regeneration and (2) develop non-invasive monitoring methods based on synchrotron X-ray imaging techniques for examining cartilage tissue constructs in situ. Based on three-dimensional (3D) printing techniques, novel biofabrication processes were developed to create constructs from synthetic polycaprolactone (PCL) polymer framework and cell-impregnated alginate hydrogel, so as to provide both structural and biological properties as desired in cartilage tissue engineering. To ensure the structural integrity of the constructs, the influence of both PCL polymer and alginate was examined, thus forming a basis to prepare materials for subsequent construct biofabrication. To ensure the biological properties, three types of cells, i.e., two primary cell populations from embryonic chick sternum and an established chondrocyte cell line of ATDC5 were chosen to be incorporated in the construct biofabrication. The biological performance of the cells in the construct were examined along with the influence of the polymer melting temperature on them. The promising results of cell viability and proliferation as well as cartilage matrix production demonstrate that the developed processes are appropriate for fabricating hybrid constructs for cartilage tissue engineering. To develop non-invasive in situ assessment methods for cartilage and other soft tissue engineering applications, synchrotron phase-based X-ray imaging techniques of diffraction enhanced imaging (DEI), analyzer based imaging (ABI), and inline phase contrast imaging (PCI) were investigated, respectively, with samples prepared from pig knees implanted with low density scaffolds. The results from the computed-tomography (CT)-DEI, CT-ABI, and extended-distance CT-PCI showed the scaffold implanted in pig knee cartilage in situ with structural properties more clearly than conventional PCI and clinical MRI, thus providing information and means for tracking the success of scaffolds in tissue repair and remodeling. To optimize the methods for live animal and eventually for human patients, strategies with the aim to reduce the radiation dose during the imaging process were developed by reducing the number of CT projections, region of imaging, and imaging resolution. The results of the developed strategies illustrate that effective dose for CT-DEI, CT-ABI, and extended-distance CT-PCI could be reduced to 0.3-10 mSv, comparable to the dose for clinical X-ray scans, without compromising the image quality. Taken together, synchrotron X-ray imaging techniques were illustrated promising for developing non-invasive monitoring methods for examining cartilage tissue constructs in live animals and eventually in human patients.

Le cartilage a une capacité de régénération très limitée car il n'est pas vascularisé. Laréparation de ce tissu est un défi et les techniques chirurgicales actuelles sont insatisfaisantes à longterme. Le cartilage est donc un bon candidat pour l'ingénierie tissulaire. La transplantation dechondrocytes autologues (TCA) a été la première thérapie cellulaire développée en rhumatologie maiscette procédure implique une amplification des cellules qui aboutit à une perte du phénotypechondrocytaire (perte de l'expression du collagène de type II, protéine majoritaire du cartilage), auprofit d'un phénotype fibroblastique (caractérisé par l'expression du collagène de type I, retrouvé dansles tissus fibreux). La TCA conduit donc à une greffe de chondrocytes dédifférenciés produisant unfibrocartilage, dont les propriétés mécaniques sont inférieures à celles du cartilage articulaire.Aujourd'hui, les agences de santé au niveau international s'accordent pour dire que cette procédurenécessite d'être améliorée, par un meilleur contrôle du phénotype cellulaire et l'utilisation debiomatériaux pour mieux combler les lésions articulaires. Il s'agit donc de passer de la thérapiecellulaire à l'ingénierie tissulaire du cartilage.L'objectif de nos travaux a été d'évaluer la capacité d'un gel innovant de peptides autoassemblants,l'hydrogel IEIK13, à jouer le rôle de support pour des chondrocytes humains afin qu'ilsproduisent une matrice cartilage sous l'action de facteurs chondrogéniques. L'objectif visé a été lacréation d'un gel cartilage implantable par arthroscopie. Le défi a été de surmonter la dédifférenciationdes chondrocytes inhérente à leur amplification et incontournable pour augmenter le réservoircellulaire. L'amplification de chondrocytes humains a été réalisée en présence de FGF-2 et d'insuline(cocktail FI) puis leur redifférenciation a été induite en gel IEIK13 sous l'action de BMP-2, d'insuline etd'hormone T3 (cocktail BIT). C'est la combinaison sélective des deux cocktails qui permet la séquencedédifférenciation-redifférenciation. Le phénotype des chondrocytes et la nature de la matriceextracellulaire synthétisée en gel ont été évalués dans un premier temps in vitro, par des analyses dePCR en temps réel, Western-blots et d'immunohistochimie. Dans un second temps, nous avonstransplanté le gel cartilage dans des lésions articulaires de genou d'un modèle original de primate nonhumain(singe cynomolgus), un type de gros animal dont la posture et le fonctionnement desarticulations s'apparentent à l'homme. Nos études d'imagerie non invasive (telle qu'elle est pratiquéechez l'homme) et immunohistochimiques trois mois après implantation montrent une réparationsatisfaisante des lésions, en comparaison avec les lésions laissées non comblées. L'ensemble de nosrésultats montre pour la première fois que l'hydrogel IEIK13 est un biomatériau favorable pourreconstruire le cartilage et que le primate non-humain est un modèle préclinique unique pour évaluerl'efficacité de l'ingénierie tissulaire du cartilage / Cartilage is not vascularized and presents poor capacity of self-regeneration. Repairing thistissue is a challenge and current surgical