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Severe traumatic injuries and surgical procedures like tumor resection often create peripheral nerve gaps, accounting for over 250,000 injuries in the US annually. The clinical "gold standard" for bridging peripheral nerve gaps is autografts, with which 40-50% of patients regain useful function. However, issues including their limited availability and collateral damage at the donor site limit the effectiveness and use of autografts. Therefore, it is critical to develop alternative bioengineered approaches that match or exceed autograft performance. With the use of guidance channels, the endogenous regeneration process spontaneously occurs when successful bridging of short gaps (< 10mm) occurs, but fails to occur in the bridging of longer gaps (≥15mm). Several bioengineered strategies are currently being explored to bridge these critical size nerve gaps. Other labs and ours have shown how filler materials that provide topographical cues within the nerve guides are able to enhance nerve growth and bridge critical length gaps in rats. However, the mechanism by which intra-luminal fillers enhance nerve regeneration has not been explored. The main goal of this dissertation was to explore the interplay between intra-luminal scaffolds and orchestrated events of provisional fibrin matrix formation, glial cell infiltration, ECM deposition and remodeling, and axonal infiltration - a sequence we term the 'regenerative' sequence. We hypothesized that the mechanism by which thin films with topographical cues enhance regeneration is by serving as physical 'organizing templates' for Schwann cell infiltration, Schwann cell orientation, extra-cellular matrix deposition/organization and axon infiltration. We demonstrate that aligned topographical cues mediate their effects to the neuronal cells through optimizing fibronectin adsorption in vitro. We also demonstrate that aligned electrospun thin films are able to enhance bridging of a critical length nerve gap in vivo by stabilizing the provisional matrix, creating a pro-inflammatory environment and influencing the maturation of the regenerating cable leading to faster functional recovery compared to smooth films and random fibers. This research will advance our understanding of the mechanisms of peripheral nerve regeneration, and help develops technologies that are likely to improve clinical outcomes after peripheral nerve injury.

L’objectif de mon travail de thèse était d’évaluer si des autogreffes de cellules souches olfactives ecto-mésenchymateuses (CSO-EMs) restauraient les capacités d’apprentissage et de mémorisation dans un modèle d’amnésie chez le rat, consécutive à une ischémie cérébrale globale (ICG). Cette dernière pouvant avoir lieu suite à un arrêt cardiaque (AC) et conduire à des conséquences neurologiques délétères telles que des atteintes cognitives et/ou sensorimotrices.C’est dans ce contexte scientifique que se sont inscrits mes travaux de recherche dont le premier objectif était de valider un modèle fiable et reproductible d’amnésie chez le rat. Nous avons sélectionné un modèle d’AC induisant une ICG caractérisé par des pertes neuronales ciblées au niveau de la zone CA1 des hippocampes dorsaux associées à des déficits d’apprentissage et de mémorisation.Enfin, la dernière étape de mon projet a consisté à évaluer l'effet des autogreffes de CSO-EMs sur la restauration des fonctions cognitives chez des rats ayant subi une ICG. Tout d’abord, j’ai dû élaborer un protocole permettant le suivi du devenir des CSO-EMs à la suite d’autogreffes, sans en altérer leurs propriétés endogènes. Puis j’ai démontré que ces autogreffes de CSO-EMs GFP+ : i) restauraient les capacités d’apprentissage et de mémorisation, ii) stimulaient la neurogénèse et iii) amélioraient la PLT chez des rats ayant subis une ICG.L’ensemble des données recueillies au cours de ma thèse accordent d’avantage de crédibilité à l’utilisation des CSO-EMs dans le cadre de thérapies menées contre les atteintes du SNC. / The main goal of my thesis was to evaluate whether autografts of ecto-mesenchymal olfactory stem cell (EM-OSCs) restored learning and memory abilities in a rats model of amnesia following global cerebral ischemia (GCI). The latter can occur following cardiac arrest (CA) and lead to deleterious neurological consequences such as cognitive and / or sensorimotor injuries.Finally, the final step in my project was to evaluate the effect of EM-OSCs autografts on restoration of cognitive functions in ischemic rats. First of all, I had to develop a protocol to monitor the fate of EM-OSCs following autografts, without altering their endogenous properties. Then, I demonstrated that these GFP+ EM-OSCs autografts: i) restored learning and memory abilities, ii) stimulated neurogenesis, and iii) improved PLT in ischemic rats. All the data gathered during my thesis give credibility to the use of EM-OSCs in the framework of therapies against the CNS damages.

