221 research outputs found
In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications
No full textWe describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications
Sunlight and carcinogenesis : expression of p53 and pyrimidine dimers in human skin following UVA I, UVA I+II, and solar simulating radiations
No full textSkin Decellularization: Development of Matrix for Burns
No full textFor patients with full thickness skin defects such as second and third degree burns, autologous skin grafts and autologous cultured epidermis are considered as the gold standard treatments (For details see Appendix 1-3). However, skin grafts can have undesirable outcomes resulting from scars, poor elasticity, and limitations in joint movement due to contractures. Many times, grafted tissue does not have the same properties as the original tissue in terms of elasticity and the scarring process can make the tissue more rigid, which is a problem for delicate regions, like the neck. The rigidity of grafted tissue can make movement and mobility difficult and it is under these circumstances, that extracellular matrix (ECM) scaffolds are usefull. In these cases skin substitutes such as extracellular matrix scaffolds can be used to improve functional outcome of delicate zones. Actually decellularized dermis seems to be the best acellular skin substitute, with no other scaffold being clinically proven to gain better results yet
Étude rétrospective de l'efficacité du PRP standardisé en fonction de son ratio avec la concentration plaquettaire dans le sang sur les tendinopathies du sportif, status actuel des exigences réglementaires en matière de production de PRP en Suisse et en Europe complété d’un guide d’implémentation pour l’utilisation de PRP dans les thérapies régénératives à destination des tissus musculo squelettiques
No full textLes préparations à base de plasma enrichi en plaquettes (PRP) sont largement utilisées en médecine du sport notamment pour le traitement des affections des ligaments et des tendons, que ce soit au sein des institutions ou des cabinets privés. Depuis 2013, les préparations de PRP sont employées au CHUV pour le traitement des tendinopathies (département de la médecine du sport).
La production du PRP consiste à prélever du sang veineux autologue, et à soumettre celui-ci a une série de centrifugations afin de concentrer les plaquettes qu’il contient. Au CHUV, il est fabriqué de manière standardisée tout en respectant les exigences édictées dans les Bonnes Pratiques de Fabrication selon un protocole mis au point par l’unité de thérapie régénérative.
Ce travail a consisté en une étude règlementaire et une étude rétrospective (2013-2020) qui ont donné lieu à la publication de deux articles. Le premier article de ce travail passe en revue la réglementation concernant l’utilisation des PRP aussi bien en Suisse qu’en Europe. Le second article est une étude rétrospective qui passe en revue les résultats thérapeutiques avec un PRP synthétisé de manière standardisée sur les tendinopathies au sein du département de médecine du sport du CHUV.
L’étude rétrospective a porté sur 48 patients (âgés de 18 à 86 ans, avec un âge moyen de 43,4 ans et différents niveaux d'activité physique) présentant des tendinopathies.
L’enjeu principal de cette étude rétrospective était de démontrer l’efficacité du PRP sur les tendinopathies produit de manière standardisée (reproductibilité du produit final) tout en respectant les exigences édictées dans les Bonnes Pratiques de Fabrication (Good Manufacturing Practices (GMP)). Cette double exigence permettrait alors de faire progresser la littérature dans le sens de la standardisation des protocoles pour obtenir une cible de concentration plaquettaire. La mise en place d’une infrastructure et d’une méthode standardisée de production ouvre la voie à la comparaison des résultats thérapeutiques de différentes études. Sans cela, toute méta analyse des données est rendue difficile tant il existe de paramètres qui interviennent entre la prise de sang et l’injection du PRP.
Conclusion du travail
Le suivi clinique a démontré que 61% des patients ont rapporté des résultats d'efficacité favorables à la suite d'une seule injection de PRP obtenu à partir de sang autologue, produit de manière standardisée selon les normes GMP et injectée de manière echo guidée par ultrasons au sein du département de médecine du sport du CHUV. L’examen des bilans sanguins des patients a mis en évidence un facteur de concentration plaquettaire compris pour la plupart des patients entre 2,0 et 2,5 permettant ainsi de réduire la variabilité du matériel biologique.
Bien que les résultats soient conformes aux études déjà publiées sur les tendinopathies en médecine du sport, dans lesquelles l'efficacité des interventions orthobiologiques à faible concentration ne semble pas liée aux niveaux d'activité sportive ou à l'âge du patient, ces résultats mettent en évidence l’intérêt de la standardisation du processus et sur l’importance du respect des normes standardisées. Cette standardisation et le respect des normes de production s’avèrent être une condition préalable à une évaluation solide, robuste et homogène de l'efficacité clinique études (comparabilité de l'efficacité/amélioration du patient).
