The aim of this project was to create a novel biodegradable, synthetic scaffold that will provide the correct topographical cues for myoblast alignment and efficient differentiation into myotubes. Skeletal muscle repair after major surgery or serious burns is often overlooked leading to poor healing and consequent loss of power in movements of affected limbs. In order to overcome this problem a tissue engineered construct could be utilised as a grafting patch to encourage further regeneration and enhance possible power to the limb. Using a biodegradable polymer can provide structural support until the tissue is established, and will be excreted by the body's natural processes as it degrades. A synthetic polymer is desirable as it can reduce the risk of immunogenic responses thus reduce risk of graft rejection. For successful in vitro growth of skeletal muscle, the cells must be encouraged to arrange themselves into parallel arrays in order for efficient fusion and consequent contraction. By incorporating the correct topographical cues into the scaffold to promote contact guidance for cellular alignment this can be achieved. Electrospinning is a reliable technique which yields highly reproducible aligned fibres from the micro- to the nanoscale. This project focuses upon creating and characterising the electrospun scaffold, checking biocompatibility with myoblasts by monitoring the topography, residual solvent within the scaffold, the mechanical properties of the scaffold, and a brief investigation into the degradation profile of the electrospun fibres. The immunogenicity of the scaffold was investigated by monitoring cytokine release from macrophages. Myoblast morphology was monitored, as was the efficiency of the cells to differentiate and their potential to become contractile myofibres. Cellular adhesion to the scaffold was also looked into by measuring the expression of integrins during early and late adhesion and on substrates with different topographies. It was found that the electrospun scaffold did not contain a significant amount of residual solvent, and macrophages were not activated any more than on tissue culture plastic. Myoblasts responded to the topography of the aligned fibres by aligning along the length of the fibres, showing elongation and bi-axial cytoskeletal arrangement after just 30 minutes culture on the aligned fibres. This elongation prompted fusion and differentiation of the myoblasts to occur faster than cells which were not exposed to the aligned topography, and this global alignment was maintained in long term culture.

O envelhecimento é caracterizado por diversas alterações no organismo, as quais acarretam em fragilidade, maior susceptibilidade a quedas, perda de autonomia e piora da qualidade de vida. O músculo esquelético também é afetado pelo envelhecimento, levando a alterações na locomoção, adaptação metabólica e em sua plasticidade. Alterações na plasticidade - prejudicam a capacidade regenerativa do músculo esquelético, desencadeando modificações em todos os estágios desse processo. Uma estratégia que tem sido bastante utilizada para minimizar/reverter o impacto do envelhecimento na função e plasticidade muscular é o treinamento físico aeróbico (TFA), o qual promove diversos benefícios à musculatura esquelética. Dessa forma, na presente dissertação investigamos a contribuição do TFA de 4 semanas em esteira rolante na capacidade regenerativa do músculo tibial anterior de camundongos jovens e idosos após lesão mecânica. A capacidade regenerativa foi avaliada por métodos histológicos e de imunofluorescência em tecido aos 2, 4 e 15 dias após a indução da lesão mecânica. Os níveis de RNAm de fatores relacionados à resposta regenerativa muscular foram avaliados por PCR em tempo real. Para confirmar a eficácia do TFA e função muscular, avaliamos a capacidade aeróbica, a deambulação e a produção de força ex vivo. Observou-se que o TFA melhorou a função muscular e a capacidade aeróbica dos animais jovens e idosos. No que diz respeito ao processo de regeneração muscular, os resultados obtidos sugerem, aumento da área necrótica, da inflamação, da deposição de colágeno e redução da área de secção transversa das fibras nos animais idosos sedentários ao longo do curso temporal estudado. Adicionalmente, observou-se redução na expressão de genes envolvidos na ativação de células satélites e atraso no processo de diferenciação dessas células nesses animais. OTFA contribuiu para a redução da área necrótica, da inflamação, levando a menor deposição de colágeno e aumento da distribuição das fibras centro nucleadas nos animais idosos. No entanto, não se observou modificações