Made available in DSpace on 2016-08-15T20:31:12Z (GMT). No. of bitstreams: 1 NayaraAC_DISSERT.pdf: 1401697 bytes, checksum: 483ad3e8e94846f136ca4ca2f701d984 (MD5) Previous issue date: 2013-08-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / At birth , domestic animals exhibit a large stock of oocytes which only a minimum reaches meiotic maturation. These oocytes contained in primordial follicles are used for the development of studies in order to clarify issues related to the activation of primordial follicles and growth as well as promote the development of new contraceptive methods and new techniques for the recovery of fertility through its cultivation. Among the ovarian culture stands out in vivo , also known as transplantation, has as main objectives to promote the return of fertility in women undergoing treatment gonadotoxic and the preservation of genetic diversity of endangered animals. In this sense the research with the in vivo culture aims to determine which methodology seems more feasible and allows better maintenance and development of the transplanted tissue. Thus , this study aimed to assess the quality of ovarian follicles after transplantation under the kidney capsule of mice. For this, we used 10 female mice ( Mus musculus ) , divided into two groups , Group 1 : transplants removed after 15 days from the insertion of the fragment , group 2 after 30 days. It has been established the control group which are the ovarian cortical fragments removed directly for histological processing. Through evaluation by light microscopy , it was observed that there was a progressive reduction in the number of morphological normal follicles with progression of duration to 15 for 30 days of transplants. Regarding the number of normal preantral, only group 2 did not differ statistically from the control. The normal antral follicles in groups 1 and 2 did not differ but were significantly lower than the control group. Regarding the evaluation of apoptotic, we obtained a higher reaction to Caspase -3 in antral follicles in the three groups. According to the results obtained in this study, subcapsular renal autotransplantation technique has proved satisfactory for maintenance and study of preantral follicles / Ao nascerem, os animais domésticos apresentam uma grande reserva de oócitos dos quais apenas um número mínimo atinge a maturação meiótica. Estes oócitos contidos em folículos pré-antrais são utilizados para o desenvolvimento de estudos com o intuito tanto de esclarecer questões relacionadas à ativação e crescimento de folículos primordiais quanto promover o desenvolvimento de novos métodos de contracepção e novas técnicas de recuperação da fertilidade através do seu cultivo. Dentre os tipos de cultivo ovariano destaca-se o in vivo, também conhecido por transplante, tem como principais objetivos promover o retorno da fertilidade em mulheres submetidas a tratamento gonadotóxico e a preservação da diversidade genética de animais ameaçados de extinção. Neste sentido, as pesquisas com o com cultivo in vivo, almejam determinar qual metodologia mostra-se mais viável e permite uma melhor manutenção e desenvolvimento do tecido transplantado. Desta forma, o presente estudo objetivou avaliar a qualidade dos folículos ovarianos após o autotransplante sob a cápsula renal de camundongas. Para isso, foram utilizadas 10 camundongas (Mus musculus), divididas em dois grupos, Grupo 1: remoção dos transplantes após 15 dias a partir da inserção do fragmento, grupo 2: após 30 dias. Ainda foi estabelecido o grupo controle que corresponde a fragmentos corticais removidos do ovário destinados diretamente para o processamento histológico. Através da avaliação por microscopia ótica, observou-se que houve uma redução no número de folículos morfologicamente normais com a progressão do tempo de duração dos transplantes de 15 para 30 dias. Com relação ao número folículos pré-antrais normais apenas o grupo 2 não diferiu estatisticamente do controle. Os folículos antrais normais dos grupos 1 e 2 não diferiram entre si mas apresentaram-se significativamente inferiores ao grupo controle. Quanto à avaliação da ocorrência de apoptose, obteve-se uma maior reação à Caspase-3 em folículos antrais nos três grupos. De acordo com os resultados obtidos no presente estudo, o autotransplante subcapsular renal demonstrou ser uma técnica satisfatória para manutenção e estudo de foliculos pré-antrais