techniques are not satisfactory in the long term. Cartilage isthus a good candidate for tissue engineering. Autologous chondrocyte transplantation (ACT) was thefirst cell therapy developed for cartilage repair. This procedure implies amplification of cells whichresults in chondrocyte dedifferentiation (loss of expression of type II collagen, the major protein ofcartilage and acquisition of expression of type I collagen, the major protein found in fibrous tissues).Thus, ACT results in implantation of fibroblastic cells producing fibrocartilage with biomechanicalproperties inferior to native articular cartilage. The international health agencies agree that ACT needsto be improved with better control of the chondrocyte phenotype and use of biomaterials. Therefore,cell therapy of cartilage needs to move towards tissue engineering of cartilage.The objective of our study was to evaluate the capacity of an innovative self-assemblingpeptide (IEIK13) to support cartilage matrix production by human chondrocytes. Our goal was to createa cartilage gel that can be implanted by arthroscopy. A main challenge was to meet the problem ofchondrocyte dedifferentiation induced by cell amplification necessary to increase the cellularreservoir. Amplification of human chondrocytes was performed in the presence of FGF-2 and insulin(cocktail FI), and redifferentiation was subsequently induced in IEIK13 gel with BMP-2, insulin, andtriiodothyronine T3 (cocktail BIT). The specific combination of these two cocktails alloweddedifferentiation-redifferentiation of chondrocytes. The status of the chondrocyte phenotype and thenature of the extracellular matrix secreted in gel were first assessed in vitro by real-time PCR, Westernblottingand immunhostochemistry analyses. With a view of clinical application, we then transplantedIEIK13-engineered cartilages into defects created in knees of an original model of non-human primate(cynomolgus monkey), a type of large animal whose anatomy and biomechanics mimic human. Ournon-invasive imaging analyses and our inmmunohistochemical studies performed three months afterimplantation show correct reparation of the lesions, in comparison with the defects left untreated.Altogether, our results demonstrate for the first time that IEIK13 is a suitable biomaterial for cartilagerepair and that cynomolgus monkey represents a unique preclinical model to evaluate efficiency ofcartilage tissue engineering.

Osteoarthritis (OA), a degenerative disease of articular joints, is the leading cause of chronic disability in the US and affects more than a third of adults over 65 years old. Due to the obesity epidemic and an aging population, the prevalence of OA is expected to rise in both young and old adults. There are no disease modifying OA drugs. Therefore, providing any treatment options that delay the onset or progression of OA is highly desirable. The scope of this dissertation examines two different strategies to promote translational therapies for OA. The first approach investigated whether Smad ubiquitin regulatory factor 2 (Smurf2), an E3 ubiquitin ligase, could be a potential therapeutic target for OA. The second approach examined the incorporation of small chemical residues to enhance the physical and bioactivity of a bioinert scaffold for cartilage tissue repair. Overexpression of Smurf2 in chondrocytes was shown to accelerate spontaneous OA development in mice. We hypothesized that reduced Smurf2 expression could slow the progression of OA and enhance the performance of cells for cartilage repair. By performing surgical destabilization of the medial meniscus (DMM) on Smurf2-deficient mice, loss of Smurf2 was shown to mitigate OA changes in young mice but this protection diminished in older mice. Assessment of Smurf2-deficient chondrocytes in vitro revealed an upregulation of chondrogenic genes compared to wild-type; however, these differences were not seen at the protein level, deterring its potential use for cell-based therapies. During the course of this study, new insights about how age and sex affects different joint compartments in response to DMM surgery were also uncovered. These results broadened existing understanding of DMM-induced OA in mice but also questioned the validity of such a model to identify disease modifying targets that are translatable to OA in humans with advanced age. Due to a lack of innate repair mechanisms in cartilage, damage to cartilage increases the risk of developing OA early. Tissue engineering provides a unique strategy for repairing damaged cartilage by delivering cells in a well-controlled environment that can promote the formation of neotissue. We hypothesized that synthetic chemical residues could enhance the mechanical properties of a bioinert scaffold and promote matrix production of encapsulated chondrocytes. Covalent incorporation of small anionic or zwitterionic chemical residues in a polyethylene glycol-based hydrogel improved its stiffness and resistance to fluid flow, however, the resulting physical environment can also exert a dominant negative effect on matrix production of encapsulated chondrocytes. These results suggest that modulating the biosynthesis of chondrocytes with biochemical signals requires a concurrent reduction in any conflicting mechanotransduction signaling, emphasizing the importance of a degradable system to promote new cartilage formation. In summary, this dissertation establishes Smurf2 as a modulator of OA progression but implies that other factors such as age or protein(s) with redundant Smurf2 functions may play a role in limiting its effect as a therapeutic target. This work also reveals fundamental biology about how chondrocytes behave in response to physical and chemical cues in their microenvironment, which will aid in the design of better scaffolds for cartilage tissue engineering.