na expressão dos genes com o TFA nesses animais. Portanto, os dados sugerem que o TFA contribui para melhora do processo de regeneração muscular em camundongos idosos / Aging is a biological process characterized by a progressive impairment in physiological systems, which leads to general frailty and reduced exercise tolerance and performance in daily living activities. Skeletal muscle is directly affected by aging, displaying changes in locomotion, metabolic adaptation, and muscle plasticity. Altered muscle plasticity affects muscle regeneration capacity in elderly. Aerobic exercise training (AET) has been used as a strategy to minimize/reverse the impact of aging on muscle function and regenerative function. Thus, we have investigated the contribution of 4-week AET (running on the treadmill) for tibialis anterior muscle regenerative response from mechanical injury in young and old muscle, which were randomly assigned into untrained and trained groups. The regenerative capacity was evaluated by histology and immunofluorescence at 2, 4 and 15 days after the mechanical injury induction. Muscle mRNA levels of regulatory genes involved in muscle regeneration were evaluated by real time PCR. To verify the effectiveness of AET and muscle function, we assessed the aerobic capacity, step length in ambulation test and ex vivo muscle force production. We observed that AE improved muscle function and aerobic capacity of young and old mice. Regarding the muscle regeneration process, our data suggest an increase in necrotic area, inflammation and collagen deposition paralleled by a reduced fiber cross sectional area in sedentary old mice. These responses were associated with changes in gene expression suggesting reduced satellite cells activation and delayed differentiation. AET contributed to reduction in both necrotic area and inflammation, leading to reduced collagen deposition and increased centronucleated fibers, suggesting improved regeneration process. However no changes were observed in mRNA levels of genes studied after AET. Altogether, our data provide evidence for AET improved regeneration process in muscle of old mice

Estudos preliminares através de microarray nos mostraram que a E3 ligase Mdm2 foi regulado positivamente no músculo de animais hipertireoideos. Dessa forma, nós inferimos uma possível relação de Mdm2 com a atrofia causada por T3. Para testar nossa hipótese, ratos foram induzidos ao hipertireoidismo para análises subsequentes. Concomitante com a perda de massa muscular foi confirmado um aumento da expressão de Mdm2 tanto no nível gênico (p<0.05) quanto protéico. Interessantemente, Mdm2 foi preferencialmente expresso em fibras tipo I, mostrando maior sensibilidade dessas fibras ao T3. Além disso, foi observado uma diminuição severa na expressão de Pax7/MyoD associado à superexpressão de Mdm2, sugerindo inatividade das células satélites. Surpreendentemente, a inibição de Mdm2 em miotubos cultivados provocou uma diminuição severa no diâmetro destes (~35%, p<0.05), ou seja, tal inibição foi incapaz de minimizar a proteólise muscular causada por T3. Portanto, nós concluímos que a responsividade de Mdm2 ao T3 agiria como um mecanismo compensatório numa tentativa de minimizar a proteólise muscular causada pelo hipertireoidismo. Esta conclusão é reforçada pela atrofia observada em miotubos durante a inibição de Mdm2 sem a presença de T3. / Previous studies in our lab through microarray assay observed Mdm2, an E3 ligase, up regulated in soleus muscle from hyperthyroid rats. In this sense, we inferred that Mdm2 could be related to muscle atrophy caused by T3. To test our hypothesis, rats were induced to experimental hyperthyroidism for subsequent analysis. Along the muscle mass loss, the increase on Mdm2 gene expression was confirmed (p<0.05) as well as protein expression by RT-PCR and Western Blot, respectively. Interestingly, Mdm2 was expressed predominantly in fiber I type during T3 treatment, demonstrating a higher sensibility when compared to type II fiber. Moreover, it was observed a severe decrease in Pax7/MyoD labeling, associated to an increase on Mdm2 labeling, suggesting that T3 could be associated with inactivation of satellite cells. Surprisingly, Mdm2 inhibition in myotubes have induced severe decrease on myotubes diameter (~35%, p<0.05), in other words, Mdm2 inhibition was not able to decrease muscle proteolysis during high levels of T3. Thus, the increase on Mdm2 levels could be a compensatory effect to reduce the muscle mass loss during T3 treatment. This conclusion is highlighted by the myotubes atrophy observed during the Mdm2 inhibition without T3 treatment.

