No description available.

Objective Skin temperatures are known to increase cutaneous sensitivity. However, it is unclear whether the amount of improved sensitivity differs depending on the protocol of heat application. Therefore, this study aimed to investigate the effects of active (treadmill walking) and passive (infrared radiator) warming of the foot sole on vibration perception thresholds. Methods Sixty healthy and injury-free subjects voluntarily participated in this study. Vibration perception thresholds (200 Hz) and plantar temperatures were measured at the hallux and 1st metatarsal head. In experiment 1, warming and mechanically stimulating the skin was achieved by walking on a treadmill for 30 min. In a follow-up study (experiment 2), external plantar heat was administered via an infrared radiator (30 min). Results In both experiments, increasing temperatures led to increased plantar sensitivity. However, the amount of improved sensitivity was greater in experiment 1, although plantar temperature increases were lower compared to experiment 2. Conclusions Warming in conjunction with mechanical stimulation seems to have a greater potential to enhance plantar sensitivity compared to external heat supply only. Significance The possible influence of mechanical stimulation and warming towards superior plantar afferent feedback highlights its importance regarding human posture and fall prevention.

Με την ηλικία, νόσοι που σχετίζονται με δομικά ελαττώματα των οστών που οφείλονται σε κατάγματα ή εκφυλισμούς, αναμένονται να αυξηθούν σε συχνότητα. Επιπλέον, η αύξηση του προσδόκιμου ζωής επιβάλλει τη χρήση βελτιωμένων συνθετικών υλικών για την αντικατάσταση νοσούντων οστών, για παράδειγμα κατά τη χρήση μεταλλικών ράβδων σε περιπτώσεις βλαβών μη-ένωσης και στις χειρουργικές επεμβάσεις αντικατάστασης ισχίου. Τα υπάρχοντα υλικά σχετίζονται με υπο-βέλτιστη οστεοενσωμάτωση και προβληματική μακροπρόθεσμη επιβίωση του σύνθετου εμφυτεύματος. Για το λόγο αυτό, η βελτίωση των υλικών επικάλυψης και των μηχανικών ιδιοτήτων των νέων, κυτταρικά συμβατών, συστατικών είναι επιτακτική. Για να αντιμετωπιστεί αυτό το πρόβλημα, υλικά νέας γενιάς είναι διαθέσιμα, ενδεχομένως με καλύτερες ιδιότητες ως υπόστρωμα προσκόλλησης για τα κύτταρα των οστών. Ο σκοπός της παρούσας εργασίας ήταν να εκτιμηθεί η ικανότητα ενός νέου, ειδικά κατασκευασμένου υλικού απο νανοσωλήνες άνθρακα ως προς τη διατήρηση της σωστής έκφρασης των χαρακτηριστικών γονιδίων των οστεοβλαστών, με έμφαση στην έκφραση των γονιδίων που εμπλέκονται στις αλληλεπιδράσεις οστεοβλαστών-υποστρώματος και έτσι προωθούν την σταθερή προσκόλληση των κυττάρων στο υπόστρωμα. Παράλληλα, ερευνήσαμε και την επίδραση της μηχανικής καταπόνησης στην έκφραση των γονιδίων αυτών σε κύτταρα που καλλιεργήθηκαν σε νανοσωλήνες άνθρακα. Χρησιμοποιήσαμε δύο ανεξάρτητες απομονώσεις οστεοβλαστών διαφοροποιημένων από ανθρώπινα μεσεγχυματικά βλαστικά κύτταρα μυελού των οστών, δηλαδή προχωρήσαμε σε δύο ανεξάρτητα πειράματα. Και στα δύο, για να γίνει ο πειραματισμός όσο εγγύτερα στις πραγματικές συνθήκες, καλλιεργήσαμε τους οστεοβλάστες υπο στατικές συνθήκες όσο και υπό συνθήκες μηχανικής καταπόνησης, για την προσομοίωση "in vivo" συνθηκών, και συγκρίθηκε η γονιδιακή έκφραση οστεοβλαστών που καλλιεργήθηκαν σε πλαστικό έναντι επιφανειών επικαλυμμένων με νανοσωλήνες άνθρακα. Απομονώσαμε το RNA από τους οστεοβλάστες μετά από την καλλιέργειά τους για 3 και 24 ώρες και προσδιορίσαμε, χρησιμοποιώντας την τεχνική real time RΤ-PCR, την έκφραση των ακόλουθων γονιδίων σε επίπεδο mRNA: κολλαγόνο-α1, αλκαλική φωσφατάση, οστεοποντίνη, βινκουλίνη και ιντεγκρίνες α4, αV, β1 και β3. Συνολικά, τα αποτελέσματα της ανάλυσης του κυτταρικού mRNA έδειξαν ότι η γονιδιακή έκφραση μετά από 3 ώρες καλλιέργειας είναι πολύ μεταβλητή, και οριστικά συμπεράσματα δεν θα μπορούσαν να εξαχθούν. Ωστόσο, αφού δίνεται η ευκαιρία στα κύτταρα να προσκολληθούν σταθερά, στις 24 ώρες, κατέστη σαφές ότι: α) η κυτταρική ταυτότητα των διαφοροποιημένων οστεοβλαστών διατηρείται, με βάση το γεγονός ότι η έκφραση αυτών των χαρακτηριστικών γονιδίων, που σχετίζονται με την προσκόλληση, συντηρείται σωστά, αν και σε διάφορα επίπεδα, β) σε στατικές συνθήκες, το επίπεδο της έκφρασης των εξετασθέντων γονιδίων είναι κατά τι χαμηλότερο σε οστεοβλάστες που καλλιεργηθήκαν σε επικαλυμμένη επιφάνεια με νανοσωλήνες άνθρακα σε