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Abstract: Providing accurate and up-to-date practical tools enabling oversight of platelet-rich plasma (PRP) legislation and of the appropriate standards to be implemented for its manufacture and use in Europe is a demanding task. This is due to rapid medico-technological advancements, slowness and disparity in legislation updates and enforcement between member states, and many reported gray-zone practices, notably for autologous PRP use. The levels of risk associated with blood manipulation processes generally dictate the manufacturing requirements for PRP preparations, which have gradually shifted toward good manufacturing practices (GMP) for standardization and overall quality enhancement. This work firstly outlines Western European and Swiss legislation for PRP products/preparations, providing key simplified information and recommendations for medical doctors seeking to implement this biological-based therapy for safe use in hospital settings, clinics, or private offices. This work secondly shows the importance of PRP-based product manufacturing standardization, which subsequently enables sound clinical evaluation of therapeutic interventions. Although the applicable legal bases provide guidelines for GMP manufacturing infrastructure and basic process design, paramount importance is set on the definition of workflows, technical specifications, and key parameters for PRP preparation and delivery. Overall, the development of simple and robust technologies and processes for PRP preparation is critical for guaranteeing the high therapeutic quality of the intervention, in collaboration with qualified GMP manufacturing platforms. Importantly, this work aims to serve as a practical tool for clinicians based in Western Europe who are willing to appropriately (i.e., administratively, and technically) implement autologous PRP treatments in musculoskeletal regenerative medicine workflows, to ensure they make informed and optimal regulatory or process-based decisions
Tailoring the Mechanical Properties of Tissue Engineering Scaffolds made from Decellularized Cartilage
No full textDue to its limited regeneration capacity, articular cartilage defects are considered a frequent
clinical problem. Initial cartilage defects, if left untreated, will progress in severity over
time and can eventually lead to degenerative joint diseases such as osteoarthritis. Hence,
orthopedic surgeons would like to aim to treat cartilage defects early on to prevent further
damage. Healthy articular cartilage consists of hyaline cartilage which assures its proper
function. However, the major drawback of current treatments such as Autologous Chondrocyte
Implantation or Microfracture is that they cannot guide the formation of a pure
hyaline cartilage. Following current treatments a fibrocartilage or a mixture of fibro- and
hyaline cartilage fills the defect in the place of hyaline cartilage. Fibrocartilage has the
disadvantage that it degrades over time due to its inferior mechanical properties compared to
hyaline cartilage. To solve this issue, future treatments should focus on creating pure hyaline
cartilage. Recently, it was hypothesized that one possibility to engineer pure hyaline cartilage
is the production of scaffolds which mimic the mechanical properties and zonal structure of
native cartilage.
The overall goal with my Ph.D. project is the development of a scaffold based on decellularised
articular cartilage, which has zone-specific mechanical properties to induce zonal
lineage commitment in chondro-progenitors. The Ph.D. project was divided into three major
sections. In the first section, the zonal mechanical properties of human articular cartilage
were measured by instrumented indentation which was information crucial to targeting the
appropriate properties in scaffolds. This resulted in finding a depth-dependent mechanical
property gradient. In the second section, a decellularisation method involving supercritical
carbon dioxide in combination with a CO2-philic detergent was developed that could
overcome the limitations of existing complex and time-consuming protocols to decellularise
articular cartilage. Using this method, bovine articular cartilage was successfully decellularised
while important cell adhesion molecules were maintained. The high matrix density of
articular cartilage makes cell infiltration challenging. For that reason, the articular cartilage
was processed into a porous scaffold, in the third section of this thesis. The porous scaffold
was produced by pepsin-digestion of the decellularised cartilage, lyophilization and covalent
crosslinking. It was demonstrated that the mechanical properties of these scaffolds could be
tailored by changing the digest concentration prior to lyophilization. However, the developed
scaffold fabrication procedure only enabled the achievement of the mechanical properties of
the superficial zone, whereas the mechanical properties were too low to target the middle,
deep and calcified zone. Further analysis was therefore only focused on superficial cartilage.
The superficial zone-specific protein lubricin was evident on the surface of the scaffolds
after 14 and 28 days of cell culture when seeded with human chondro-progenitors. This
confirms that mimicking the zone-specific mechanical properties in these prepared scaffolds
can produce zonal lineage commitment.