O hormônio tireoideano (T3) é um potente regulador das funções cardíacas e musculares esqueléticas. Desse modo, o presente trabalho identificou o efeito de tal hormônio nas proteínas da banda M sarcomérica, as quais fazem parte das proteínas estruturais cardíacas e musculares esqueléticas. O T3 diminuiu a expressão da proteína M no coração, uma das proteínas da banda M, e agiu diretamente no gene dessa proteína M. No músculo esquelético, T3 aumentou a expressão de EH-miomesina no músculo sóleo e reduziu a expressão de proteína M no músculo extensor digital longo (EDL). Portanto, pode-se concluir que o T3 possui uma importante função na regulação da expressão de proteínas estruturais musculares e a ausência dessas proteínas pode ocasionar lesões das estruturas musculares cardíacas e esqueléticas. / Thyroid hormone (T3) regulates many functions of the heart and skeletal muscle. In this way, the present work identified the effect of T3 on the sarcomeric M band proteins, which are structural proteins from the heart and skeletal muscle. T3 down regulated M protein expression in the heart, one of the M band proteins, and, moreover, T3 could regulate M protein gene directly. In the skeletal muscle, T3 up regulated EH-myomesin expression in soleus muscle and T3 down regulated M protein expression in the extensor digitorum longus muscle (EDL). Therefore, we could conclude that T3 has an essential function in the regulation of muscle structural protein and the absence of these proteins could cause lesions at cardiac and skeletal muscle structures.

Neste estudo, testamos dois novos moduladores seletivos do receptor de glicocorticóide, nomeados L5 e L7, em comparação com o dexametasona, sobre aspectos estruturais, funcionais e moleculares no músculo sóleo. Ratos Wistar foram tratados com doses progressivas de dexametasona, L5 e L7 em 1 ou 7 dias. A massa corporal e a ingestão alimentar apresentaram queda após o tratamento com dexametasona em todas as doses; os tratamentos com L5/L7 mostraram resposta semelhante aos controles. O peso do músculo foi diminuído pelo dexametasona, efeito não observado nos tratamentos com L5/L7. Apenas o tratamento com dexametasona causou uma diminuição na área de secção transversa dos tipos de fibra muscular analisada. A força tetânica do sóleo foi diminuída pela dexametasona, nos tratamentos com L5/L7 este parâmetro também não foi afetado. A expressão gênica de MAFbx/Atrogin-1 e MuRF-1 foi elevada pela dexametasona; por outro lado, L5/L7 não elevaram a expressão destes genes. Concluímos que o L5/L7, em contraste com o dexametasona, preveniu o músculo esquelético da atrofia. / In this study, we have tested two new selective modulators named L5 and L7 along with dexamethasone in skeletal muscle structural, functional and molecular aspects. Male Wistar rats were treated with progressive doses of dexamethasone, L5 and L7 for 1 and 7 days. While body weight and food intake were decreased by the dexamethasone treatment in all doses, L5/L7 treatments induced gain in body weight similarly to controls. Muscle weight was decreased by dexamethasone, while L5/L7 were ineffective. Only the dexamethasone treatment caused a decrease in the analyzed cross sectional area of the skeletal muscle fiber types. Soleus tetanic force was decreased by the dexamethasone treatment, while L5/L7 treatments did not alter this parameter. MAFbx/Atrogin-1 and MuRF-1 gene expressions were elevated by dexamethasone; on the other hand, L5/L7 did not modulate any expression of those genes. We conclude that L5/L7, in contrast to dexamethasone, spare skeletal muscle from structural and functional loss, and molecular changes, reinforcing their role as a therapeutic device.

