La pulpe dentaire est un tissu connectif innervé et vascularisé, contenu dans une structure minéralisée inextensible formée par l’émail, la dentine et le cément. Ce tissu assure l’homéostasie et la sensibilité de la dent. Il est sujet à des lésions sévères faisant suite à une carie ou à un traumatisme. La thérapeutique conventionnelle préconisée alors est le traitement endodontique, qui consiste en l’exérèse de la totalité du tissu pulpaire et le comblement de l’espace évidé par un matériau synthétique bioinerte. Malgré les résultats cliniques satisfaisants, cette thérapeutique induit une fragilisation de la dent et une plus grande susceptibilité aux infections, qui peuvent conduire à terme à la perte de la dent. En se basant sur la présence de cellules souches mésenchymateuses dans la pulpe dentaire, des stratégies de régénération alternatives au traitement endodontique traditionnel sont à l’étude, afin de permettre le maintien des fonctions de nutrition et de sensibilité de la pulpe, garantes de la pérennité de la dent sur l’arcade. Elles s’inscrivent dans deux approches: la régénération de novo, en cas de nécrose du tissu pulpaire et la régénération partielle, lorsque seul le tissu pulpaire endommagé est éliminé et régénéré. Nos travaux portent sur la faisabilité de cette dernière approche dans un modèle préclinique. En effet, dans la perspective d’un transfert vers la clinique humaine, des modèles chez le gros animal doivent être développés afin de tester la faisabilité et le succès de cette thérapie, dans des conditions proches de la clinique. Du fait de ses similitudes avec l’homme en termes d’anatomie et de physiologie, le miniporc représente un modèle de choix pour les études précliniques d’ingénierie pulpaire. L’objectif principal de cette étude est de tester la faisabilité de la régénération pulpaire partielle, en implantant des cellules pulpaires porcines (pDPCs) contenues des hydrogels injectables dans des défauts pulpaires artificiellement créés chez le miniporc. Au cours ce travail, différentes techniques d’imagerie d’évaluation du processus de régénération ont été développées. En particulier, un protocole d’angiographie tridimensionnelle in- pour la visualisation du réseau vasculaire pulpaire a été mise au point. Par ailleurs, en utilisant des paramètres morphométriques spécifiques, initialement développés pour caractériser l’os, une analyse tridimentionnelle par micro-CT des tissus minéralisés de réparation a été élaborée. En appliquant un “split mouth model”, les hydrogels injectables ensemencés ou non par des pDPCs ont été implantés dans des molaires et des prémolaires, après amputation de la pulpe camérale. À 21 jours après la chirurgie, les analyses d’imagerie, d’histologie et d’immunologie ont mis en évidence, qu’indépendamment de la présence des pDPCs, l’implantation des hydrogels a induit la formation d’un pont d’ostéodentine. La caractérisation morphométrique tridimensionnelle a montré que la microarchitecture de ce pont différait largement de la dentine native. De plus, en présence des pDPCs, le processus de réparation était modifié, avec une moins bonne étanchéité du pont. Au cours de ce travail, une technique de suivi non invasive de la régénération a tenté d’être mise au point. Une angiographie tridimensionnelle par soustraction a été réalisée avant et après la procédure de régénération pulpaire partielle. Si les angiographies ont révélé l’entière vascularisation des mâchoires et des dents à croissance continue, l’apport vasculaire des dents matures traitées, du fait de son faible flux, n’a pas pu être mis en évidence. L’absence de régénération partielle de la pulpe dans les conditions testées souligne l’importance des modèles précliniques pour identifier les facteurs promouvant un environnement favorable à la régénération, dans la perspective d’un transfert vers la clinique humaine. / The dental pulp is a connective tissue, which is highly innervated and vascularized, encapsulated in a mineralized inextensible structure formed by enamel, dentin and cementum, ensuring the homeostasis and sensibility of the tooth. The pulp is often damaged by caries and trauma, resulting in infection or necrosis. In such situations, the routine clinical treatment is a root canal therapy, which consists in the elimination of the affected tissue and filling of the endodontic canal system with bioinert synthetic materials. In spite of satisfactory clinical outcomes, none of the original functions is restored and the lack of sensitivity as well as natural defence may lead to tooth fracture and reinfection. Cell-based pulp regeneration could provide a valid alternative to traditional endodontic treatment of damaged teeth. This strategy focuses, in fact, on the preservation of the healthy pulp tissue and the regeneration of the damaged one, by combining stem cells, scaffolds and growth factors. In case of trauma or carious lesion, as the pulp inflammatory reaction is compartmentalized in first instance, such conservative approach could be indicated. Regarding non-rodent animal model, to our knowledge, only Iohara et al. (2009) reported the achievement of partial pulp regeneration in canine tooth by implantation of subfractions of autologous pulp cells; however, in the perspective of a transfer to the human clinic, larger animal models should be developed to test the feasibility and the success of the therapy mimicking the clinical conditions of pulpotomy. Due to dental anatomical and physiological similarities with human, the minipig constitutes a model of choice for preclinical pulp engineering studies. The aim of this study was to develop a preclinical model of partial dental pulp regeneration in minipig, by implanting a pulp construct, made by self-assembling nano-peptide injectable hydrogel and porcine minipig dental pulp cells (pDPCs), in artificially created pulp defects. Secondarily, in the context of this preclinical model, two different techniques of analysis of the regeneration process have been developed. In particular, an in vivo 3D subtraction angiography has been set for the visualization of dental pulp vascular network. Indeed, further developments of this modality open promising perspectives of its application for the morphometric characterization of angiogenesis process in newly formed dental tissues and bone defects. Moreover, using specific morphometric parameters, initially developed to characterize bone, a micro-CT morphometric analysis of the mineralized reparative tissues, obtained by the partial pulp regeneration protocol, has been elaborated. By split mouth model, pulp constructs made with self-assembling injectable nano-peptide hydrogel with and without porcine dental pulp cells (pDPCs) were implanted, after pulp chamber amputation in premolars and molars. At day 21 after surgery, three-dimensional morphometric characterization, Masson’s trichrome and immunolabeled for DSP and BSP were performed on treated teeth. 3D subtraction angiographies have been performed before and after partial pulp regeneration procedure. Regardless of the presence of pDPCs, the implantation of pulp construct induces the formation of an osteodentin bridge, whose microarchitecture sensibly differs from the native dentin. Furthermore, the presence of pDPCs in the construct slightly impairs this reparative process. The latter was led the remaining pulp cells, instead of the pDPCs in the scaffold. Angiographies could show entire vascularization of jaws and continuously growing teeth but blood supply of treated mature permanent teeth could not be displayed. The failure of partial pulp regeneration cell based strategy, in these near-real clinical conditions, highlights the importance of preclinical models, to identify the factors promoting a favourable regenerative environment, in the perspective of a transfer to the human clinics.