These results show a promising concept to induce zonal lineage commitment in chondro-progenitors, a valuable
feature to engineer pure hyaline cartilage with natural structure in future cartilage treatments.LB
Fetal bone cells for tissue engineering
No full textIn this thesis, the potential use of fetal bone cells for tissue engineering was evaluated. At first, fetal bone cells were characterized in vitro regarding their proliferation rate, the induction of their osteoblastic phenotype, and their antigenicity. They showed rapid proliferation and were able to differentiate into mature osteoblasts as shown by the expression of bone differentiation markers on the protein (alkaline phosphatase, ALP)) as well on the gene level (cbfa-1, ALP, alpha1 chain of type I collagen, and osteocalcin). Furthermore, they were able to mineralize their extracellular matrix in vitro. Antigenicity was investigated with respect to their immune profile on the protein and gene expression level and their interaction with lymphocytes in chemotaxis and immuno modulation assays, showing the immunoprivilege of fetal bone cells. In a second phase, fetal bone cells were seeded on porous scaffolds made of poly(L-lactic acid) (PLA) reinforced with 5 %wt β-tricalcium phosphate (β-TCP) or with 5 %wt hydroxyapatite (HA) to test the behaviour of such a new bone graft substitute in vitro. When seeded on these scaffolds, fetal bone cells were still able to differentiate as shown by the induction of alkaline phosphatase enzyme activity, osteocalcine production and in vitro mineralization. Next, host tissue reaction and bone repair were evaluated in vivo with scaffolds implanted in rats. Host tissue reaction to scaffolds was shown to decrease with time of exposure and osteoconductive properties of PLA/ceramic scaffolds were assessed. Finally, the new bone graft substitute consisting of fetal bone cells and PLA/TCP scaffolds was integrated in trabecular and cortical bone defects in rats to evaluate their potential for tissue repair in vivo. Histological and radiographic analysis showed improved trabecular bone infiltration in a drill defect model in the femoral condyles as well as enhanced porous ossification of the implant in critical size defects in the parietal bone when compared with the scaffold material alone. We conclude that fetal bone cells reveal an interesting potential for therapeutic use in bone tissue engineering.LB
Epiphyseal Chondro-Progenitor Cell Therapy For Articular Cartilage Regeneration
No full textArticular cartilage regeneration poses particularly tough challenges for implementing cell-based therapies. Many cell types have been investigated looking for a balanced combination of responsiveness and stability, yet techniques are still far from defining a gold standard. The work presented here focuses on the reliable expansion and characterization of a clinical-grade human epiphyseal chondro-progenitor [ECP] cell bank from a single tissue donation. ECPs were investigated at both low and high cumulative population doublings, tracking morphology, monolayer expansion kinetics and resistance to cryogenic shock. Three dimensional micro-pellet assays were used to determine spontaneous cartilage-like extracellular matrix deposition. Differentiation studies were undertaken to assess the propensity for commitment into other lineages and their stability. ECPs exhibited remarkable homogeneity in expansion with a steady proliferative potential and a stable population surface marker profile (CD14-, CD34-, CD45-, HLA-DP, DQ, DR-, and CD26+, CD44+, CD73+, CD90+, CD105+, CD166+, HLA-A,B,C+). ECPs also exhibit a stable spontaneous chondrogenic potential, depositing glycosaminoglycan rich matrix as well as Collagen I, Collagen II and display an inherent resistance to multilineage differentiation. ECPs were photoencapsulated in methacrylated hyaluronic acid hydrogels and subjected to dynamic compression. In response to 3D mechanostimulation, ECPs modulated the presentation of surface receptors for TGF|3, a potent chondrogenic morphogen. When co-encapsulated with bone marrow derived MSCs, the trend was reversed, pointing to potential crosstalk regulation between ECPs prone to shifting their baseline expression and the modulating MSCs. As a first step in defining a novel strategy for cartilage regeneration, we have conducted a GLP-grade pre-clinical safety study in goats to assess the effect of implanted ECPs in a full thickness cartilage defect. ECPs were delivered within a collagen-based matrix to optimize therapeutic cellular localization. The cell-laden construct is delivered in combination with microfracture to direct new tissue repair and remodeling. The results from the 3-months pre-clinical study highlight the safety of ECPs, the feasibility of the proposed treatment protocol as well as early indications as to their regenerative role and potential. The findings presented herein demonstrate the reliability, stability and responsiveness of Epiphyseal Chondro-Progenitor cells, granting them clear advantages for their use in defining novel strategies for cartilage regeneration.LB