As lesões dos nervos periféricos, que inervam os músculos esqueléticos, evoluem para perdas da propriocepção e alterações na morfologia e função das fibras musculares, causando um impacto negativo na qualidade de vidas dos indivíduos. Tais lesões implicam em alteração na expressão de genes específicos do músculo, como por exemplo, na MyoD e Miogenina, atuantes na ativação de células satélites e reguladores da massa muscular A técnica cirúrgica de tubulização é um recurso empregado na prática clínica para tratamento de músculos que sofreram desnervação. O objetivo do presente estudo foi analisar se a técnica de tubulização com o preenchimento de gordura altera a expressão de Myod e Miogenina, a morfometria do músculo sóleo de ratos e localização da Myod e Miogenina. Para isso, 57 ratos Wistar foram separados em grupos: controle inicial (GCI); final 45 (GCF45), final 150 (GCF150), desnervado 45 dias (GD45), desnervado 150 dias (GCD150) e grupos experimentais com veia vazia 45 dias (GESP45) e 150 dias (GESP150) e com veia preenchida de gordura 45 dias (GEG45) e 150 dias (GEG150). Para os procedimentos cirúrgicos de desnervação e reinervação e coleta do músculo os animais foram profundamente anestesiados. Após os devidos tempos experimentais, os animais foram sacrificados, o músculo sóleo foi dissecado, envolvido em meio de criopreservação e estocado a -80°C. A quantificação de mRNA do MyoD e Miogenina foi realizada por amplificação por reação em cadeia de polimerase (PCR) em tempo real (RealTimePCR) e a localização da produção de Myod e Miogenina foi realizada por microscopia confocal a laser e imunofluorescência. A morfometria foi realizada em lâminas coradas com HE, observadas em microscópio ótico e calculadas pelo software Image Pro-Plus 6.2. Os resultados do presente estudo mostraram que houve aumento da expressão do Myod e Miogenina nos grupos experimentais 45 dias quando comparados ao grupo controle inicial e um decréscimo da expressão de Myod e Miogenina para os grupos experimentais com 150 dias. A área da secção transversa nos grupos experimentais com 45 dias (GESP45 e GEG45) não apresentaram diferença estatística, quando comparado com grupo desnervado 45 dias (GCD45), enquanto que o grupo experimental com preenchimento de gordura 150 dias (GEG150) obteve os melhores resultados na medida da área da secção transversal do músculo sóleo. As lâminas observadas no microscópio confocal mostram a MyoD e Miogen localizadas no mionúcleo. Concluiu-se que o uso da gordura na técnica de tubulização do nervo ciático de ratos, interfere na regeneração do músculo sóleo. / Peripheral nerve injuries can result in the loss of propioception, morphological and functional alterations of muscle fibers which causes a negative impact on the quality of life. These injuries elicit an alteration on the expression of muscle specific genes, like MyoD and Myogenin, involved in the satellite cell activation and muscle mass regulation. The vein graft tubulization is a well known technique for treatment of denervated muscle. The aim of this work was to investigate if vein graf tubulization filled with fat tissue changes the expression and localization of MyoD and Myogenin and to study if it can modify the morphometry of soleus muscle. Fifty seven Wistar rats were divided in initial control group (ICG), final control group 45 days and 150 days (FCG45; FCG150), denervated 45 days and 150 days (D45; D150) and experimental groups with vein graft 45 days and 150 days (VG45; VG150). and vein graft filled with fat tissue 45 days and 150 days (VF45; VF150). For denervation and reinervation procedures and muscle biopsy the animals were submitted to anaesthesia and after the experimental time they were euthanized. Soleus muscle was dissected, involved in criopreservation medium and stored at -80oC. It was performed RealTime polymerase chain reaction (RealTimePCR) for MyoD and Myogenin mRNA quantification. The localization of its production was analysed by laser confocal microscopy and immunofluorescence staining. The morphometric analysis were done by Hematoxilin-Eosin staining and examined at optical microscopy using the Image ProPlus 6.2 software. There was an upregulation on the expression of MyoD and Myogenin for the experimental groups at 45 days when compared to the initial control group. On the other hand, we found a downregulation on the MyoD and Myogenin expression in the same groups with 150 days. The area of transversal section in the 45 days experimental groups (VF45, VG45) did not show statistical difference compared with denervated group with 45 days (D45). Moreover, the group filled with fat tissue at 150 days (VF150) presented the best results in the transversal section area of soleus muscle. In addition, the slides analysed under confocal microscopy showed the localization of MyoD and Myogenin in the mionuclei. In conclusion, the application of vein graft filled fat tissue improves the soleus muscle regeneration.