Enxertos livres de pele de espessura total são indicados para cobrir grandes defeitos de pele em superfícies flexoras ou em extremidades distais e sofrem lesão por isquemia e reperfusão pela própria natureza do procedimento cirúrgico. O objetivo deste trabalho foi testar a associação de células tronco mesenquimais de origem adiposa (ADSCs) heterólogas a enxertos cutâneos autólogos de espessura total em ratos Wistar. Enxertos de 12 mm de diâmetro foram executados no dorso de 30 ratos, em dois locais: cranial e caudal. Os ratos foram distribuídos em seis grupos (n=5): grupo ADSC_G recebeu, no enxerto, 1x106 ADSCs em 200 μL de Solução Salina 0,9% (SS); grupo ADSC_B recebeu 1x106 ADSCs em 200 μL de SS na borda do leito receptor; grupo ADSC_GB, metade da mesma suspensão na borda e outra metade no enxerto. Os grupos controle, SS_G e SS_B, receberam apenas SS no enxerto ou nas bordas respectivamente. No grupo S, o enxerto não recebeu nenhum tratamento durante as cirurgias. Curativos do tipo tie-over permaneceram até o 5º dia. Na cirurgia (d0), aos 5 (d5) e 14 (d14) dias de pós-operatório, os desenhos dos enxertos foram digitalizados e suas áreas mensuradas (software ImageJ). As avalições clínicas consideraram peso, presença de secreções e ocorrência de epidermólise. A planimetria demonstrou a taxa de pele normal, de pele avermelhada e de ulceração, assim como a contração dos enxertos entre os intervalos d0–d5, d5-d14 e d0-d14. As amostras dos enxertos foram obtidas em d14 para coloração com Hematoxilina e eosina e Tricrômico de Masson. A epiderme foi avaliada para espessamento, ceratose, acantose, degeneração hidrópica, infiltrado inflamatório. A derme foi avaliada quanto a rarefação pilosa, tecido de granulação, infiltrado inflamatório, deposição de colágeno. Os resultados foram expressos em média e desvio padrão, e a análise estatística utilizou as Equações de Estimações Generalizadas (GEE), com nível de significância de 5%. O grupo ADSC_G apresentou melhores aspectos macroscópicos, menos ocorrência de epidermólise e de rarefação pilosa e uma das menores médias de área de ulceração, juntamente com os outros grupos tratados com ADSCs. O grupo ADSC_G demonstrou as menores médias de alterações histopatológicas como: espessamento da epiderme, degeneração hidrópica, tecido de granulação. O mesmo grupo ficou entre as menores médias de acantose e infiltrado inflamatório na derme, ao mesmo tempo que apresentou as maiores médias de VEGF no subcutâneo e no tecido de granulação. Dessa forma, pode-se concluir que as ADSCs protegeram os enxertos dos efeitos deletérios da isquemia, assim como e sugerem-se novos estudos em fases iniciais da cicatrização para compreensão dos mecanismos envolvidos. / Evaluation of adipose derived stem cells (ADSC) effect on full-thickness skin graft (FTSG) as a wound healing model in ischemic conditions. Two 12 mm diameter FTSGs were harvested and placed onto dorsal recipient beds of twenty-four Wistar rats, in two anatomic regions: cranial and caudal. Rats were randomized into five groups. Before grafting, group E FTSGs received subfascial injection of 1X106 ADSCs diluted in 200 μL of physiologic saline. Group EC FTSGs received only physiologic saline. Group B received ADSCs in the recipient bed edges; group C received physiologic saline in the edges. Group ADSC_GB received the same ADSCs number and volume, half in the graft and half in the edges. Using planimetry, grafts were analyzed for graft´s contraction rate (d0, d5, d14), normal skin rate and occurrence of epidermolysis and failure rate (d14). FTSGs samples were obtained on the d14 to hematoxylin-eosin and Masson’s Trichrome staining for epidermal analysis (epidermal thickening, keratosis, acanthosis, hydropic degeneration, inflammatory infiltrate) and dermal analysis (hairless, granulation tissue, inflammatory infiltrate, collagen deposition). The obtained results were expressed by mean (n=5). Statistical significance (p<0,05) was calculated using Generalized Estimating Equations. The ADSC_G group had better macroscopic aspects, less occurrence of epidermolysis and hairless and one of the lowest averages of ulceration area, along with the other groups treated with ADSCs. The ADSC_G group showed the lowest mean on the histopathological changes measured such as thickening of the epidermis, hydropic degeneration, less granulation tissue. The same group was among the lowest acanthosis and inflammatory infiltrate averages in the dermis, while presented with the greatest of VEGF average in the subcutaneous and in the granulation tissue. Thus it can be concluded that the ADSCs may have protected the grafts from the deleterious effects of ischemia and suggest new studies in the early stages of healing to understand the mechanisms involved.