Fetal cells for intervertebral disc regeneration
No full textDegeneration of the intervertebral disc (IVD) is thought to be one main factor in the development of back pain. It is not a symptom by itself but can lead to painful pathological conditions. Current treatments aim at relieving pain by conservative care, medications or surgical removal of the painful part of the disc but do not treat the causes. Disc degeneration is a progressive process which begins in the center of the disc by the loss of water due to impaired cell activity. Our hypothesis is that regeneration of the disc at the first stages of degeneration is likely to delay the progression and the need for surgery. Regeneration could be achieved by a cell-based therapy, which would bring active matrix producing and/or growth factor delivering cells. The up-scaling from bench-side to clinic for a cell therapy is limited due to safety and consistency matters. Thus, the choice of cell-type is of utmost importance to assure consistent efficacy and safety of a therapeutic application. The aim of this work was to characterize human fetal cells isolated from fetal cartilaginous tissues regarding their potential for the correction of IVD degeneration. In a first step and first chapter, a methodology for creating consistent and safe fetal cell banks from only one single tissue donation was developed. This method was illustrated by the example of fetal skin cells, which dedicated cell banks have already been tested in clinical trials. Secondly, in the second chapter, the isolation and chondrogenic potential of fetal spine cells was presented. Fetal cells were isolated from spine units (2 IVDs and 1 vertebra) from 5 donors aged from 12 to 16 weeks of gestation and amplified in monolayer to establish dedicated and consistent cell banks. Alginate bead cultures of fetal spine cells showed heterogenous matrix synthesis ability. Two fetal cell donors (14 and 15 weeks of gestation), showed good aggrecan and type II collagen production with very low type X collagen production. Mesenchymal stem cells (MSCs) can be differentiated into several phenotypes and show good regeneration capacity. Fetal cells also show good regeneration property but there is little information concerning their plasticity. Thus, in chapter three, the plasticity of fetal spine cells was investigated and compared it to that of MSCs. Cells isolated from fetal articular cartilage were also investigated as they also represent a potential source of fetal cartilaginous cells. Similarly to MSCs, fetal cells from both origins were positive for surface markers CD44, CD73, CD105 and CD166 and were negative for CD34 and CD45. Fetal cells exhibited much lower adipogenic and osteogenic differentiation levels than MSCs. As expected, fetal cells showed high chondrogenic differentiation, showed by aggrecan and type II collagen production, in TGF-β3 stimulated high cell mass density system. A mild level of type X collagen was detected in fetal spine cell pellets, whereas fetal cartilage cell pellets were highly positive. In chapter four, the response of fetal spine cells to glucose deficiency and hypoxia was assessed since these stress conditions would be similar to those of the in vivo disc. Viability of fetal cells cultured in monolayer was not altered by glucose level in normoxia and under 5% oxygen but was decreased by low glucose levels under 2.2% oxygen. Hypoxia induced an increase in SOX-9 gene expression independently of glucose level. However, a trend for a decreased gene expression of aggrecan and type I collagen under hypoxia and deficiency in glucose was observed. Finally in chapter five, fetal spine cell conditioned media (FSCCM) was tested for anti-inflammatory properties in a model of interleukin-1 (Il-1) stimulated nucleus pulposus (NP) cells. Il-1 stimulated bovine NP cells showed decreased cycloxygenase-2 (COX-2) expression and increased Prostaglandin E2 (PGE2) production in response to FSCCM, whereas human NP cells showed a decreased COX-2 gene expression as well as decreased PGE2 production. Molecular weight fractions of FSCCM had the same inhibitory effect than whole FSCCM, indicating that activity of FSCCM is more likely due to a combination of compounds rather than to a single molecule. In conclusion, fetal spine cells have been shown to be a good candidate for IVD regeneration. Fetal cell banks can easily be established and provide consistent cell lots with the extensive safety testing accomplished to assure necessary security for the patient. However, donor to donor variation in the matrix synthesis capacity will impose a strict donor selection, which could be based on spontaneous matrix synthesis in alginate bead culture. Type X collagen production was very low in unstimulated cells but could be induced by chondrogenic factors, indicating a potential risk for endochondral ossification, risk which should be assessed in an in vivo model. On the other hand, the ability of fetal spine cells to survive in low oxygen and glucose conditions, their low plasticity and their anti-inflammatory properties are interesting advantages for IVD regeneration.LB
