Skeletal muscle regeneration is hindered in severe injuries and degenerative diseases, including volumetric muscle loss (VML), due to the failure of current treatments to induce functional tissue growth. Various biological functions in skeletal muscle are supported by the extracellular matrix (ECM), a collection of proteins and glycosaminoglycans. <i>In vivo</i> studies on murine plantaris muscle hypertrophy indicate that ECM remodeling facilitates muscle growth, but a global analysis of ECM protein dynamics during skeletal muscle hypertrophy and repair are unknown. Understanding this influence of the ECM can establish instructive cues for regenerative therapies. Here, we define global proteomic changes throughout stages of plantaris muscle hypertrophy, with an emphasis on characterizing ECM proteins. Synergistic ablation of the gastrocnemius and soleus muscles induced a compensatory hypertrophic effect causing a 40% mass increase in the plantaris muscle 28 days post injury. Liquid chromatography-tandem mass spectrometry revealed the differential abundance of 1233 proteins, including 99 ECM proteins, across five time points. After two days of injury, a significant increase of ECM glycoproteins was observed although the overall collagen abundance decreased. Throughout the duration of injury, the relative abundance of type I collagen decreased while there was an increase of proteins associated with type I collagen fibrillogenesis (types III and V) and basement membrane (types IV and VI). Collectively, these results provide a better understanding of ECM dynamics throughout skeletal muscle hypertrophy. Future studies will evaluate protein synthesis by using non-canonical amino acids to identify newly synthesized proteins. Temporal analysis of protein dynamics symbolic to injury and tissue growth will provide tissue engineers with precise information to develop successful regenerative therapies to restore functional muscle in VML.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2018-06-14T20:50:43Z No. of bitstreams: 1 Stella Maris Lins Terena.pdf: 2089344 bytes, checksum: 21a72af34c64206cc3ab2d9b66964a22 (MD5) / Made available in DSpace on 2018-06-14T20:50:43Z (GMT). No. of bitstreams: 1 Stella Maris Lins Terena.pdf: 2089344 bytes, checksum: 21a72af34c64206cc3ab2d9b66964a22 (MD5) Previous issue date: 2016-06-28 / Skeletal muscle is a tissue of great adaptive capacity and is able to change its characteristics to meet various functional demands. This adaptation can be caused by mechanical overloading which results in an increase in the size of the area of muscle fiber and increase muscle mass. This process is known as muscle hypertrophy and during numerous modifications hypertrophy occur in both the muscle tissue and tendons. Studies with low-level laser (LLL) in skeletal muscle have demonstrated that its effects are positive with respect to the reduction process of inflammation, myonecrosis and reduced influence on the collagen fibers during the remodeling process.The present study aimed to analyze the effects of LLL (780nm) during the compensatory hypertrophy process on the muscular aspects and tendon of the plantaris muscle of mice (in vivo) and also evaluate its effects on human endothelial cells (HUVECs) in vitro. They were used for the in vivo experiment 22 Wistar rats were divided into control group, non-irradiated group and irradiated group hypertrophy hypertrophy. The parameters used for the irradiation were λ = 780nm, beam area 0.04 cm2, 40mW output power, energy density of 10 J / cm2. periods of 7 and 14 days were analyzed. To cause hypertrophy model was used for ablation of synergists being taken for this study, the gastrocnemius medial and lateral muscles and the soleus muscle, leaving the plantaris muscle suffered overload. the cross-sectional area were analyzed, collagen area, the number of myonuclei and mature blood vessels in muscle tissue and the organization and arrangement of collagen fibers in the tendon. For the experiment in vitro endothelial cells were used (HUVECs) were divided into control group and the other groups irradiated also with λ = 780nm, 0.04 cm2 beam area, 40mW output power and energy density of 1 J / cm2, 5J / cm2, 10 J / cm2 to 20J / cm2. The different application time was calculated for all groups received the same dose of energy. They assessed cell viability, total protein and vessel formation in Matrigel. The in vivo results showed that there was increased cross-sectional area of the