σύγκριση με τα κύτταρα που καλλιεργηθήκαν σε πλαστικό, και γ) σε σύγκριση με τις στατικές συνθήκες, το μηχανικό ερέθισμα ενισχύει την έκφραση αυτών των γονιδίων οστεοβλαστών όταν καλλιεργούνται σε νανοσωλήνες άνθρακα, για την επίτευξη υψηλών επιπέδων mRNA έκφρασης των γονιδίων κυτταρικής προσκόλλησης. Τα αποτελέσματα της τελευταίας ανάλυσης της γονιδιακής έκφρασης είναι επίσης συμβατά με τις συνολικές ποσότητες RNA που λαμβάνονται, υποστηρίζοντας έμμεσα τη σταθερή προσκόλληση και επιβίωση των οστεοβλαστών σε νανοσωλήνες άνθρακα υπο συνθήκες μηχανικής καταπόνησης. Συμπεραίνουμε λοιπόν ότι το νέο υπόστρωμα από νανοσωλήνες άνθρακα που αναλύθηκε σε μηχανικές συνθήκες διέγερσης που προσομοιάζουν, κατά το δυνατόν, συνθήκες καταπόνησης in vivo, συνιστά ένα κατάλληλο κυτταρικό υπόστρωμα, συμβατό με την επιβίωση των οστεοβλαστών, τη διαφοροποίηση, την ανάπτυξη και την σταθερή προσκόλλησή τους στο υπόστρωμα νανοσωλήνων. Η εργασία αυτή υποστηρίζει την πιθανότητα της χρήσης αυτών των νέων υλικών στο μέλλον για την επικάλυψη σκελετικών προσθέσεων, με σκοπό την απόκτηση βέλτιστης οστεοενσωμάτωσης. / As population ages, diseases related to bone structural defects due to fracture or degeneration are expected to increase in frequency. In addition, the increase in life expectancy necessitates better composite materials for replacement of diseased/fractured bones, for example during the use of metal rods for non-union defects and in hip replacement surgery. The existing materials are associated with sub-optimal osseointegration and problematic long-term survival of the composite graft. For this reason, improvement of coating materials and engineering of novel cell-compatible components is imperative. To address this problem, new-generation materials are available, with possibly better bone cell adherence properties. The Aim of this work was to evaluate the ability of a novel, specially-constructed carbon nanotube material to sustain proper expression of characteristic osteoblast genes, with emphasis on the expression of genes that are functionally involved in osteoblast-matrix interactions and promote firm cell adherence to substrate. We used two independent isolates of osteoblasts differentiated from human bone marrow mesenchymal stem cells, ie we proceeded to two independent experimental runs. In both, to make the experimentation more context-relevant, we grew the osteoblasts in static as well as under mechanical strain, to simulate in vivo conditions, and also compared gene expression in osteoblasts grown on plastic versus carbon nanotube-coated surface. We isolated RNA from the osteoblasts at 3 hours and 24 hours after seeding them on the culture vessels and determined, using real-time RT-PCR techniques, the level of expression of the following genes at the mRNA level: α1-collagen, alkaline phosphatase, osteopontin, vinculin, and integrins α4, αV, β1 and β3. All in all, the results on cell mRNA analysis indicated that gene expression at 3h post-plating is too variable and no firm conclusions could be drawn. However, once the cells are given a chance to firmly adhere, at 24h, it became clear that: a) osteoblast cell identity is maintained, based on the fact that the expression of these characteristic matrix- and adhesion-related genes is properly maintained, albeit in various levels, b) in static conditions, the level of expression of the examined genes is lower in cells grown on nanotube-coated surface compared to cells grown on plastic, and c) in comparison to static conditions, mechanical stimulation enhances expression of these genes in osteoblasts grown on nanotubes, to attain robust levels of cell adherence gene mRNA expression. The results of the latter gene expression analysis are also compatible with total RNA quantities obtained, indirectly arguing firm osteoblast adhesion/survival on nanotubes under mechanical strain conditions. We therefore conclude that the novel carbon nanotubes assayed herein in lifelike mechanical stimulation conditions, constitute an appropriate cell-bearing surface, compatible with osteoblast survival, differentiation, growth and firm adherence to substrate. This work raises the possibility of using this novel material in the future to coat skeletal prostheses, in order to obtain improved osseointegration.