A New Ex Vivo Human Skin Burn Model
No full textActuellement, la plupart des modèles de brûlures utilisés pour les essais précliniques sont des animaux. Pour des raisons éthiques, anatomiques et physiologiques évidentes, ces modèles pourraient être remplacés par des systèmes ex vivo optimisés. La création d'un modèle de brûlure sur la peau humaine à l'aide d'un laser à colorant pulsé pourrait représenter un modèle pertinent pour la recherche préclinique. Six échantillons de peau abdominale humaine excédentaire ont été prélevés dans l'heure suivant l'intervention chirurgicale. Des brûlures ont été provoquées sur de petits échantillons de peau nettoyée à l'aide d'un laser à colorant pulsé sur des échantillons de peau, à des fluences, des nombres d'impulsions et des durées d'illumination variables. Au total, 70 brûlures ont été provoquées sur la peau ex vivo avant d'être analysées histologiquement et dermato-pathologiquement. Les échantillons de peau brûlée irradiée ont été classés selon un code spécifique représentant les degrés de brûlure. Ensuite, une sélection d'échantillons a été inspectée après 14 et 21 jours pour évaluer leur capacité à guérir spontanément et à se réépithélialiser. Nous avons déterminé les paramètres d'un laser à colorant pulsé induisant des brûlures du 1er, 2ème et 3ème degré sur la peau humaine et avec des paramètres fixes, en particulier des brûlures superficielles et profondes du 2ème degré. Après 21 jours avec le modèle ex vivo, un néo-épiderme s'est formé. Nos résultats ont montré que ce processus simple, rapide et indépendant de l'utilisateur crée des brûlures reproductibles et uniformes de degrés différents et prévisibles, proches de la réalité clinique. Les modèles de peau humaine ex vivo peuvent remplacer et compléter l'expérimentation animale, en particulier pour le criblage préclinique à grande échelle. Ce modèle pourrait être utilisé pour favoriser l'essai de nouveaux traitements sur des degrés standardisés de brûlures et ainsi améliorer les stratégies thérapeutiques.
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Currently, most burn models for preclinical testing are on animals. For obvious ethical, anatomical, and physiological reasons, these models could be replaced with optimized ex vivo systems. The creation of a burn model on human skin using a pulsed dye laser could represent a relevant model for preclinical research. Six samples of excess human abdominal skin were obtained within one hour after surgery. Burn injuries were induced on small samples of cleaned skin using a pulsed dye laser on skin samples, at varying fluences, pulse numbers and illumination duration. In total, 70 burn injuries were performed on skin ex vivo before being histologically and dermato-pathologically analyzed. Irradiated burned skin samples were classified with a specified code representing burn degrees. Then, a selection of samples was inspected after 14 and 21 days to assess their capacity to heal spontaneously and re-epithelize. We determined the parameters of a pulsed dye laser inducing first, second, and third degree burns on human skin and with fixed parameters, especially superficial and deep second degree burns. After 21 days with the ex vivo model, neo-epidermis was formed. Our results showed that this simple, rapid, user-independent process creates reproducible and uniform burns of different, predictable degrees that are close to clinical reality. Human skin ex vivo models can be an alternative to and complete animal experimentation, particularly for preclinical large screening. This model could be used to foster the testing of new treatments on standardized degrees of burn injuries and thus improve therapeutic strategies
Human fetal progenitor tenocytes for the treatment of tendon afflictions
No full textLes blessures au niveau des tendons, qu'elles soient aiguës ou chroniques, sont très fréquentes. Malheureusement, la guérison est très lente et les résultats sont souvent insuffisants. Dans le cas des blessures aiguës, la qualité de la structure originelle n'est jamais retrouvée et un tissu cicatriciel se développe avec de potentielles pertes de mobilité et un risque augmenté de nouvelles ruptures. Les cellules foetales sont connues pour induire une régénération des tissus en l'absence de cicatrice. Dans cette thèse, une lignée cellulaire de ténocytes progéniteurs fœtaux humains (hFPTs) a été testée comme une source pour améliorer la guérison des tendons. Les caractéristiques des cellules ont été évaluées, ainsi que leur capacité à être utilisées dans différents supports tels que des hydrogels ou des matrices extracellulaires décellularisées. La thèse présente aussi les résultats d'une première phase d'évaluation in vivo sur un modèle de tendon rotulien de lapin
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