INTRODUCTION. La guérison rapide des sites donneurs des greffes cutanées favorise la survie des victimes de brûlures graves (>50 % de superficie brûlée). La mortalité élevée de ces patients est attribuable au fait que la superficie des brûlures excède celle de la peau saine. Des cultures épithéliales autologues (CEA) sont des feuillets de kératinocytes produits en culture à partir de la peau du patient. Cette étude a évalué l’effet des CEA sur l'épithélialisation des sites donneurs chez les grands brûlés. MÉTHODES. Tous les patients recevant des CEA ont été prospectivement inclus. Les plaies des sites donneurs ont été recouvertes de CEA, sauf pour une région contrôle randomisée de 7 x 7 cm. Des biopsies faites sur la greffe de peau ont permis de contrôler la profondeur des plaies sur les sites donneurs. Il y avait deux types de contrôles, avec gaze non adhérente trempée dans le milieu de culture ou dans le salin. L’épithélialisation était quantifiée globalement (% d’épithélialisation par photographie) et histologiquement (par biopsie au poinçon) à simple insu. La guérison des zones de contrôle et CEA était comparée par analyse de variance et par le test de Student. RÉSULTATS. Entre 2008 et 2009, 6 patients furent recrutés avec un total de 11 sites donneurs. Ces patients avaient en moyenne 43.5 ans, 56 % de superficie brûlée, 45% de brûlure pleine épaisseur, 66% avaient une brûlure d’inhalation, 75 jours de séjour. Il n’y a aucune corrélation entre le pourcentage d’épithélialisation et l’épaisseur du prélèvement des greffes (Pearson 0.19). Le score photographique est significativement influencé par le traitement (CEA vs Contrôle; p = 0,039) et par le jour postopératoire (p < 0,001). Le temps moyen pour atteindre un score photographique de guérison pour les zones contrôles fut de 10.2 jours contre 8.6 jours pour le CEA (p = 0,021). A l’évaluation histologique, les sites donneurs traités par le milieu de culture ont évolué aussi favorablement que ceux traités par des feuillets de CEA. CONCLUSION. L’utilisation de CEA sur les sites donneurs semble accélérer leur épithélialisation chez les victimes de brûlures graves. Cet effet est probablement le résultat d’une stimulation de la réépithélialisation innée de la plaie, plutôt que par une adhérence des feuillets de kératinocytes cultivés à la surface de la plaie. / RATIONALE: Prompt healing of split thickness skin graft donor sites is primordial to the survival of severely burned patients. Increased mortality of patients with >50 % TBSA is attributable to the limited availability of donor sites. This study evaluated the effect of Cultured Epithelial Autograft (CEA) application on skin graft donor site healing. METHODS: All burn patients receiving CEA were prospectively included. Donor site wounds were covered with CEA except a randomly designated 7x7 cm control region. Autograft biopsies were taken to document graft harvest thickness. One half of the controls were covered with non-adherent gauze soaked in the culture media and the other controls only received a non-adherent gauze dressing. Epithelialization was objectively evaluated by scoring blinded photographs with an analogue scale. Punch biopsies of the donor sites were evaluated histologically. Repeated measures ANOVA and T-test were used. RESULTS: Between 2008 and 2009, 6 patients were enrolled for a total of 11 donor sites. The patients averaged 43.5 years, 56 % TBSA, 45 % FT-TBSA, 66 % had inhalation injury and mean length of stay was 75 days. As expected, dermatome settings and autograft thickness measured by microscope did not correlate (Pearson 0.19). There was no correlation between the percentage of epithelialization of the punch biopsies of the donor sites and the thickness of the harvest. Photographic score was significantly influenced by its treatment CEA vs Control (p=0.039) and by postoperative day (p<0.001). Mean time to healing was 8.6 days for CEA compared to 10.2 days for controls (p=0.021). Infection was noted on only one donor site. On histologic analysis, the control sites dressed with gauze soaked in the culture media healed as nicely and promptly as the CEA sheet treated region. CONCLUSION: Use of CEA on donor sites appears to stimulate epithelialization. This effect is probably mediated by stimulation of local wound healing processes rather than by engraftment of keratinocytes from the CEA sheets.