irradiated group after 14 days (26.3%) when compared to non-irradiated group. There was also increased number of myonuclei in the irradiated group after 14 days. The total area of muscle collagen increased (4.2%) in the irradiated group in 7 days and decreased (6.4%) in the irradiated group after 14 days when compared to non-irradiated group in both periods. Analysis by birefringence in the tendons showed better organization of the fibers 7 and 14 days when compared to non-irradiated group. In vitro results have shown decrease in cell viability in all irradiated all groups compared to the control and counting mature vessels was increased number of vessels in the irradiated group even after 14 days. In conclusion laser irradiation increased muscle mass, the number of myonuclei and the number of mature vessels and decreased collagen total area of muscle tissue after 14 days; improved tendon collagen organization for 7 and 14 days and interfere with the viability and total protein concentration in a dose-dependent endothelial cells HUVECs. / O músculo esquelético é um tecido de grande capacidade adaptativa e que é capaz de alterar suas características para atender às diversas demandas funcionais. Essa adaptação pode ser causada por uma sobrecarga mecânica que resulta num aumento do tamanho da área da fibra muscular e aumento de massa muscular. Esse processo é conhecido como hipertrofia muscular e durante a hipertrofia inúmeras modificações ocorrem tanto no tecido muscular quanto nos tendões. Os trabalhos recentes com laser de baixa potência (LBP) no músculo esquelético tem demonstrado que seus efeitos são positivos no que diz respeito ao processo de redução da inflamação, redução da mionecrose e influência sobre as fibras de colágeno durante o processo de remodelamento. Nesse contexto o presente estudo teve por objetivo analisar os efeitos do LBP (780nm) durante o processo de hipertrofia compensatória sobre os aspectos músculares e tendíneos do músculo plantar de ratos (in vivo) e também avaliar seus efeitos em células endoteliais humanas (Huvecs) in vitro. Foram utilizados para o experimento in vivo 22 ratos Wistar, divididos em 3 grupos: controle, não irradiado com hipertrofia e irradiado com hipertrofia. Os parâmetros utilizados para a irradiação foram λ= 780nm, área do feixe 0,04 cm2, potência de saída 40mW, densidade de energia de 10J/cm2. Foram analisados os períodos de 7 e 14 dias. Para causar a hipertrofia foi utilizado o modelo de ablação dos sinergistas sendo retirados para este estudo os músculos gastrocnêmio lateral e medial e o músculo sóleo, restando o músculo plantar que sofreu a sobrecarga. Foram analisados a área de secção transversa, a área do colágeno, o número de mionúcleos e vasos sanguíneos maduros no tecido muscular e organização e disposição das fibras de colágeno no tendão. Para o experimento in vitro foram utilizadas células endoteliais (Huvecs) divididas em grupos controle e os demais grupos irradiados também com λ= 780nm, área do feixe 0,04 cm2, potência de saída 40mW e densidades de energia de 1J/cm2, 5J/cm2, 10J/cm2 e 20J/cm2. O tempo de aplicação foi diferente, calculado para que todos os grupos recebessem a mesma dose de energia. Foram avaliados a viabilidade celular, proteína total e a formação de vasos em matrigel. Os resultados in vivo obtidos demonstraram que houve aumento da área de secção transversa do grupo irradiado após 14 dias (26,3%) quando comparado ao grupo não irradiado. Houve também aumento do número de mionúcleos no grupo irradiado após 14 dias. A área total do colágeno muscular aumentou (4,2%) no grupo irradiado em 7 dias e diminui (6,4%) no grupo irradiado após 14 dias quando comparado ao grupo não irradiado nos dois períodos. A análise por birrefringência nos tendões demonstrou maior organização das fibras em 7 e em 14 dias quando comparadas ao grupo não irradiado. Os resultados in vitro demonstraram que as diferentes fluências do laser interferiram de maneira diferente na viabilidade de células Huvecs em todos os períodos avaliados. Com relação a contagem de vasos maduros houve aumento no grupo irradiado após 14 dias. Em conclusão a irradiação laser aumentou a massa muscular, o número de mionúcleos e o número de vasos maduros e diminuiu a área total de colágeno no tecido muscular após 14 dias; melhorou a organização do colágeno tendíneo em 7 e em 14 dias e interferiu na viabilidade e concentração de proteína total de maneira dose dependente das células endoteliais Huvecs.

Abstract Achilles tendon rupture (ATR) conservative treatment result usually good clinical outcome, but despite the treatment method calf muscle strength deficit persist. Recent evidence suggests that surgery might surpass conservative treatment in restoring strength after ATR, but structural explanations for surgery-related improved strength remain uncertain. The purposes of this thesis were to compare calf muscle isokinetic strength recovery, calf muscle volume, fatty degeneration and AT elongation after conservative treatment or after open surgical repair of ATR. An additional aim was to assess the role of fascial augmentation in terms of calf muscle isokinetic strength recovery, AT elongation, calf muscle volume atrophy and fatty degeneration, and their relationship with calf muscle isokinetic strength in long-term follow-up after ATR surgery. Surgery resulted in 10% to 18% greater plantar flexion strength (P = 0.037) compared to conservative treatment. The mean differences between affected and healthy soleus muscle volumes were -18% after surgery and -25% after conservative treatment (P = 0.042). At 18 months, AT were, on average 19 mm longer in patients treated conservatively compared to surgery (P &#60; 0.001). At 18 months, patients with greater (2–3) fatty degeneration had lower soleus muscle volumes and plantar flexion strength in the healthy leg. In long term, augmentation did not affect any of the strength variables, but the injured side showed 12% to 18% strength deficit compared with the healthy side (P &#60; 0.001). The AT was, on average, 12 mm longer in the affected leg than in the healthy leg (P &#60; 0.001). The mean soleus muscle volume was 13% lower in the affected leg than in the healthy leg (P &#60; 0.001). The mean volumes of the medial- and lateral gastrocnemius muscles were 12% and 11% lower in the affected leg than in the healthy leg, respectively (P &#60; 0.001). AT elongation correlated substantially with plantar strength deficit (ρ = 0.51, P &#60; 0.001) and with both gastrocnemius (ρ = 0.46, P = 0.001) and soleus muscle atrophy (ρ = 0.42, P = 0.002). Calf muscle fatty degeneration was more common in the affected leg compared healthy leg (P &#8804; 0.018). In conclusion, surgery of ATR restored calf muscle isokinetic strength earlier and more completely than conservative treatment. Conservative treatment resulted in greater soleus muscle atrophy and AT elongation compared surgery, which may partly explain the surgery related better strength results. Augmentation provided no long-term benefits compared with simple suturation, and a 12 to 18% plantar flexion strength deficit compared to the healthy side persisted. AT elongation may explain the smaller calf muscle volumes, greater fatty degeneration, and plantar flexion strength deficit observed in long-term follow-up after surgical repair of ATR. / Tiivistelmä Akillesjännerepeämän (ATR) konservatiivisella ja leikkaushoidolla hoidolla saavutetaan hyvät kliiniset tulokset. Viimeisimmät tutkimukset kuitenkin viittaavat leikkaushoidolla saavutettavan paremmat voimat kuin konservatiivisella hoidolla, mutta rakenteelliset selitykset leikkaushoidon paremmalle pohjelihaksen voimille ovat epäselviä. Työn tarkoituksena oli verrata pohjelihaksen isokineettisten voimien palautumista, pohjelihastilavuuksia, rasvadegeneraatiota ja akillesjänteen (AT) pidentymistä ATR:n konservatiivisen- ja leikkaushoidon jälkeen. Tarkoituksena oli arvioida lihaskalvovahvikkeen merkitystä pohjelihaksen isokineettisten voimien palautumisessa pitkäaikaisseurannassa. Lisäksi tutkimme AT pidentymisen, pohjelihastilavuuksien ja rasvadegeneraation suhdetta pohjelihaksen isokineettisiin voimiin ATR:n leikkaushoidon jälkeen 14 v seurannassa. Leikkaushoidolla saavutettiin 10–18 % paremmat pohjelihaksen voimat verrattuna konservatiiviseen hoitoon. Leikkaushoidon jälkeen soleuslihasten tilavuuksien puoliero terveen jalan hyväksi oli 18 % ja konservatiivisen hoidon jälkeen 25 %. 18 kk kohdalla konservatiivisesti hoidettujen AT oli 19 mm pidempi verrattuna leikkauksella hoidettuihin. 18 kk kohdalla potilaat, joilla vamma jalan soleuslihaksen rasva-degeneraatio oli korkea (2–3), kärsivät suuremmasta soleuslihaksen atrofiasta ja pohjelihaksen voima puolierosta. Voimat eivät muuttuneet 12 kk ja 14 v kontrollien välillä. Lihaskalvovahvikkeella ei ollut merkitystä voimien palautumisessa pelkkään suoraan ompeluun verrattuna, mutta vammapuoli jäi 10–18 % heikommaksi verrattuna terveeseen jalkaan. Vammajalan akillesjänne oli 12 mm pidempi terveeseen jalkaan verrattuna. Vammajalan kolmipäisen pohjelihaksen tilavuus oli 11–13 % pienempi verrattuna terveeseen jalkaan. Akillesjänteen pituus korreloi pohjelihaksen voimapuolieron sekä pohjelihasatrofian kanssa. Akillesjännerepeämän leikkaushoidolla pohjelihaksen isokineettiset voimat palautuvat nopeammin ja täydellisemmin kuin konservatiivisella hoidolla. Leikkaushoitoon verrattuna konservatiivinen hoito johtaa suurempaan soleuslihaksen atrofiaan ja akillesjänteen pidentymään, mikä selittää osittain leikkaushoidon paremmat voimatulokset. 14 v seurannassa lihaskalvovahvikkeesta ei ole etua akillesjännerepeämän leikkaushoidossa. Akillesjännerepeämän leikkaushoidosta huolimatta potilaalle jää terveeseen jalkaan verrattuna 10–18 % pohjelihasten voimapuoliero. Akillesjänteen pidentyminen mahdollisesti selittää pohjelihasten atrofian, rasvadegeneraation ja pysyvän pohjelihasten voimapuolieron akillesjännerepeämän leikkaushoidon jälkeen 14 v seurannassa.