The progress of cell therapy can be greatly facilitated by using suitable experimental models. It is essential to verify the clinical usefulness of new healing procedures obtained in studies on laboratory animals by using a large animal model. One of suitable models well acceptable in medical community is undoubtedly the miniature pig, which resembles humans in terms of physiology and body proportions. This PhD thesis presents the summary of our experimental studies relating to possible exploitation of mesenchymal and neural stem cells in the healing of locomotive apparatus and neural tissue disorders in humans or animals. The first part of the thesis briefly describes the current issue of cell therapy and animal models, mesenchymal cells and/or their combination with new types of scaffolds, neurogenesis, neural stem cells and their potential application in therapy of spinal cord injury. The second part is focused on the goals and methodology, the individual publications being listed in the third part. Our experiments with iatrogenic physeal defect in rabbits, which served as a model of the occurrence of valgous deformation in the clinical practice, showed the positive preventive and therapeutical effects of a new type of scaffolds seeded with allogeneic mesenchymal stem cells in animals without...

Huntington's disease (HD) belongs to neurodegenerative disorders. It is a monogenic disease caused by trinucleotic CAG expansion in exon 1 of gene coding protein huntingtin. Even though the cause of HD is known since 1993, the pathophysiology and cure for HD reminds to be found. The animal models are being used for better understanding of HD. The most common animal models for HD are rodents, especially mice but it was also important to create large animal models, which will be more like human. Therefore, TgHD minipig was created in Academic of Science in Liběchov in 2009. This model was created by microinjection of lentiviral vector carrying N-terminal part of human HTT with 124 repetitive CAG in exon 1. This model is viable and in every generation, is part of the offspring transgenic. In this thesis, I specialized to biochemical and behavioral changes of this model. I compared transgenic and wild type siblings. I found that biochemical changes are manifested mostly by increased level of mtHtt fragments in testes and brain. In behavioral part of this thesis I established new methods for testing behavioral changes in this model. The introduced methods showed some changes between wild type and transgenic animals at the tested ages but these changes were not significant due to the low number of...

The shortage of human tissues and organs for allotransplantation can be overcome by xenotransplantation. As a source of organs, the miniature pig is convenient. However, the presence of pathogens transmissible to the recipients, especially porcine endogenous retrovirus (PERV), represents a threat for successfull xenotransplantation. Infectious PERVs contain three classes of envelope glycoprotein. Two classes, PERV-A and PERV-B are polytropic, they can infect human, pig and mink cells in vitro. PERV-C is evolutionary young, ecotropic isolate that can infect pig only. We previously detected a new full-lenght, but replication-defective PERV-A isolate dubbed (MAMBA) with high transcriptional activity in Large-White pig from a Czech breed. To support our results with PERV-MAMBA epigenetic regulation in pig tissues, in vitro DNA methylation essay was accomplished. Methylated or non-methylated reporter plasmids containing provirus 5' LTR were transfected into 293T cells and luciferase activity was measured. In both cases, methylated LTR decreased significantly expression of luciferase. Thus, PERV LTR-driven transcription is sensitive to DNA methylation. We also used PERV-A MAMBA provirus to study recombination between two pig endogenous retroviruses. We prepared 293T and BeWo cell clones harboring PERV-A...