Mechanical stimulation plays a crucial role in promoting cell differentiation. However, applying physical force directly to cells requires complex equipment and a sterile environment, posing challenges. To overcome this, stimuli-responsive biomaterials or 4D scaffolds can serve as an alternative platform for mechanical stimulation. These scaffolds, fabricated using advanced 3D printing techniques, can apply the necessary force to cells. To optimize their functionality, bioactive molecules or extracellular matrices can be incorporated or decorated on their surfaces. This thesis proposal focuses on developing a versatile material platform that allows customization through systematic composition adjustment and on-demand printing, while also offering surface modification capabilities. The primary objective is to create a novel cell carrier platform using thermo-responsive polymers. By manipulating the additive monomer compositions, we can finely adjust properties such as the transition temperature of the polymers, tailoring them to specific requirements. Furthermore, this platform will enable the fabrication of complex three-dimensional biomaterial structures with controllable porosity, a critical aspect of biomaterial design. Leveraging the capabilities of three-dimensional printing technology, we can program and achieve desired porosity levels in the printed structures, providing enhanced flexibility for biomaterial design. The development of thermo-responsive scaffolds involved three distinct stages aimed at designing an optimized platform that effectively operates within the physiological range while ensuring cell viability. One of the key challenges was to achieve a balance between thermoresponsive behavior and biocompatibility. In the initial stage, we investigated the interplay between a crosslinkable three-armed macromer (trimethylolpropane triacrylate-TMPTA) and various monomers (N-isopropylacrylamide-NiPAAm, methyl methacrylate-MMA, dimethylaminoethyl acrylate-DMAEA, 4-acryloylmorpholine-AMO) using thermally induced solution polymerization. NiPAAm, known for its thermoresponsive properties, was selected despite its limited biocompatibility. DMAEA was chosen to adjust the polymer network transition temperature by introducing cationic charge, which disrupts the coil-globule effect of PNiPAAm and provides cell adhesiveness of the composition. Additionally, the hydrophilic monomer AMO was incorporated to further fine-tune the polymeric network. We examined the behavior of these components within the physiological range and their integration into the PNiPAAm network, establishing significant correlations between the transition temperature of the polymer and the crosslinker and monomers in their soluble condition. In the second stage of our research, we introduced photo-induced polymerization to enhance the crosslinking ratio. By utilizing this method, we successfully fabricated photo-polymerized mixtures (photoresists) into thermo-responsive discs, enabling us to study their swelling behavior between 37℃ and 25℃. Our findings revealed that the swelling behavior could be adjusted by varying the ratios of the crosslinker and monomers in the experimental groups. Through careful experimentation, we identified a suitable composition (3% w/w TMPTA, 80% w/w NiPAAm, 15% w/w DMAEA, 5% w/w AMO, and 4% w/w photo-initiator(PI)) that required minimal crosslinking incorporation while still retaining thermo-responsiveness. Furthermore, we conducted a preliminary biocompatibility study by fabricating the mixture into thin-films and cultivating them with L929 fibroblast cells. In the third and final stage, we utilized the optimized formulations from the previous stage to build thermo-responsive 3D scaffolds using continuous Digital Light Processing (cDLP) printing. We investigated the effects of various parameters, such as curing time and monomer composition, on the swelling property of the scaffolds. Additionally, we introduced glycofurol (GF) as a photo-polymerization solvent, which allowed us to produce scaffolds with improved resolution and reduced printing time. The resulting optimized scaffolds, with a composition of 3% w/w TMPTA, 80% w/w NiPAAm, 15% w/w DMAEA, 5% w/w AMO, 4% w/w PI, and 10 seconds per layer, exhibited the desired thermo-responsiveness. To further understand the mechanical properties and thermal dependencies of