Enxertos livres de pele de espessura total são indicados para cobrir grandes defeitos de pele em superfícies flexoras ou em extremidades distais e sofrem lesão por isquemia e reperfusão pela própria natureza do procedimento cirúrgico. O objetivo deste trabalho foi testar a associação de células tronco mesenquimais de origem adiposa (ADSCs) heterólogas a enxertos cutâneos autólogos de espessura total em ratos Wistar. Enxertos de 12 mm de diâmetro foram executados no dorso de 30 ratos, em dois locais: cranial e caudal. Os ratos foram distribuídos em seis grupos (n=5): grupo ADSC_G recebeu, no enxerto, 1x106 ADSCs em 200 μL de Solução Salina 0,9% (SS); grupo ADSC_B recebeu 1x106 ADSCs em 200 μL de SS na borda do leito receptor; grupo ADSC_GB, metade da mesma suspensão na borda e outra metade no enxerto. Os grupos controle, SS_G e SS_B, receberam apenas SS no enxerto ou nas bordas respectivamente. No grupo S, o enxerto não recebeu nenhum tratamento durante as cirurgias. Curativos do tipo tie-over permaneceram até o 5º dia. Na cirurgia (d0), aos 5 (d5) e 14 (d14) dias de pós-operatório, os desenhos dos enxertos foram digitalizados e suas áreas mensuradas (software ImageJ). As avalições clínicas consideraram peso, presença de secreções e ocorrência de epidermólise. A planimetria demonstrou a taxa de pele normal, de pele avermelhada e de ulceração, assim como a contração dos enxertos entre os intervalos d0–d5, d5-d14 e d0-d14. As amostras dos enxertos foram obtidas em d14 para coloração com Hematoxilina e eosina e Tricrômico de Masson. A epiderme foi avaliada para espessamento, ceratose, acantose, degeneração hidrópica, infiltrado inflamatório. A derme foi avaliada quanto a rarefação pilosa, tecido de granulação, infiltrado inflamatório, deposição de colágeno. Os resultados foram expressos em média e desvio padrão, e a análise estatística utilizou as Equações de Estimações Generalizadas (GEE), com nível de significância de 5%. O grupo ADSC_G apresentou melhores aspectos macroscópicos, menos ocorrência de epidermólise e de rarefação pilosa e uma das menores médias de área de ulceração, juntamente com os outros grupos tratados com ADSCs. O grupo ADSC_G demonstrou as menores médias de alterações histopatológicas como: espessamento da epiderme, degeneração hidrópica, tecido de granulação. O mesmo grupo ficou entre as menores médias de acantose e infiltrado inflamatório na derme, ao mesmo tempo que apresentou as maiores médias de VEGF no subcutâneo e no tecido de granulação. Dessa forma, pode-se concluir que as ADSCs protegeram os enxertos dos efeitos deletérios da isquemia, assim como e sugerem-se novos estudos em fases iniciais da cicatrização para compreensão dos mecanismos envolvidos. / Evaluation of adipose derived stem cells (ADSC) effect on full-thickness skin graft (FTSG) as a wound healing model in ischemic conditions. Two 12 mm diameter FTSGs were harvested and placed onto dorsal recipient beds of twenty-four Wistar rats, in two anatomic regions: cranial and caudal. Rats were randomized into five groups. Before grafting, group E FTSGs received subfascial injection of 1X106 ADSCs diluted in 200 μL of physiologic saline. Group EC FTSGs received only physiologic saline. Group B received ADSCs in the recipient bed edges; group C received physiologic saline in the edges. Group ADSC_GB received the same ADSCs number and volume, half in the graft and half in the edges. Using planimetry, grafts were analyzed for graft´s contraction rate (d0, d5, d14), normal skin rate and occurrence of epidermolysis and failure rate (d14). FTSGs samples were obtained on the d14 to hematoxylin-eosin and Masson’s Trichrome staining for epidermal analysis (epidermal thickening, keratosis, acanthosis, hydropic degeneration, inflammatory infiltrate) and dermal analysis (hairless, granulation tissue, inflammatory infiltrate, collagen deposition). The obtained results were expressed by mean (n=5). Statistical significance (p<0,05) was calculated using Generalized Estimating Equations. The ADSC_G group had better macroscopic aspects, less occurrence of epidermolysis and hairless and one of the lowest averages of ulceration area, along with the other groups treated with ADSCs. The ADSC_G group showed the lowest mean on the histopathological changes measured such as thickening of the epidermis, hydropic degeneration, less granulation tissue. The same group was among the lowest acanthosis and inflammatory infiltrate averages in the dermis, while presented with the greatest of VEGF average in the subcutaneous and in the granulation tissue. Thus it can be concluded that the ADSCs may have protected the grafts from the deleterious effects of ischemia and suggest new studies in the early stages of healing to understand the mechanisms involved.

INTRODUÇÃO: A paratireoidectomia total com auto-enxerto imediato heterotópico (PTH+AE) é uma das técnicas cirúrgicas hoje usadas no tratamento do hiperparatireoidismo secundário à doença renal crônica e do hiperparatireoidismo persistente após o transplante renal. Os níveis adequados de paratormônio sistêmico (PTHs) no pós-operatório ainda são controversos e o perfil funcional do auto-enxerto de tecido paratireóideo, pouco esclarecido. No presente estudo, nós analisamos a função do tecido paratireóideo implantado pacientes com hiperparatireoidismo secundário e terciário. MÉTODO E CASUÍSTICA: Em um estudo prospectivo observacional, 19 pacientes portadores de doença renal crônica (PDRC) e quatro pacientes transplantados renais (PTR) foram submetidos à PTX+AE com e seguidos por um ano. Todos os pacientes apresentaram PTHs indetectável no pós-operatório imediato (POi). Os níveis séricos de PTH em ambos os membros superiores, cálcio, fósforo, fosfatase alcalina e reposição de cálcio elementar e calcitriol foram verificados com um mês, dois, três, quatro, seis, nove e 12 meses após a cirurgia. A 25-Hidroxivitamina D (25OHD) foi medida no POi, seis e 12 meses após a cirurgia. A função do auto-enxerto foi classificada em estados funcionais (EF) de acordo com os níveis de PTHs. RESULTADOS: A maioria dos PDRC e PTR mostraram níveis detectáveis de PTHs já no primeiro mês. No segundo mês pósoperatório, todos os pacientes apresentaram níveis detectáveis de PTHs quando também houve estabilização dos níveis séricos em ambos os grupos. Os gradientes de concentração de PTH não mostraram correlação com o PTHs. No pós-operatório, a hipercalcemia foi observada em 73,7% dos PDRC em pelo menos um episódio, reduzindo ou inibindo a secreção de paratormônio em oito pacientes. Não houve melhora dos níveis de 25OHD em ambos os grupos, permanecendo com níveis insuficientes. Oito PDRC regrediram de EF ao final do estudo. CONCLUSÃO: O perfil funcional do AE é semelhante entre os PDRC e PTR. A função do AE, inicia-se no primeiro mês pós-operatório, atingindo um maior EF até o quarto mês pós-operatório na maioria dos pacientes, porém pode haver declínio da função ao longo do tempo / BACKGROUND: Total parathyroidectomy with immediate forearm transplantation (PTX+AT) is employed in renal hyperparathyroidism. Appropriate postoperative systemic parathyroid hormone (sPTH) levels are still controversial and autograft functional behavior is unclear. In the present study, we analyzed the function of autotransplanted parathyroid tissue in secondary and tertiary hyperparathyroidism. METHODS: In a prospective observational study, 19 dialysis patients (DPs) and 4 kidney transplant patients (KTPs) who underwent PTX+AT were followed. All patients had undetectable PTH in the early postoperative period (ePO). Autograft function and the following biochemical variables were assessed at one, two, three, four, six, nine and 12 months following the operation: serum calcium, phosphorus, alkaline phosphatase, and intact PTH before and after the operation. 25-Hidroxyvitamin D levels (25OHD) were measured in the ePO, six and twelve months. Oral doses of calcium and calcitriol were recorded. Autograft function was stratified into four functional status (FS) according to sPTH. RESULTS: All functioning grafts presented sPTH until the second month. On the second postoperative month, all patients had sPTH detectable levels also when serum levels were steady in both groups. PTH gradients showed no significant correlation with sPTH levels. In at least one occasion in the postoperative period, hypercalcemia was observed in 73.7% of DPs, and it reduced or inhibited PTH secretion in 8 patients. There was no improvement in levels of 25OHD in both groups, resulting in insufficient levels. Eight DPs changed to a lower FS at the end of the study. CONCLUSION: The function of the autotransplanted parathyroid tissue usually initiates during the first month following operation and is similar in patients. The most of the patients reachs FS2 during the fourth month but autograft function can decrease over time

Enxertos livres de pele de espessura total são indicados para cobrir grandes defeitos de pele em superfícies flexoras ou em extremidades distais e sofrem lesão por isquemia e reperfusão pela própria natureza do procedimento cirúrgico. O objetivo deste trabalho foi testar a associação de células tronco mesenquimais de origem adiposa (ADSCs) heterólogas a enxertos cutâneos autólogos de espessura total em ratos Wistar. Enxertos de 12 mm de diâmetro foram executados no dorso de 30 ratos, em dois locais: cranial e caudal. Os ratos foram distribuídos em seis grupos (n=5): grupo ADSC_G recebeu, no enxerto, 1x106 ADSCs em 200 μL de Solução Salina 0,9% (SS); grupo ADSC_B recebeu 1x106 ADSCs em 200 μL de SS na borda do leito receptor; grupo ADSC_GB, metade da mesma suspensão na borda e outra metade no enxerto. Os grupos controle, SS_G e SS_B, receberam apenas SS no enxerto ou nas bordas respectivamente. No grupo S, o enxerto não recebeu nenhum tratamento durante as cirurgias. Curativos do tipo tie-over permaneceram até o 5º dia. Na cirurgia (d0), aos 5 (d5) e 14 (d14) dias de pós-operatório, os desenhos dos enxertos foram digitalizados e suas áreas mensuradas (software ImageJ). As avalições clínicas consideraram peso, presença de secreções e ocorrência de epidermólise. A planimetria demonstrou a taxa de pele normal, de pele avermelhada e de ulceração, assim como a contração dos enxertos entre os intervalos d0–d5, d5-d14 e d0-d14. As amostras dos enxertos foram obtidas em d14 para coloração com Hematoxilina e eosina e Tricrômico de Masson. A epiderme foi avaliada para espessamento, ceratose, acantose, degeneração hidrópica, infiltrado inflamatório. A derme foi avaliada quanto a rarefação pilosa, tecido de granulação, infiltrado inflamatório, deposição de colágeno. Os resultados foram expressos em média e desvio padrão, e a análise estatística utilizou as Equações de Estimações Generalizadas (GEE), com nível de significância de 5%. O grupo ADSC_G apresentou melhores aspectos macroscópicos, menos ocorrência de epidermólise e de rarefação pilosa e uma das menores médias de área de ulceração, juntamente com os outros grupos tratados com ADSCs. O grupo ADSC_G demonstrou as menores médias de alterações histopatológicas como: espessamento da epiderme, degeneração hidrópica, tecido de granulação. O mesmo grupo ficou entre as menores médias de acantose e infiltrado inflamatório na derme, ao mesmo tempo que apresentou as maiores médias de VEGF no subcutâneo e no tecido de granulação. Dessa forma, pode-se concluir que as ADSCs protegeram os enxertos dos efeitos deletérios da isquemia, assim como e sugerem-se novos estudos em fases iniciais da cicatrização para compreensão dos mecanismos envolvidos. / Evaluation of adipose derived stem cells (ADSC) effect on full-thickness skin graft (FTSG) as a wound healing model in ischemic conditions. Two 12 mm diameter FTSGs were harvested and placed onto dorsal recipient beds of twenty-four Wistar rats, in two anatomic regions: cranial and caudal. Rats were randomized into five groups. Before grafting, group E FTSGs received subfascial injection of 1X106 ADSCs diluted in 200 μL of physiologic saline. Group EC FTSGs received only physiologic saline. Group B received ADSCs in the recipient bed edges; group C received physiologic saline in the edges. Group ADSC_GB received the same ADSCs number and volume, half in the graft and half in the edges. Using planimetry, grafts were analyzed for graft´s contraction rate (d0, d5, d14), normal skin rate and occurrence of epidermolysis and failure rate (d14). FTSGs samples were obtained on the d14 to hematoxylin-eosin and Masson’s Trichrome staining for epidermal analysis (epidermal thickening, keratosis, acanthosis, hydropic degeneration, inflammatory infiltrate) and dermal analysis (hairless, granulation tissue, inflammatory infiltrate, collagen deposition). The obtained results were expressed by mean (n=5). Statistical significance (p<0,05) was calculated using Generalized Estimating Equations. The ADSC_G group had better macroscopic aspects, less occurrence of epidermolysis and hairless and one of the lowest averages of ulceration area, along with the other groups treated with ADSCs. The ADSC_G group showed the lowest mean on the histopathological changes measured such as thickening of the epidermis, hydropic degeneration, less granulation tissue. The same group was among the lowest acanthosis and inflammatory infiltrate averages in the dermis, while presented with the greatest of VEGF average in the subcutaneous and in the granulation tissue. Thus it can be concluded that the ADSCs may have protected the grafts from the deleterious effects of ischemia and suggest new studies in the early stages of healing to understand the mechanisms involved.

Malgré l’évolution des outils de bio-ingénierie tissulaire et la sophistication des recherches visant à développer de nouveaux substituts cutanés, la prise en charge clinique des brûlures sévères a relativement peu évolué depuis plus de 30 ans. Si la survie est quasiment garantie par l’utilisation de cultures d’épidermes autologues (CEA), le traitement des grands brûlés n’en reste pas moins traumatisant, avec l’émergence de séquelles physiques (cicatrices hypertrophiques, dyschromies, fragilité cutanée), pathophysiologiques (désordres métaboliques et immunitaires), et neuropsychologiques (neuropathies, douleurs chroniques, syndrome de stress post-traumatique). Depuis leur découverte dans les années 1960, les Cellules Stromales Mésenchymateuses (CSM) ont suscité un intérêt grandissant dans le domaine de la thérapie cellulaire, en raison de leurs propriétés trophiques et immunomodulatoires. La découverte de leur haute plasticité face à divers stimuli environnementaux a plus récemment ouvert le champ de nouvelles stratégies de thérapie ciblée appelées « préconditionnement ». Ainsi, cette thèse a pour objet l’évaluation d’une stratégie de préconditionnement de CSM, afin d’en potentialiser l’action thérapeutique, dans le cadre du traitement de brûlures par CEA. Ce travail de thèse a été partagé en trois unités expérimentales. Tout d’abord la stratégie de préconditionnement a été mise au point sur des modèles de cicatrisation in vitro, permettant ainsi de souligner le rôle intéressant de l’interleukine-1β (IL-1β) et de la substance P (SP). Ensuite, l’efficacité et les mécanismes d’action de ces facteurs de préconditionnement ont été évalués in vitro, puis in vivo sur un modèle de plaie excisionnelle, en utilisant les CSM ou leurs produits de sécrétion. Il a ainsi pu être démontré que l’IL-1β améliorait l’efficacité des CSM en promouvant leur activité anti-inflammatoire, pro-migratoire et de remodelage. Il a également été montré que cet effet était en partie lié à un mécanisme impliquant la voie du TGF-β1. Enfin, la plus-value de cette stratégie thérapeutique a fait l’objet d’une étude in vivo sur un modèle de brûlure profonde mimant la prise en charge du patient sévèrement brûlé. Malgré divers problèmes techniques limitant la prise de greffe de CEA dans ce modèle, l’effet anti-inflammatoire et pro-angiogénique des CSM a pu être confirmé. Ces résultats semblent donc montrer l’intérêt d’une thérapie par CSM préconditionnées. Des études précliniques complémentaires sont maintenant requises pour vérifier la plus-value d’une telle thérapie dans le contexte de la brûlure cutanée. / Since the 1980’s, little progress has been made in the management of major burns, in spite of several research advances in the field of skin tissue engineering and regenerative medicine. Developed in 1975, Cultured Epithelial Autografts (CEA) are the last-in-date significant breakthrough, allowing patient survival in most critical cases. However, patients still have to cope with debilitating sequelae including hypertrophic scars, skin fragility, immunometabolic dysfunctions, chronic pain and post-traumatic stress disorder. Mesenchymal Stromal Cells (MSC) have raised an increasing interest during the past 50 years due to their trophic and immunomodulatory properties. Recent findings about their high plasticity to external stimuli have fostered the development of new targeted therapies known as “preconditioning strategies”. This PhD work thus aimed to assess a MSC preconditioning strategy for the treatment of major burns using CEA. The present work was divided into three main experimental parts. First, in vitro experiments were developed in order to set up the preconditioning strategy, and revealed the interesting role of both interleukin-1β (IL-1β) and substance P (SP). Then, both effectiveness and mechanism of action of these preconditioning modalities were assessed in vitro and in vivo, either using MSC or their secretory products. It was thus shown that IL-1β could potentiate MSC effectiveness through the promotion of pro-migratory, anti-inflammatory and pro-remodeling activities. This effect was shown to be partly mediated by the TGF-β1 signaling pathway. At last, this preconditioning strategy was evaluated in a third degree burn rat model mimicking the surgical treatment applied to severe burn patients. Despite technical hurdles limiting CEA engraftment, anti-inflammatory and pro-angiogenic properties of MSCs were confirmed in this model. These preliminary results underline the potential of a preconditioned MSC therapy in wound healing. Additional preclinical studies are now required to corroborate the benefit of such a therapy in major burns.

Osteoarthritis is one of the most common causes of disability in adults in America. It is a progressive and degenerative disease where the articular cartilage is broken down and lost from the surfaces of bones causing chronic pain and swelling in the joints, and currently has no cure. The most commonly osteoarthritis starts from a focal lesion on the cartilage surface, which will expand on the surface and downwards through the thickness of the tissue. The current gold standard for correcting cartilage focal lesions is the osteochondral autograft/allograft transplantation (OAT), which replaces the defect with a fresh osteochondral graft. The main limiting factor for using the OAT comes from the limited number of autograft and allografts that are available for implantation. To address the concern of graft availability, this study will look at the development of a porcine osteochondral xenograft (OCXG). The first aim of this research is to establish a decellularization protocol that will remove the antigens and cellular debris, which are the leading causes of graft rejection when implanting animal tissue in humans. The second aim of this study is restoring the mechanical strength of the OCXG that was lost during the decellularization process through crosslinking the tissue using genipin and epigallocatechin gallate (EGCG). The third aim is comparing the performance of the complete crosslinked OCXG at different degrees of crosslinking in a long-term goat animal model. The final aim is an alternative way to correct focal lesions through the development of an injectable collagen stabilizing treatment with genipin and punicalagin that will slow or stop the growth of a lesion and prevent osteoarthritis.

Introduction : Les conduits nerveux synthétiques représentent une alternative chirurgicale aux autogreffes dans la réparation des traumatismes aux nerfs périphériques. Afin d’améliorer la régénération nerveuse périphérique, plusieurs biomatériels, tels que la multicouche polyélectrolyte de soie (MPE), et modèles ont été étudiés. Dans le cadre de ma maitrise, nos objectifs de recherche sont d’établir si la MPE de soie permet d’améliorer la régénération nerveuse périphérique in vivo et si notre nouveau modèle de conduit (« jelly roll ») peut mener à une meilleure régénération du nerf sciatique chez le rat que le modèle de conduit creux. Méthodes : Dans cette étude, une technique chirurgicale in vivo de lacération et de réparation du nerf sciatique chez le rat fut utilisé. Cinq conditions expérimentales de conduits (autogreffe, conduit creux avec et sans MPE de soie et « jelly roll » avec et sans MPE de soie) furent implantées (n= 2 rats par condition). Après 4 semaines, les conduits furent récupérés et marqués par immunohistochimie avec le neurofilament et la protéine basique de la myéline (MBP). La performance de chaque conduit fut évaluée par sa capacité à supporter l’excroissance axonale à travers le long du conduit et à travers la largeur de ce dernier à divers endroits. Résultats : Chaque condition expérimentale a supporté une régénération axonale avec différents degrés de succès. Globalement, l’autogreffe a supporté une plus longue croissance de fibres. De plus, la surface de fibres obtenue était plus large que les autres conditions. Les conduits avec la MPE de soie ont eu une performance similaire à leurs homologues sans soie. De plus, le modèle de conduit creux a mené à une meilleure régénération axonale que le modèle du « jelly roll ». Conclusion : L’autogreffe demeure le meilleur conduit pour supporter la régénération nerveuse périphérique. Les conduits avec la MPE de soie peuvent supporter une régénération nerveuse similaire aux conduits sans soie tandis que le modèle de « jelly roll » a généré des performances inférieures au modèle de conduit creux. / Background: Synthetic nerve conduits constitute alternative surgical options to autografts in the repair of peripheral nerve injuries. Silk polyelectrolyte multilayer (PEM) as a biomaterial and novel conduit designs have been proposed to improve peripheral nerve regeneration. In my master’s project, my objective is to assess whether silk PEM can improve peripheral nerve regeneration in vivo and to assess whether our novel conduit design (“jelly roll”) can better support rat sciatic nerve regeneration than a hollow conduit design. Methods: In this study, an in vivo rat model of sciatic nerve laceration and repair was used. Five experimental conduit conditions (autograft, hollow conduit with and without silk PEM, and jelly roll with and without silk PEM) were implanted (n=2 rats per condition). After 4 weeks, the conduits were harvested and immuno-stained for neurofilament and myelin basic protein (MBP). Conduit performance was assessed by its ability to support axonal outgrowth throughout the conduit’s length and at various locations along its width. Results: Each condition supported axonal regeneration at varying levels of success. Overall, the autograft group outperformed all other groups by supporting the longest and widest occupying regenerating fibers. Conduits with silk PEM performed similarly to conduits without silk PEM. In addition, the hollow conduit design demonstrated better regenerative outcomes than the jelly roll design. Conclusion: The autograft remains the superior conduit to support peripheral nerve regeneration. Conduits with silk PEM support nerve regeneration in the same capacity as non silk-coated conduits while the jelly roll design underperformed in comparison to the hollow conduit design.

Recombinant human bone morphogenic proteins (rhBMPs) are extensively studied and employed clinically for treatment of various bone defects. Current clinical delivery vehicles suffer wasteful burst releases that mandate supra-physiological dosing driving concerns over safety and cost. It was therefore investigated whether a unique drug delivery vehicle sequestered within a composite scaffold could lower the burst release of rhBMP-2. PLGA-calcium phosphate tri-phasic composite scaffolds delivered model protein BSA with burst release of ~13% and sustained kinetics of 0.5-1.5% BSA/day up to 45 days. rhBMP-2 was delivered with zero burst release however at much lower levels, totaling 0.09% to 0.9 % release over 10 days, but had up to 6.3-fold greater bioactivity than fresh rhBMP-2 (p<0.05). In conclusion, the three-phase composite scaffold can deliver bioactive proteins with a reduced burst release and sustained secondary kinetics.

Recombinant human bone morphogenic proteins (rhBMPs) are extensively studied and employed clinically for treatment of various bone defects. Current clinical delivery vehicles suffer wasteful burst releases that mandate supra-physiological dosing driving concerns over safety and cost. It was therefore investigated whether a unique drug delivery vehicle sequestered within a composite scaffold could lower the burst release of rhBMP-2. PLGA-calcium phosphate tri-phasic composite scaffolds delivered model protein BSA with burst release of ~13% and sustained kinetics of 0.5-1.5% BSA/day up to 45 days. rhBMP-2 was delivered with zero burst release however at much lower levels, totaling 0.09% to 0.9 % release over 10 days, but had up to 6.3-fold greater bioactivity than fresh rhBMP-2 (p<0.05). In conclusion, the three-phase composite scaffold can deliver bioactive proteins with a reduced burst release and sustained secondary kinetics.