O enxerto gengival livre (EGL) promove aumento do tecido queratinizado, mas apresenta contração tecidual, problemas estéticos e dor pós-operatória. Recentemente, uma técnica modificada de EGL visa a menor contração e melhor coloração. O objetivo deste estudo foi comparar a eficácia da técnica modificada de EGL com a técnica original de EGL, em relação à largura ápico-cervical do tecido queratinizado após 1 ano de seguimento. Para isso, foi realizado um ensaio clínico randomizado, multicêntrico, em que 40 indivíduos foram submetidos a uma cirurgia de aumento de tecido queratinizado na região de incisivos inferiores com a técnica original (Grupo controle; n=20) ou com a técnica modificada (Grupo teste; n=20). O preparo do leito receptor deu-se de forma idêntica em ambas as técnicas. No grupo controle, o EGL foi estabilizado com suturas e deixado exposto. No grupo teste, o EGL foi recoberto pelo retalho. O desfecho primário foi a largura ápico-cervical do tecido queratinizado (LTQ) e, juntamente com a espessura do tecido queratinizado (ETQ) e os parâmetros clínicos: retração gengival (RG), profundidade clínica de sondagem (PS), nível clínico de inserção (NCI), índice PASS (IPASS) e índice de sangramento à sondagem (ISS), foram analisados antes da cirurgia e aos 3, 6 e 12 meses após a cirurgia. O tempo transcirúrgico (TT), a dor pós-operatória (DPO) na área doadora e receptora, a quantidade de medicação consumida (QM), a contração vertical do enxerto (CV), a correspondência de cor da gengiva (COR) e a satisfação estética do paciente (SE), também foram avaliadas. O teste t e ANOVA de medidas repetidas, seguido do teste post-hoc de Newman-Keuls foram utilizados para análise dos desfechos. O nível de significância foi estabelecido em 5% (p<0,05). Ambas as técnicas promoveram aumento de LTQ e ETQ. Não houve diferença significativa entre as técnicas para LTQ, ETQ, CV, COR e SE. O grupo teste apresentou significativamente menos dor, tanto na área doadora quanto na receptora, aos 7 dias. Ademais, consumiu significativamente menos analgésicos durante o período pós- operatório. Portanto, a técnica modificada de EGL foi tão eficaz quanto a técnica original no aumento da largura ápico-cervical de tecido queratinizado, apresentando como vantagem um menor desconforto pós-operatório. / Free gingival graft (FGG) promotes keratinized tissue augmentation, though graft shrinkage, esthetic issues and postoperative pain may occur. Recently, a modified technique was proposed aiming less shrinkage and better color matching. The objective of this study was to compare the efficacy of the modified FGG technique with the original technique, in relation to the apico-coronal width of keratinized tissue after 1 year of follow-up. A multicentric randomized clinical trial was designed and included 40 subjects who were submitted to a surgery for keratinized tissue augmentation in lower incisor area. Control group (n=20) received the original technique and test group (n=20) received the modified technique. Recipient area was prepared identically for both groups. In control group, FGG was sutured and left exposed whereas in test group, FGG was recovered by the flap. Primary outcome was the apico-coronal width of keratinized tissue (WKT). Additionally, thickness of keratinized tissue (TKT) and clinical parameters: gingival recession (GR), probing depth (PD), clinical attachment level (CAL), PASS index (PI) and bleeding on probing (BoP) were measured before surgery and after 3, 6 and 12 months. Surgery time (ST), postoperative pain (POP) at recipient and donor site, number of pain killers consumed (PC), vertical shrinkage (VS), color matching (CM) and patient\'s esthetic satisfaction (ES) were also analyzed. Statistical analysis of the outcomes was performed with t test and ANOVA for repeated measures, followed by the Newman-Keuls post-hoc test. Significance level was stablished at 5% (p<0.05). Both techniques were effective in augmenting WKT and TKT. There was no statistically difference between techniques for WKT, TKT, VS, CM and ES. Test group showed less pain at recipient and donor site at 7 days of follow-up. Also, test group consumed significant less pain-killers. Therefore, the modified technique of FGG was as effective as original technique in augmenting apico-coronal width of keratinized tissue, but presented less postoperative discomfort as an advantage.

INTRODUCTION. La guérison rapide des sites donneurs des greffes cutanées favorise la survie des victimes de brûlures graves (>50 % de superficie brûlée). La mortalité élevée de ces patients est attribuable au fait que la superficie des brûlures excède celle de la peau saine. Des cultures épithéliales autologues (CEA) sont des feuillets de kératinocytes produits en culture à partir de la peau du patient. Cette étude a évalué l’effet des CEA sur l'épithélialisation des sites donneurs chez les grands brûlés. MÉTHODES. Tous les patients recevant des CEA ont été prospectivement inclus. Les plaies des sites donneurs ont été recouvertes de CEA, sauf pour une région contrôle randomisée de 7 x 7 cm. Des biopsies faites sur la greffe de peau ont permis de contrôler la profondeur des plaies sur les sites donneurs. Il y avait deux types de contrôles, avec gaze non adhérente trempée dans le milieu de culture ou dans le salin. L’épithélialisation était quantifiée globalement (% d’épithélialisation par photographie) et histologiquement (par biopsie au poinçon) à simple insu. La guérison des zones de contrôle et CEA était comparée par analyse de variance et par le test de Student. RÉSULTATS. Entre 2008 et 2009, 6 patients furent recrutés avec un total de 11 sites donneurs. Ces patients avaient en moyenne 43.5 ans, 56 % de superficie brûlée, 45% de brûlure pleine épaisseur, 66% avaient une brûlure d’inhalation, 75 jours de séjour. Il n’y a aucune corrélation entre le pourcentage d’épithélialisation et l’épaisseur du prélèvement des greffes (Pearson 0.19). Le score photographique est significativement influencé par le traitement (CEA vs Contrôle; p = 0,039) et par le jour postopératoire (p < 0,001). Le temps moyen pour atteindre un score photographique de guérison pour les zones contrôles fut de 10.2 jours contre 8.6 jours pour le CEA (p = 0,021). A l’évaluation histologique, les sites donneurs traités par le milieu de culture ont évolué aussi favorablement que ceux traités par des feuillets de CEA. CONCLUSION. L’utilisation de CEA sur les sites donneurs semble accélérer leur épithélialisation chez les victimes de brûlures graves. Cet effet est probablement le résultat d’une stimulation de la réépithélialisation innée de la plaie, plutôt que par une adhérence des feuillets de kératinocytes cultivés à la surface de la plaie. / RATIONALE: Prompt healing of split thickness skin graft donor sites is primordial to the survival of severely burned patients. Increased mortality of patients with >50 % TBSA is attributable to the limited availability of donor sites. This study evaluated the effect of Cultured Epithelial Autograft (CEA) application on skin graft donor site healing. METHODS: All burn patients receiving CEA were prospectively included. Donor site wounds were covered with CEA except a randomly designated 7x7 cm control region. Autograft biopsies were taken to document graft harvest thickness. One half of the controls were covered with non-adherent gauze soaked in the culture media and the other controls only received a non-adherent gauze dressing. Epithelialization was objectively evaluated by scoring blinded photographs with an analogue scale. Punch biopsies of the donor sites were evaluated histologically. Repeated measures ANOVA and T-test were used. RESULTS: Between 2008 and 2009, 6 patients were enrolled for a total of 11 donor sites. The patients averaged 43.5 years, 56 % TBSA, 45 % FT-TBSA, 66 % had inhalation injury and mean length of stay was 75 days. As expected, dermatome settings and autograft thickness measured by microscope did not correlate (Pearson 0.19). There was no correlation between the percentage of epithelialization of the punch biopsies of the donor sites and the thickness of the harvest. Photographic score was significantly influenced by its treatment CEA vs Control (p=0.039) and by postoperative day (p<0.001). Mean time to healing was 8.6 days for CEA compared to 10.2 days for controls (p=0.021). Infection was noted on only one donor site. On histologic analysis, the control sites dressed with gauze soaked in the culture media healed as nicely and promptly as the CEA sheet treated region. CONCLUSION: Use of CEA on donor sites appears to stimulate epithelialization. This effect is probably mediated by stimulation of local wound healing processes rather than by engraftment of keratinocytes from the CEA sheets.

Σκοπός αυτής της πειραματικής μελέτης είναι ο έλεγχος βιολογικής συμπεριφοράς διαφόρων τύπων μοσχευμάτων που σήμερα χρησιμοποιούνται ευρέως ως υποκατάστατα οστοών. Υλικό–Μέθοδος: Στην παρούσα μελέτη χρησιμοποιήθηκαν 90 κουνέλια Νέας Ζηλανδίας, ηλικία 3.5 μηνών και βάρους 4(0.25)kg, τα οποία χωρίσθηκαν σε 6 ομάδες, η κάθε μία εκ των οποίων περιελάμβανε 15 κουνέλια. Υπό γενική αναισθησία, με ενδομυϊκή χορήγηση κεταμίνης 35mg/kg και ξυλαζίνης 5mg/kg, δημιουργήθηκε, με πλάγια χειρουργική προσπέλαση, μια οπή με φρέζα διαμέτρου 4.5mm και βάθους 8mm, στους μηριαίους κονδύλους των 2 οπισθίων άκρων του κάθε κουνελιού (σύνολο 180 οπές). Στις οπές αυτές τοποθετήθηκαν τα ακόλουθα μοσχεύματα: Ομάδα Ι-αυτομόσχευμα, Ομάδα ΙΙ-αλλομόσχευμα (Grafton®), Ομάδα ΙΙΙ-ξενομόσχευμα (Lubboc®), Ομάδα ΙV-συνθετικό υποκατάστατο οστικών μοσχευμάτων (Ceraform®), Ομάδα V- συνθετικό υποκατάστατο οστικών μοσχευμάτων (Οsteoset®), Ομάδα VI-χωρίς μόσχευμα. Μετά την τοποθέτηση των μοσχευμάτων, τα κουνέλια θυσιάστηκαν με ενδοφλέβια νατριούχο θειοπεντάλη 5ml (pentothal) 10%, σε 1, 3 και 6 μήνες όπου έγινε λήψη δειγμάτων (το κάτω τριτημόριο του μηριαίου) για ιστολογική μελέτη. Τα δείγματα αξιολογήθηκαν με μια ιστολογική κλίμακα βαθμολόγησης 15-point για να καθοριστεί η ποιότητα της πώρωσης, η παρουσία οστικού ελλείμματος, η νέοαγγειογένεση και η αντιδραστική παρουσία κυττάρων φλεγμονής, καθώς και ο βαθμός ενσωμάτωσης και ανακατασκευής του πώρου. Αποτελέσματα: Σύμφωνα με την ιστολογική κλίμακα το αυτομόσχευμα έδειξε τα καλύτερα αποτελέσματα σε όλες τις χρονικές στιγμές. Όλοι οι άλλοι τύποι μοσχεύματος έδειξαν σημαντικά κατώτερα αποτελέσματα σε σχέση με το αυτόλογο μόσχευμα (p≤0.05). Το Lubboc είχε σημαντικά καλύτερα αποτελέσματα σε σχέση με τα άλλα τρία μοσχεύματα (Grafton, Ceraform και Osteoset). Το Ceraform είχε τα κατώτερα αποτελέσματα σε όλες τις κατιγορίες Συμπεράσματα: Το αυτόλογο μόσχευμα παραμένει το πρότυπο αναφοράς “gold standard” των μοσχευμάτων, επιδεικνύοντας εξαιρετικές ικανότητες ενσωμάτωσης. Το βόειο ξενομόσχευμα (Lubboc®) συνέβαλλε στη σύνθεση του πεταλιώδους οστού πιο αποτελεσματικά από το αλλομόσχευμα (Grafton®). Τα υποκατάστατα οστών (Ceraform® και Οsteoset®) ήταν κατώτερα από τα αλλομοσχεύματα και τα ξενομοσχεύματα / Background: Different types of bone-graft substitutes have been developed and are on the market worldwide to eliminate the drawbacks of autogenous grafting. This experimental animal study was undertaken to evaluate the different histological properties of various bone graft substitutes utilized in this hospital. Material/Methods: Ninety New Zealand white rabbits were divided into six groups of 15 animals. Under general anesthesia, a 4.5 mm-wide hole was drilled into both the lateral femoral condyles of each rabbit, for a total of 180 condyles for analysis. The bone defects were filled with various grafts, these being 1) autograft, 2) DBM crunch allograft (Grafton(R)), 3) bovine cancellous bone xenograft (Lubboc(R)), 4) calcium phosphate hydroxyapatite substitute (Ceraform(R)), 5) calcium sulfate substitute (Osteoset(R)), and 6) no filling (control). The animals were sacrificed at 1, 3, and 6 months after implantation and tissue samples from the implanted areas were processed for histological evaluation. A histological grading scale was designed to determine the different histological parameters of bone healing. Results: The highest histological grades were achieved with the use of cancellous bone autograft. Bovine xenograft (Lubboc) was the second best in the histological scale grading. The other substitutes (Grafton, Ceraform, Osteoset) had similar scores but were inferior to both allograft and xenograft. Conclusions: Bovine xenograft showed better biological response than the other bone graft substitutes; however, more clinical studies are necessary to determine its overall effectiveness.

Over 600,000 bone-grafting operations are performed each year in the United States. The majority of the bone used for these surgeries comes from autografts that are limited in quantity or allografts with high failure rates. Current synthetic bone grafting materials have poor mechanical properties, handling characteristics, and bioactivity. The goal of this dissertation was to develop a clinically translatable bone tissue engineering scaffold with improved handling characteristics, bioactivity, and smart delivery modalities. We hypothesized that this could be achieved through the rational selection of Food and Drug Administration (FDA) approved materials that blend favorably with hydroxyapatite (HA), the principle mineral component in bone. This dissertation describes the development of smart bone tissue engineering scaffolds composed of the biodegradable amphiphilic polymer poly(D,L-lactic acid-co-ethylene glycol-co- D,L-lactic acid) (PELA) and HA. Electrospun nanofibrous HA-PELA scaffolds exhibited improved handling characteristics and bioactivity over conventional HApoly( D,L-lactic acid) composites. Electrospun HA-PELA was hydrophilic, elastic, stiffened upon hydration, and supported the attachment and osteogenic differentiation of rat bone marrow stromal cells (MSCs). These in vitro properties translated into robust bone formation in vivo using a critical-size femoral defect model in rats. Spiral-wrapped HA-PELA scaffolds, loaded with MSCs or a lowdose of recombinant human bone morphogenetic protein-2, templated bone formation along the defect. As an alternate approach, PELA and HA-PELA were viii rapid prototyped into three-dimensional (3-D) macroporous scaffolds using a consumer-grade 3-D printer. These 3-D scaffolds have differential cell adhesion characteristics, swell and stiffen upon hydration, and exhibit hydration-induced self-fixation in a simulated confined defect. HA-PELA also exhibits thermal shape memory behavior, enabling the minimally invasive delivery and rapid (>3 sec) shape recovery of 3-D scaffolds at physiologically safe temperatures (~ 50ºC). Overall, this dissertation demonstrates how the rational selection of FDA approved materials with synergistic interactions results in smart biomaterials with high potential for clinical translation.