these scaffolds, we conducted rheological analysis. This analysis helped establish a relationship between the mechanical properties of the scaffolds and their response to temperature changes. To investigate the potential of cell stimulation through periodic changes, we conducted an experiment involving the seeding of L929 fibroblasts and C2C12 myoblasts on thermo-responsive 3D scaffolds. Our objective was to assess the ability of cells to proliferate on scaffolds with different compositions. Specifically, we examined two types of scaffolds: lattice scaffolds, characterized by a porous structure with a periodic network that enables cells to inhabit a 3D environment, and raft scaffolds, which feature a dense 3D structure designed for cells to reside on the surface for observation and evaluation. The lattice scaffolds were composed of ≥2% w/w DMAEA, while the raft scaffolds consisted of ≥5% w/w DMAEA. To evaluate cell proliferation, we conducted direct contact experiments and employed live/dead assays, subjecting the scaffolds to temperature switching conditions at 31℃ and 37℃. These experimental setups aimed to provide insights into the response and behavior of cells in the presence of thermo-responsive scaffolds with varying compositions. The results revealed favorable adhesion and spreading of the cells on the scaffolds. Interestingly, in our dynamic temperature experiment, we observed that myoblasts seeded on the scaffolds exhibited both proliferation and spreading, whereas myoblasts subjected to constant-temperature conditions did not show the same behavior. This suggests that the expansion and contraction of the scaffold, observed in previous experiments, may impact cell viability. Further investigation is needed to better understand this phenomenon. Additionally, we enhanced cell adhesiveness of the scaffolds by impregnating the scaffolds with poly-L-lysine and tested them with hASCs (human adipose-derived stem cells). Significant differences were observed between scaffolds with and without poly-L-lysine, highlighting the effectiveness of this approach. In conclusion, we have successfully developed a thermo-responsive 3D scaffold that exhibits a transition temperature within the physiological range, ensuring cell survival, and provides mechanical stimulation to the cells through the coil-globule effect without causing cell detachment. Among the formulations tested, the GF-printed formulation (3% w/w TMPTA, 80% w/w NiPAAm, 15% w/w DMAEA, 5% w/w AMO, and 4% w/w photo-initiator) with an exposure time of 10 seconds per layer showed the most promising results for cell cultivation under periodic changes in temperature, with a transition temperature of 36.3 °C ± 0.9 °C. Furthermore, we conducted direct cell contact experiments and confirmed the biocompatibility of the thermo-responsive macromer-based scaffolds. These findings demonstrate that this material platform offers a versatile and responsive material for mechanical stimulation of cells on three-dimensional scaffolds. These promising results suggest that this approach holds significant potential for tissue engineering applications and can be utilized to develop mechanical stimulation devices for various biomedical applications.:CHAPTER 1……………………..……………...…………………………..…4 Introduction CHAPTER 2……………………..…………………………..……………….29 Material and Methods CHAPTER 3……………………………………..…..……………………….52 Thermo-Responsive Polymer from Thermal Synthesis Studies CHAPTER 4…………………………………..……………………………...70 An Adjustable Thermo-Responsive Polymer from Photo Synthesis CHAPTER 5……………………………………………………………....….88 Fabrication of Thermo-Responsive Scaffolds from DLP Printing CHAPTER 6…………………...…………………………………………....107 3D Scaffold Biocompatibility Studies CHAPTER 7…………………...……………………………………………139 Discussions CHAPTER 8…………………...……………………………………………161 Summery APPENDIX…………………...………………………………………….…166 Bibliography, List of Publications, CV, Declaration of Authorship, Acknowledgements, Related publication