Osteoarthritis is a prevalent disease that affects the articular cartilage of the joints. Millions of people suffer worldwide and it is a major cause of disability in the United States. Current research for treatments of osteoarthritis are studying tissue-engineered cartilage in vitro generated by articular chondrocytes. A challenge faced in vitro for cartilage tissue engineering is the failure of chondrocytes to produce adequate expression of type II collagen. Surprisingly, the media commonly used in vitro lacks 14 vitamins and minerals present in the physiological environment of chondrocytes. Therefore, studying the interactions between micronutrients and chondrocytes may help in potentially increasing the amount of type II collagen expressed by these cells. This project studied the combinatorial effects of vitamins and minerals in defined chondrogenic media on type II collagen expression. Linolenic acid was determined to have predominantly negative effects on chondrogenesis and Vitamin B7 to have beneficial effects. Multiple vitamins and minerals displayed significant interactions, both positive and negative.

Les cellules stromales/souches mésenchymateuses de la gelée de Wharton humaines (CSM-WJ) représentent une source abondante et intéressante de cellules souches pour des applications en ingénierie cellulaire et tissulaire. Leur origine fœtale leur confère des caractéristiques spécifiques par rapport aux cellules stromales/souches mésenchymateuses isolées à partir de moelle osseuse humaine (CSM-MO). Tout d'abord, le but de ce travail est d'optimiser les conditions de culture des CSM-GW pour leur utilisation clinique ultérieure. Nous nous concentrons sur l'influence de la concentration en oxygène lors de l'expansion en monocouche de P1 à P7 sur plusieurs paramètres permettant de caractériser les CSM. Les résultats obtenus sont comparés à ceux obtenus avec les CSM-MO. Notre travail a montré des différences entre les deux sources cellulaires en termes de prolifération et de différenciation adipocytaire. D’après nos résultats, l'hypoxie, au cours de l'expansion, est un paramètre important à prendre en compte en ce qui concerne la prolifération et le potentiel de différenciation chondrocytaire. L'influence des facteurs obstétricaux sur les caractéristiques des CSM-GW est également explorée. Cette étude se situant également dans le cadre de l’ingénierie tissulaire du cartilage, la seconde phase du projet consiste à induire la différenciation des cellules en chondrocytes en ensemençant ces dernières dans un biomatériau à base d’alginate et d’acide hyaluronique, et sur une cinétique de 28 jours. Les résultats obtenus sont comparés à ceux obtenus avec les CSM-MO. Après 4 semaines de culture, les CSM-GW sont capables de s'adapter à leur environnement et d’exprimer des gènes et des protéines matriciels spécifiques du cartilage tels que le collagène de type 2, qui se trouve plus exprimé après différenciation à partir des CSM-GW qu’à partir de CSM-MO / Mesenchymal Stromal/Stem Cells from human Wharton’s jelly (WJ-MSC) are an abundant and interesting source of stem cells for applications in cell and tissue engineering. Their fetal origin confers specific characteristics compared to Mesenchymal Stromal/Stem Cells isolated from human bone marrow (BM-MSC). First, the aim of this work is to optimize WJ-MSC culture conditions for their subsequent clinical use. We focus on the influence of oxygen concentration during monolayer expansion on several parameters to characterize MSC. The results are compared to those obtained with BM-MSC. Our work distinguishes WJ-MSC from BM-MSC in terms of proliferation and adipogenic differentiation. Considering our results, hypoxia during cell expansion is an important parameter to take into account regarding proliferation potential but also chondrogenic differentiation potential. The influence of obstetric factors on WJ-MSC characteristics is also explored. In cartilage tissue engineering context, the second phase of the project is to induce cell differentiation into chondrocytes by seeding them in Alginate/Hyaluronic Acid hydrogel scaffold, and during 28 days. The results obtained are compared to those obtained with BM-MSC. After 4 weeks of culture, WJ-MSC are able to adapt to their environment and express specific cartilage-Related genes and matrix proteins such as type 2 collagen, which is found more expressed after differentiation fromWJ-MSC, than from BM-MSC