This study investigates the skeletal muscle repair and regeneration process following blunt trauma injury in murine models. Skeletal muscle injury is recorded most often in sports injuries and include strains and sprains, contusions, and bruising, however, there is growing consensus about the role skeletal muscle plays in the reparative process of bone fractures. Skeletal muscle stem cells or satellite cells are mesenchymal stem cell derived cells that exist between the basal lamina and cell membrane of muscle fibers usually in close proximity to capillary beds. After a traumatic injury, satellite cells respond to the influx of signaling from immune cells, oxygen tension, and myogenic proteins which influence differentiation into myoblasts for repair of tissue damage. Research continues to elucidate the relationship between bone and skeletal muscle following trauma injuries. Skeletal muscle stem cells play a vital role in fracture healing, and in certain conditions, are even induced into the osteogenic pathway. The goals of this study are to characterize the temporal progression of myogenesis during muscle repair that will be used with future studies of muscle and bone injury. And to identify potential crosstalk mechanisms between muscle and bone repair during trauma. In our experiment model trauma was introduced to mice with a modified muscle contusion device where a weight was dropped onto the femoral quadriceps muscles and the quadriceps and biceps muscle tissues were harvested at post-operative days (POD) 2, 4, 12, 16, and 24. Reverse-Transcriptase Quantitative Polymerase Chain Reaction was used to analyze gene expression profiles for satellite/stem cells (Pax7 and Prx1), muscle regeneration (MyoD, Myf5, Myl2, and Myh1), angiogenesis (VegfA, VegfR2), myokine (Myostatin and IL6), and BMP signaling (ID1). Our findings indicate that both Pax7 and Prx1 expression slightly decreased after injury but showed a significant (p<0.05) increase and peak of expression at POD 16 in the femoral quadriceps muscles. The early myogenic genes, MyoD and Myf5 peaked early at POD 4 while the adult myofiber markers, Myl2 and Myh2, peaked later at POD 16 in the femoral quadriceps muscles. Only slight changes were observed in the femoral biceps muscles. The angiogenic genes peaked at POD16 in the femoral quadriceps muscles and POD 12 in the femoral biceps muscles. The expression of Myostatin, an inhibitor of muscle mass, decreased early (POD 4 and 12) however showed a non-significant increase at POD 16 in the femoral quadriceps muscles. Lastly, the expression of ID1, which is downstream target of BMP signaling peaked early at POD 4 in the femoral quadriceps muscles. These data indicates that stem/satellite cells decrease in response to muscle injury but by POD 4, myogenic commitment and programming occurs. While early myogensis occurs, BMP signaling peaks and Myostatin expression decreases suggesting a coordinated event. Adult myofiber regeneration occurs in parallel to angiogenesis. The myogenic events were primarily isolated to the injured femoral quadriceps muscles. This model of muscle injury can be used to study muscle regeneration within context to bone injury.

Le muscle strié squelettique possède de grandes capacités de régénération grâce à ses cellules souches, les cellules satellites. Après une lésion, le processus de régénération musculaire qui se met en place est finement régulé dans le temps et l’espace par le microenvironnement, constitué de cellules avoisinantes mais également par des éléments de la matrice extracellulaire (MEC). Cette dernière se compose de molécules structurales comme les collagènes et de composants possédant un rôle trophique comme les glycosaminoglycanes (GAGs). La MEC musculaire est peu étudiée à cause d’une organisation tridimensionnelle complexe rendant son exploration difficile. Lors d’une lésion avec perte de substance musculaire, la régénération est altérée, associée à une fibrose et une inflammation chronique. Ce type de lésion est fréquemment rencontré en traumatologie mais survient également chez le blessé de guerre. Malgré un traitement optimal, une invalidité fonctionnelle persiste chez ces patients. L’utilisation d’un biomatériau décellularisé, constitué de MEC pourrait fournir ce support physique et trophique faisant défaut dans ce type de lésion. Dans ce travail, nous avons entrepris l'établissement d'une MEC d’origine musculaire et nous avons établi un protocole de décellularisation permettant d’obtenir un biomatériau conservant l’architecture spécifique de la MEC musculaire avec une élimination de la majorité des antigènes cellulaires afin d'éviter une réponse immunitaire délétère après implantation. Néanmoins, le protocole retenu ne permet de conserver certaines