Encore en 2015, un grand nombre d’individus décèdent de pathologies du rythme cardiaque non contrôlées ou d’un manque de disponibilité de donneurs d’organes compatibles. Le génie tissulaire en créant, réparant ou améliorant la fonction des tissus est une option prometteuse afin de diminuer la mortalité associée à ces pathologies. L’objectif global de mon projet de recherche était de développer des outils et d’étudier l’impact fonctionnel des différents stimuli (mécanique et électrique) de l’environnement cardiaque dans le but de définir des conditions optimisées de culture pour la fabrication de tissu de remplacement par génie tissulaire. Cette thèse présente le développement d’un bioréacteur; un système qui optimise les conditions pour la culture cellulaire. L’efficacité du bioréacteur est validée par des expériences de culture cellulaire qui se concentrent sur la prolifération cellulaire, l’organisation cellulaire, l’expression génique et protéique de même que sur l’activité contractile spontanée. En premier lieu, nos résultats montrent, bien que la fréquence de contraction moyenne mesurée reste inchangée, une augmentation significative du nombre de cas de réentrées pour les cultures sur verre comparativement aux cultures sur Polydimethylsiloxane. Une augmentation de l’instabilité spatiotemporelle a été démontrée lorsque les cardiomyocytes étaient déposés sur un support de Polydimethylsiloxane et cette dernière corrèle avec une diminution non-significative de l’ARNm de la connexine-43 et une augmentation significative de l’ARNm pour CaV3.1 et HCN2. La culture sur Polydimethylsiloxane est également associée avec une plus forte réponse à l’isoprotérénol (β-adrénergique) et à l’acétylcholine (parasympathique). En second lieu, nous présentons les résultats du développement de notre bioréacteur en mettant l’emphase sur les caractéristiques (composantes accessibles, étirement uniaxial, électrode de carbone, stimulation biphasique) tout en validant notre approche pour optimiser les conditions de culture et améliorer la rentabilité des étapes de production du tissu de remplacement. Pour finir, nous partageons une nouvelle approche d’évaluation des caractéristiques contractiles de cellules cardiaques en culture. Nous avons développé des algorithmes qui utilisent les données de vidéomicroscopie pour valider l’impact de stimuli, évaluer l’hétérogénéité du signal enregistré et détecter des conditions favorables au développement d’arythmies. / In 2015, there are still a large number of people who die due to diseases of uncontrolled heart rhythm or due to lack of availability of compatible donor organs. Tissue engineering aim to create, repair or improve the function by different techniques. Tissue engineering is a viable option to reduce the mortality associated with many heart conditions. The overall goal of my PhD research was to study the functional impact of different stimuli in cardiac environment (mechanical and electrical stimulation) on cardiac cell cultures. This, in order to define optimized culture conditions for the production of replacement tissue using tissue engineering. This thesis presents the stages of creation and development of a bioreactor; a system that permits the culture of cardiac cells by integrating various stimuli. The optimization of culture conditions by using the bioreactor was confirmed by cell culture experiments that focus on cell proliferation, cell organization, gene and protein expression as well as on spontaneous activity. In the first place, our results show that although mean frequency of spontaneous activity remained unaltered, incidence of reentrant activity was significantly higher in samples cultured on glass compared to PDMS substrates. Higher spatial and temporal instability of the spontaneous rate activation was found when cardiomyocytes were cultured on PDMS, and correlated with decreased connexin-43 (unsignificant) and a significant increased CaV3.1 and HCN2 mRNA levels. Compared to cultures on glass, cultures on PDMS were associated with the strongest response to isoproterenol (β-adrenergic) and acetylcholine (parasympathetic). Secondly, we present the design of our bioreactor with an emphasis on its characteristics and by putting in perspective the relevance of our approach to optimize culture conditions and to improve profitability culture experiences and production stages of replacement heart tissue. Finally, a new approach is proposed to evaluate the characteristics of the contractile cells in culture which allows to validate the functional impact of stimuli, evaluate the heterogeneity in the beating behavior of the cells and to detect localized abnormal activity that could favour arrhythmia.

Healthy bone is bombarded with many different mechanical strain derived signals during normal daily activities. One of these signals is present as a direct connective tissue strain on the cells. However, there is also the presence of an electrically charged streaming potential during this straining. The electrical potential is created from the movement of charged fluid through the small bone porosities. To date, little focus has been applied to elucidating the possible synergistic effects of these two stimulants. The aim of this project was to evaluate the effects of mechanical strain and indirect electrical stimulation upon the development of bone forming osteoblast cells and any possible synergistic effects of the two stimulants. This aim was achieved by using a novel device, designed and developed with the capability of creating a cell substrate surface strain along with an exogenous electrical stimulant individually or at the same time. Proliferation and differentiation were determined as a measure of cellular development. The indirect electrical stimulation was achieved through the use of a pulsed electromagnetic field (PEMF) while the mechanical strain was produced from dynamic stretching of a deformable cell substrate. Strain and strain rate were modelled from recent studies proposing that relatively high frequency, low strain osteogenic mechanical stimulants are more indicative of what healthy bone would be experiencing during normal activities. The PEMF signal mimicked a clinically available bone growth stimulator signal. Results showed a PEMF stimulus on monolayers of SaOS-2 and MG-63 osteoblast-like cells leads to a depression in proliferation. A concomitant increase in alkaline phosphatase production was also observed for the SaOS-2 cultures, but not for the MG-63 cell line. It was hypothesised that this was due to the MG-63's lack of phenotypic maturity compared to the SaOS-2 cells. Mechanical strain of the cell substrate alone, at a relatively high frequency (5Hz) but small strain, did not significantly effect either cell proliferation or differentiation for the MG-63 cells. However, when the electrical and mechanical stimulants were combined a significant increase in cellular differentiation occurred with MG-63 cultures, revealing a possible synergistic effect of these two stimulants on the development of bone cells.

Résumé soumis à confidentialité / Not available

Objective Skin temperatures are known to increase cutaneous sensitivity. However, it is unclear whether the amount of improved sensitivity differs depending on the protocol of heat application. Therefore, this study aimed to investigate the effects of active (treadmill walking) and passive (infrared radiator) warming of the foot sole on vibration perception thresholds. Methods Sixty healthy and injury-free subjects voluntarily participated in this study. Vibration perception thresholds (200 Hz) and plantar temperatures were measured at the hallux and 1st metatarsal head. In experiment 1, warming and mechanically stimulating the skin was achieved by walking on a treadmill for 30 min. In a follow-up study (experiment 2), external plantar heat was administered via an infrared radiator (30 min). Results In both experiments, increasing temperatures led to increased plantar sensitivity. However, the amount of improved sensitivity was greater in experiment 1, although plantar temperature increases were lower compared to experiment 2. Conclusions Warming in conjunction with mechanical stimulation seems to have a greater potential to enhance plantar sensitivity compared to external heat supply only. Significance The possible influence of mechanical stimulation and warming towards superior plantar afferent feedback highlights its importance regarding human posture and fall prevention.