molécules trophiques d’intérêt comme les GAGs. Les « ReGeneRaTing Agent®» (RGTA®) sont des mimétiques fonctionnels de ces GAGs, utilisés en clinique pour améliorer la cicatrisation cutanée et cornéenne. Ces mimétiques conservent une capacité de liaison aux facteurs de croissance avec une résistance aux dégradations enzymatiques. Nous avons évalué l’utilisation de ces molécules au cours de la réparation musculaire, dans un modèle in vivo chez le rongeur. Nous avons réalisé une analyse histologique précoce (8e jour de régénération) mettant en évidence une augmentation du nombre de noyaux par myofibre en faveur d’une augmentation de la fusion, validée également in vitro sur des progéniteurs musculaires. Nous avons également observé une augmentation du nombre de vaisseaux, suggérant une amélioration de l’angiogenèse. Le nombre de gouttelettes lipidiques, marqueur d’une mauvaise régénération, était en diminution. L’exploration histologique plus tardive (28e jour de régénération) n’a retrouvé que l’augmentation du nombre de vaisseaux en faveur d’un effet durable sur l’angiogenèse. Ces RGTA® peuvent être couplés aux biomatériaux et sont particulièrement résistants dans un environnement inflammatoire pouvant être rencontré dans les lésions avec perte de substance musculaire. Des chimiokines et des facteurs de croissance pourront également être ajoutés au biomatériau matriciel afin de favoriser la migration des différents progéniteurs nécessaires à une néoformation musculaire. L’efficacité thérapeutique de ces biomatériaux optimisés nécessitera d’être évaluée dans un modèle in vivo de perte de substance / Skeletal muscle exhibits high capacity for regeneration after an injury that relies on resident stem cells. Muscle regeneration is tightly regulated by both the immune response and other resident cells, as well as by cues from the local extracellular matrix (ECM), contributing to a coordinated repair process. Muscle ECM is a network of structural macromolecules with a large majority of collagens and trophic molecules such as glycosaminoglycans (GAGs). In the skeletal muscle tissue, ECM was overlooked due to its complex organization making investigations difficult. Muscle regenerative ability can be overtaken in large muscle wasting, such as in volumetric muscle loss (VML), leading to fibrosis formation and chronic inflammation. This type of injury predominantly occurs in traumatology and in war-wounded patients, with functional disability despite an optimal treatment. The use of biomaterials could provide the biochemical and physical cues that are missing in this pathologic repair. In this work we have focused on obtaining a biomaterial composed of skeletal muscle ECM. We have tested several decellularization protocols both to preserve the three-dimensional architecture of the muscle ECM and to completely remove cell components in order to avoid a deleterious immune response after implantation. However, the protocol did not allow the preservation of trophic molecules such as GAGs, in the scaffold.“ReGenerating Agents” (RGTA®) are functionally analogous of GAGs with a crucial property to resist enzymatic degradation. They function to restore a proper microenvironment for tissue healing with already a clinical application in skin and corneal repair. We have explored the effects of RGTA® in muscle regeneration using an in vivo model in mouse. At early time of regeneration (day 8), we performed histologic analysis. We showed that regenerating myofibers contained more nuclei in the treated animals, in favor of an increase of progenitor fusion, which has been validated in vitro in myogenic cultures. The number of capillaries was higher in favor of a better angiogenesis. Lipid droplets, a marker of impaired regeneration, were reduced by RGTA® administration. At later time of regeneration (day 28), capillary number was still improved in favor of a durable effect of RGTA® on angiogenesis. RGTA® could be incorporated into biomaterials and are particularly resistant in an inflammatory environment, such as that occurring after a VML injury. Chemokines and growth factors could also be added in ECM-based scaffolds to promote the migration of progenitors that are essential for myofiber neoformation. Therapeutic efficacy of these optimized biomaterials will require to be evaluated in an in vivo model of VML

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第22533号 / 人博第936号 / 新制||人||223(附属図書館) / 2019||人博||936(吉田南総合図書館) / 京都大学大学院人間・環境学研究科共生人間学専攻 / (主査)教授 林 達也, 教授 石原 昭彦, 教授 久代 恵介 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第22671号 / 人博第957号 / 新制||人||227(附属図書館) / 2020||人博||957(吉田南総合図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 川本 卓男, 教授 宮下 英明, 教授 高田 穣 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM