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Yes / Articular cartilage, a connective tissue, contains chondrocytes and glycosaminoglycans (GAGs) which aid in water retention, providing the tissue with its magnificent ability to prevent friction, withstand loads and absorb compressive shocks however, cartilage, does not have the ability to regenerate and repair. Osteoarthritis (OA) is a progressive degenerative disease, which includes reduction of cartilage thickness between two bones in a joint, causing painful bone-to-bone contact. OA affects over 8 million people in the UK alone. , and as the primary causes are unknown, available treatments including surgical and non-surgical techniques which only reduce the symptoms created by the disorder instead of providing a cure. This project focused on utilizing TGF-β1, a cytokine found in elevated amounts in healthy cartilage when compared to degraded cartilage, in order to observe the effects of the growth factor on both healthy and osteoarthritic chondrocytes. The healthy and the osteoarthritic chondrocytes were cultured in two different media (DMEM with and without TGF- β1) before utilizing the SpectraMax M2/M2e plate reader to observe and analyze the effect of TGF-β1 on water retention properties of cells. This has been achieved by quantifying the GAG content using DMMB dye. Results showed that although TGF-β1 did displayed an increase in glycosaminoglycan synthesis, the statistical increase was not vast enough for the alternative hypothesis to be accepted; further experimentation with TGF-β1, alongside other cytokines within the growth factor family is needed to perceive the true influence of the growth factor on un cured degenerative diseases. It was concluded that both the healthy and osteoarthritic cells treated with TGF-β1 absorbed considerably more DMMB in comparison to the cells, suggesting that TGF-β1 indeed works to aid in water retention. TGF-β1 is a key factor to be exploited when constructing treatments for osteoarthritis

Osteoarthritis is a debilitating joint disease characterized by the degradation of articular cartilage due to long term wear or acute injury. OA can lead to pain, limited mobility, and stiffness in the joint, and current treatment options often require invasive surgery or are limited to corrective attempts at mitigating pain. Due in part to the complexity of the disease and lack of holistic understanding of its advancement, there is no known treatment to halt or reverse the effects of OA progression in the joint. In order to address this need, the underlying mechanisms that drive the mechanical degradation of cartilage structure in its progression must be determined. The objective of this dissertation is to (1) investigate the mechanical breakdown of cartilage through fatigue failure in physiologically relevant models and (2) to introduce a minimally invasive method for increasing the mechanical integrity of cartilage in an effort to reverse the effects of OA. In order to classify the mechanical mediation of wear in OA disease pathology, wear progression in human articular cartilage must be fully characterized. Human articular cartilage exhibits a remarkable resilience to wear during frictional sliding, making it difficult to induce damage in the tissue in experimental models. Previous work established reciprocal compressive stresses, and not frictional stresses, as the primary initiator of delamination fatigue wear in immature bovine cartilage. In Chapter 2, we tested the hypothesis that reciprocal compressive stresses could induce fatigue wear in human articular cartilage and thus establish a reproducible and characterizable model of wear induction in human tissue. Human articular cartilage was subjected to 24 hours of frictional sliding in two contact configurations: stationary contact area (SCA), and migrating contact area (MCA). Five samples were tested in the SCA configuration, which induces frictional stresses, and five were tested in the MCA configuration, which induces reciprocal compressive stresses and frictional stresses. The SCA samples showed no conclusive damage after 24 hours of sliding, and recovered 99.3% ± 2.34% of their original thickness after testing. Three out of five MCA samples showed conclusive signs of damage, one in the form of tissue splitting, one in the form of blister formation, and one in the form of complete tissue tearing. The average friction coefficient in the SCA group (μ_SCA= 0.090 ± 0.008) was higher than the average friction coefficient in the MCA group (μ_MCA= 0.066±0.020; p=0.03). Although conducted as two separate studies, the results in Chapter 2 provide a preliminary data set to suggest that reciprocal compressive stresses are responsible for fatigue failure in human tissue, coherent with the results in the immature bovine model. Additionally, results of Chapter 2 establish a reproducible and physiologically relevant protocol for damage induction in human tissue. Future work will investigate this hypothesis with directly paired SCA and MCA human articular cartilage tissue samples of similar OA grade. To further understand cartilage damage mechanics in physiologically relevant conditions, Chapter 3 and 4 investigate the role of synovial fluid in fatigue failure of immature bovine cartilage. Synovial fluid is often incorrectly identified as the source of low friction in cartilage sliding. In fact, it has an effect on the friction coefficient that is far secondary to interstitial fluid load support. Further, reciprocal compressive stresses, not frictional stresses, have been shown to be responsible for fatigue failure. It is imperative to understand the function of synovial fluid in wear mechanics. We tested the hypothesis that synovial fluid reduces the rate of fatigue failure in immature bovine articular cartilage due to the protective effects of its molecular constituents. Eight paired medial and lateral tibial plateaus were tested in MCA sliding in phosphate buffer saline (n=8) or synovial fluid (n=8) to directly compare fatigue rate in synovial fluid versus phosphate buffer saline. An additional study evaluated the effect of molecular constituents on wear rate by testing medial and lateral tibial plateaus in 50% (n=8) and 25% (n=8) synovial fluid diluted with phosphate buffer saline. All eight samples tested in phosphate buffer saline damaged after 24 hours of reciprocal sliding, and none of the samples tested in pure synovial fluid became damaged over the same duration. After an additional two days of sliding, two of eight samples tested in pure synovial fluid got damaged. In the samples tested in 50% and 25% synovial fluid-phosphate buffer saline dilutions, one sample and five samples got damaged after 72 hours of sliding respectively. The results of this study confirmed the hypothesis that synovial provides a protective effect against fatigue failure. The study also suggests that dilution of the synovial below a critical value reduces the concentration of molecular constituents available to protect the cartilage against damage. Chapter 4 investigates the mechanism of synovial fluid’s protective effect further, by examining its potential to extend the duration of elevated fluid load support under compression and thereby reduce cartilage susceptibility to fatigue. The results of Chapter 4 illustrated that synovial fluid had no effect on the stress relaxation response of the cartilage to unconfined compression, disproving the presented hypothesis. Therefore, future work will investigate the function of synovial fluid in reducing the rate of fatigue, independent of its effect on friction. The final two studies of this dissertation present a novel treatment modality to induce collagen crosslinks that enhance the cartilage equilibrium modulus. The technique introduced is presented as a minimally invasive alternative to current surgical interventions and proposes to increase the integrity of early-OA tissue. In Chapter 5, we investigate the hypothesis that low-level femtosecond laser treatment of cartilage can increase the stiffness of the equilibrium modulus without damaging tissue integrity or cell viability. In the first experiment, six immature bovine cartilage samples were treated with the laser and the equilibrium modulus was found to increase in stiffness (p<10⁻³). The technique was also applied to human articular cartilage tissue with “low” and “high” OA, and tissue was found to have an increase in equilibrium modulus (p=0.003 and p=0.03, respectively). Cell viability was preserved under these treatment conditions. Chapter 6 further outlines a safe envelope of laser treatment parameters through evaluation of the effect of thermal heating on the equilibrium modulus of cartilage samples. The results of this study found that temperatures above 65 ℃ (p<10⁻³) increase the tissue modulus, but no change in modulus occurs below 65 ℃ (p=1.00). The results of Chapter 6 provide an insight to the mechanical effect of thermal exposure, and informed the laser treatment parameters presented in Chapter 5, which were confirmed to produce thermal heating far below temperatures that result in thermal stiffening. Through the results presented in Chapter 5 and 6, preliminary data is provided to introduce a novel method for crosslink induction in the superficial zone of articular cartilage. In future work, this technique can be applied as a potential strategy to increase fatigue wear resistance, and to reduce the progression of OA in diseased tissue. The work presented in this dissertation seeks to contribute to the understanding of fatigue wear in articular cartilage under physiologically relevant conditions, as well as introduce a method for enhancing cartilage tissue properties with laser treatment. In the first half of the dissertation, the effect of reciprocal compressive stresses was evaluated in human articular cartilage tissue and in immature bovine cartilage immersed in synovial fluid in an effort to understand the mechanism of delamination fatigue failure in OA progression. In the second half, a laser treatment modality was shown to increase tissue equilibrium modulus stiffness without compromising tissue viability.

[EN] Articular cartilage is a tissue that consists of chondrocytes surrounded by a dense extracellular matrix (ECM). The ECM is mainly composed of type II collagen and proteoglycans. The main function of articular cartilage is to provide a lubricated surface for articulation. Articular cartilage damage is common and may lead to osteoarthritis. Articular cartilage does not have blood vessels, nerves or lymphatic vessels and therefore has limited capacity for intrinsic healing and repair. Tissue engineering (TE) is a powerful approach for healing degenerated cartilage. TE uses three-dimensional (3D) scaffolds as cellular culture supports. The scaffold provides a structure that facilitates chondrocyte adhesion and expansion while maintaining a chondrocytic phenotype and limiting dedifferentiation, which is a problem in two-dimensional (2D) systems. Cell attachment to the scaffolds depends on the physical and chemical characteristics of their surface (morphology, rigidity, equilibrium water content, surface tension, hydrophilicity, presence of electric charges). The primary aim of this thesis was to study the influence of different kinds of biomaterials on the response of chondrocytes to in vitro culture. 3D scaffold constructs must have an interconnected porous structure in order to allow cell development through the network, to maintain their differentiated function, as well as to allow the entry and exit of nutrients and metabolic waste removal. Therefore, the effect of the hydrophilicity and pore architecture of the scaffolds was studied. A series of polymer and copolymer networks with varying hydrophilicity was synthesised and biologically tested in monolayer culture. Cell viability, proliferation and aggrecan expression were quantified. When human chondrocytes were cultured on polymer substrates in which the hydrophilic groups were homogeneously distributed, adhesion, proliferation and viability decreased with the content of hydrophilic groups. Nevertheless, copolymers in which hydrophilic and hydrophobic domains alternate showed better results than the corresponding homopolymers. Biostable and biodegradable scaffolds with different hydrophilicity and porosity were synthesised using a template of sintered microspheres of controlled size. This technique allows the interconnectivity between pores and their size to be controlled. Periodic and regular pore architectures and reproducible structures were obtained. The mechanical behaviour of the porous samples was significantly different from that of the bulk material of the same composition. Cells fully colonised the scaffolds when the pores' size and their interconnection were sufficiently large. Another objective was to assess the chondrogenic redifferentiation in a biodegradable 3D scaffold of polycaprolactone (PCL) of human autologous chondrocytes previously expanded in monolayer. This study demonstrated that chondrocytes cultured in PCL scaffolds without fetal bovine serum (FBS) efficiently redifferentiated, expressing a chondrocytic phenotype characterised by their ability to synthesise cartilage-specific ECM proteins. The influence that pore connectivity and hydrophilicity of caprolactone-based scaffolds has on the chondrocyte adhesion to the pore walls, proliferation and composition of the ECM produced was studied. The number of cells inside polycaprolactone scaffolds increased as porosity was increased. A minimum of around 70% porosity was necessary for this scaffold architecture to allow seeding and viability of the cells within. The results suggested that some of the cells inside the scaffold adhered to the pore walls and kept the dedifferentiated phenotype, while others redifferentiated. In conclusion, the findings of this thesis provide valuable insight into the field of cartilage regeneration using TE techniques. The studies carried out shed light on the right composition, porosity and hydrophilicity of the scaffolds to be used for optimal cartilage production. / [ES] El cartílago articular es un tejido compuesto por condrocitos rodeados por una densa matriz extracelular (MEC). La MEC se compone principalmente de colágeno tipo II y de proteoglicanos. La función principal del cartílago articular es proporcionar una superficie lubricada para las articulaciones. Las lesiones en el cartílago articular son comunes y pueden derivar a osteoartritis. El cartílago articular no tiene vasos sanguíneos, nervios o vasos linfáticos y, por tanto, tiene una capacidad limitada de auto-reparación. La ingeniería tisular (IT) es un área prometedora en la regeneración de cartílago. En la IT se utilizan "andamiajes" (scaffolds) tridimensionales (3D) como soportes para el cultivo celular y tisular. Los scaffolds proporcionan una estructura que facilita la adhesión y la expansión de los condrocitos, manteniendo un fenotipo condrocítico limitando su desdiferenciación; que es el mayor problema en los sistemas bidimensionales (2D). La adhesión celular a los scaffolds depende de las características físicas y químicas de su superficie (morfología, rigidez, contenido de agua en equilibrio, tensión superficial, hidrofilicidad, presencia de cargas eléctricas). El objetivo general de esta tesis fue estudiar la influencia de diferentes tipos de biomateriales en la respuesta de los condrocitos en cultivo in vitro. Los scaffolds deben tener una estructura porosa interconectada para permitir el desarrollo celular a través de toda la estructura 3D, potenciando que los condrocitos mantengan su fenotipo, así como permitiendo entrada de nutrientes y eliminación de desechos metabólicos. Se estudió el efecto de la hidrofilicidad y de la arquitectura de poro. Se cuantificó la viabilidad celular, la proliferación y la expresión de agrecano. Cuando los condrocitos humanos se cultivaron en sustratos poliméricos donde los grupos hidrófilos se distribuyeron de manera homogénea, la adhesión, la proliferación y la viabilidad disminuyó con el contenido de grupos hidrófilo. Sin embargo, los copolímeros en los que los dominios hidrófilos e hidrófobos se alternaban mostraron mejores resultados que los homopolímeros correspondientes. Se sintetizaron series de scaffolds bioestables y series biodegradables con diferente hidrofilicidad y porosidad utilizando plantillas de microesferas sinterizadas. Se obtuvieron arquitecturas de poros regulares y reproducibles. Las células colonizaron el scaffold en su totalidad cuando los poros y la interconexión entre ellos era lo suficientemente grande. Se evaluó la rediferenciación condrogénica de condrocitos autólogos humanos, previamente expandidos en monocapa, sembrados en un scaffold biodegradable de policaprolactona (PCL). Se demostró que los condrocitos cultivados en scaffolds de PCL con medio sin suero bovino fetal (FBS), se rediferenciaban de manera eficiente; expresando un fenotipo condrocítico, caracterizado por su capacidad de sintetizar proteínas de la MEC específicas de cartílago hialino. Se estudió la influencia de la hidrofilicidad y la conectividad de los poros de los scaffolds de caprolactona sobre la adhesión de los condrocitos a las paredes de los poros, su capacidad proliferativa y la composición de MEC sintetizada. Se observó que un mínimo de 70% de porosidad era necesario para permitir la siembra de los condrocitos en el scaffold y su posterior viabilidad. El número de células aumentaba a medida que aumentaba la porosidad del scaffold. Los resultados sugieren que parte de las células que se adherían a las paredes internas de los poros mantenían el fenotipo desdiferenciado de condrocitos cultivados en monocapa, mientras que otros se rediferenciaban. En conclusión, los resultados de esta tesis aportan un avance en el campo de la regeneración de cartílago articular utilizando técnicas de IT. Los estudios realizados proporcionan directrices sobre la composición, la porosidad y la hidrofilicidad más adecuada para l / [CA] El cartílag articular és un teixit format per condròcits envoltats per una densa matriu extracel·lular (MEC). La MEC es compon principalment de col·lagen tipus II i de proteoglicans. La funció principal del cartílag articular és proporcionar una superfície lubricada a les articulacions. Les lesions en el cartílag articular són comuns i poden derivar en osteoartritis. El cartílag articular no té vasos sanguinis, nervis ni vasos limfàtics i, per tant, té una capacitat limitada d'auto-reparació. L'enginyeria tissular (IT) és una àrea prometedora en la regeneració del cartílag. A la IT s'utilitzen "bastiments" (scaffolds) tridimensionals (3D) com a suports per al cultiu cel·lular i tissular. Els scaffolds proporcionen una estructura que facilita l'adhesió i l'expansió dels condròcits, mantenint un fenotip condrocític limitant la seua desdiferenciació; que és el major problema en els sistemes bidimensionals (2D). L'adhesió cel·lular als scaffolds depèn de les característiques físiques i químiques de la superfície (morfologia, rigidesa, contingut d'aigua en equilibri, tensió superficial, hidrofilicitat i presència de càrregues elèctriques). L'objectiu general d'aquesta tesi va ser estudiar la influència de diferents tipus de biomaterials en la resposta dels condròcits en cultiu in vitro. Els scaffolds han de tindre una estructura porosa interconnectada per a permetre el desenvolupament cel·lular a través de tota l'estructura 3D, potenciant que els condròcits mantinguen el seu fenotip així com permetent l'entrada de nutrients i l'eliminació de productes metabòlics. S'ha estudiat l'efecte de la hidrofilicitat i de l'arquitectura de porus dels scaffolds. Es va quantificar la viabilitat cel·lular, la proliferació i l'expressió de agrecà. Quan els condròcits humans es van cultivar en substrats polimèrics en els quals els grups hidròfils es van distribuir de manera homogènia, l'adhesió, la proliferació i la viabilitat van disminuir amb el contingut de grups hidròfils. No obstant això, els copolímers en els quals els dominis hidròfils i hidròfobs s'alternaven van mostrar millors resultats que els homopolímers corresponents. Es van sintetitzar sèries de scaffolds bioestables i sèries biodegradables amb diferent hidrofilicitat i porositat utilitzant plantilles de microesferes sinteritzades. Es van obtindre arquitectures de porus regulars i reproduïbles. Les cèl·lules van colonitzar el scaffold en la seua totalitat quan els porus i la interconnexió entre ells era suficientment gran. Es van avaluar la rediferenciació condrogènica de condròcits autòlegs humans, prèviament expandits en monocapa, en un scaffold biodegradable de policaprolactona (PCL). Es va demostrar que els condròcits cultivats en scaffolds de PCL sense sèrum boví fetal (FBS) es rediferenciaven de manera eficient, expressant un fenotip condrocític caracteritzat per la seua capacitat de sintetitzar proteïnes de la MEC específiques de cartílag hialí. També es va estudiar la influència de la hidrofilicitat i la connectivitat dels porus dels scaffolds de caprolactona sobre l'adhesió dels condròcits a les parets dels porus, la seua capacitat proliferativa i la composició de MEC sintetitzada. Es va observar que un mínim del 70% de porositat sembla ser necessari per permetre la sembra dels condròcits i la seua posterior viabilitat en el scaffold. El nombre de cèl·lules augmentava a mesura que augmentava la porositat del scaffold. Els resultats suggereixen que part de les cèl·lules que s'adherien a les parets internes dels porus mantenien el fenotip desdiferenciat de condròcits cultivats en monocapa, mentre que altres es rediferenciaven. En conclusió, els resultats d'aquesta tesi proporcionen informació valuosa en el camp de la regeneració de cartílag utilitzant tècniques d'IT. Els estudis realitzats proporcionen directrius sobre la composició, la porositat i la hidrofilicitat m / Pérez Olmedilla, M. (2015). Tissue engineering techniques to regenerate articular cartilage using polymeric scaffolds [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58987

Please note: we were unable to immediately open the spreadsheet below. We repaired the spreadsheet file with Excel, and have a copy of it in storage. If you have difficulty opening the spreadsheet, please write to us at open-help@bu.edu. / Osteoarthritis (OA) is a painful, chronic, non-inflammatory disease affecting 140 million people worldwide that alters synovial joint structure and function. OA progressively breaks down hyaline cartilage, the hydrated tissue that provides a smooth, nearly frictionless surface and distributes loads applied to articulating joint surfaces. The loss of glycosaminoglycans (GAGs) from the extracellular matrix of cartilage is an early marker of OA. Therefore, imaging methods that quantify the GAG content of cartilage are of interest. This work investigates the synthesis and development of three cationic contrast agents (CAs) for imaging articular cartilage (AC): CA4+, an iodinated small molecule, and tantalum oxide nanoparticles (Ta2O5 NPs) for x-ray Computed Tomography (CT) imaging; and Gadopentetate-dilysine (Gd(DTPA)Lys2), a gadolinium small molecule for Magnetic Resonance (MR) imaging. These cationic contrast agents are attracted to the strong negative fixed charge of extracellular GAG and, therefore, infiltrate cartilage. This work begins with an overview of CT and MR imaging basic principles, current clinical CAs and contrast enhanced imaging of AC. First, the large-scale (50 g) synthesis of CA4+ is described and the partitioning over time of CA4+ into ex vivo AC is correlated to GAG content and cartilage mechanical properties. Similar partitioning studies are applied to anionic, neutral and cationic Ta2O5 NPs, where the cationic NP exhibited substantially greater affinity for AC. Moreover, by maintaining the positive charge on the NP surface and introducing a polyethylene glycol coating, a NP formulation is described for successful in vivo cartilage imaging. Next described is the MRI CA, Gd(DTPA)Lys2, which affords an equivalent T1 signal in cartilage at 1/10th the effective dosage of anionic gadopentetate. Finally, the equilibrium partitioning of the small molecule CT and MRI CAs is directly compared to GAG content and mechanical properties in human finger AC. In summary, results show cationic CAs strongly correlate to both GAG and mechanical properties and distribute in direct proportion to GAG unlike anionic CAs. The use of cationic CAs to quantify the biochemical and mechanical changes of AC may aid drug discovery and improve clinical assessment and intervention of OA for future patients. / 2017-11-03T00:00:00Z

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25186号 / 医博第5072号 / 新制||医||1072(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 安達 泰治, 教授 森本 尚樹, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

A longitudinal study was designed to characterize developmental patterns of plasma (PL) and synovial fluid (SF) total insulin-like growth factor-I (IGF-I) concentrations, as well as their association with measurements of skeletal growth in Thoroughbred horses. Horses were randomly assigned to one of two dietary treatment groups and fed diets with either a high or low starch content to examine the effects of dietary energy source on PL and SF IGF-I. At 3, 6, 9, 12 and 15 mo of age, PL and carpal SF samples were collected for analysis of total IGF-I. Body weight gain, wither height gain and forearm length gain were calculated for the 90 day periods between SF and PL sampling. No influence of diet on PL or SF IGF-I was detected (P > 0.05). Average SF IGF-I concentrations were 30.1 ± 1.8% of that found in PL, and PL and SF IGF-I were positively correlated (r = 0.48, P = 0.0003) There was an effect of month of age on both PL and SF IGF-I concentrations (P < 0.05). There was a positive correlation between all measures of gain except forearm length gain with PL and SF IGF-I (r = 0.41 to 0.55, P < 0.05). In our second study, we evaluated the use of a liquid-nitrogen cooled mortar and pestle, motorized freezer mill and rotor-stator homogenizer for homogenization of small (<50mg) articular cartilage samples. The rotor-stator homogenizer produced quanitfiable RNA yields, and was used to evaluate three different RNA isolation protocols. Two of the protocols were commercially available RNA extraction kits, with the third a modified guanidinium isothiocyanate/acid-phenol extraction procedure. The combined average yield for all protocols was 91.9 ng RNA/mg of cartilage. All protocols yielded a sufficient quantity of quality RNA suitable for gene expression analysis. / Master of Science

Tightly focused femtosecond laser pulses are widely used in the biomedical field due to their nonlinear multiphoton precision and minimal thermal side effects. Below the threshold of optical breakdown, light energy contributes to photochemical reactions that introduce more chemical bonding in the form of collagen crosslinking (CxL) in extracellular matrices of transparent tissues such as corneal stroma. Previously, based on the principles of ultrafast laser-tissue interaction, a novel collagen CxL method relying on low-density plasma (LDP) generating reactive oxygen species (ROS) was proposed and applied to cornea tissue for vision correction by the Vukelic Group and extended to articular cartilage tissue for early osteoarthritis treatment in collaboration with Musculoskeletal Biomechanics Research Laboratory. Despite the efficiency and safety of the procedure, LDP was elusive and challenging to control due to its potential dependence on a cascade of intertwining factors such as ultrafast laser wavelength, power, pulse duration, repetition rate, and ionization resonance. This thesis has two aims: the first is to investigate the photochemical laser-tissue interaction with femtosecond nanojoule energy pulses, and the second is to develop robust and practical laser parameter envelopes for treating corneal ectatic diseases and osteoarthritis. Chapter 2 proposes a corneal epithelium-stromal level wound healing treatment. Relying on the interaction between reactive oxygen species (ROS) created by low-density plasma (LDP) therapy and inflammatory cytokines, epithelium recovery is accelerated on in vivo rabbit corneas. Chapters 3 to 5 focus on photochemical reaction-based morphological correction and biomechanical enhancement for corneal diseases such as keratoconus and astigmatism. A wavelength-independent, nonenzymatic CxL technique based on oxygen-independent, pentose-mediated glycation and ROS acceleration is developed; collagen CxL efficiency is tested through autofluorescence microscopy and nanoindentation. Subsequently, the combined effects of simultaneous external mechanical loading and nonenzymatic collagen CxL, achieved by both traditional CxL that involves soaking eyes with riboflavin solution, a photosensitizer, and then activating it with ultraviolet A light (UVA-Riboflavin-CxL) and new ROS catalyzed glycation CxL (ROS-Glycation-CxL) techniques, are investigated on ex vivo rabbit corneas. Through X-Ray Diffraction, permanent adjustments to the ultrastructure of collagen fibril packing are observed, ultimately contributing to refractive power changes in corneal topography. Furthermore, with the addition of melanin application that increases absorption and ionization efficiency, a robust method for generating plasma and reactive oxygen species (ROS) is proposed and implemented on ex vivo corneas to address ectatic diseases. Chapter 6 discusses the effect of plasma-guided laser collagen CxL on articular cartilages’ compressive equilibrium modulus and chondrocyte viability. Stemming from the melanin-assisted protocol and ultrafast pulses' high peak power, a plasma spark-mediated laser treatment is hypothesized to biomechanically enhance both bovine and human articular cartilage superficial zone for the treatment of osteoarthritis. Chapter 7 concludes this thesis and proposes future directions.

A síndrome fêmoro-patelar (SFP) acomete adultos jovens e caracteriza-se por dor no joelho durante a realização de atividades funcionais. Os fatores etiológicos incluem o aumento do ângulo Q e desequilíbrios musculares e para seu tratamento normalmente são realizados exercícios de fortalecimento do músculo quadríceps femoral, não sendo encontradas referências quanto ao uso de alongamento muscular no reequilíbrio da articulação fêmoro-patelar. Dessa forma, os objetivos deste estudo foram comparar a eficácia de exercícios de fortalecimento do músculo quadríceps femoral e alongamento dos músculos da cadeia posterior na recuperação funcional de pacientes com SFP. Para isso, foram selecionadas 40 mulheres sedentárias com idade entre 18 e 32 anos com SFP. Antes do tratamento foram avaliados: medida da flexibilidade, do encurtamento dos músculos isquiotibiais, do ângulo Q, aplicação de escalas da capacidade funcional (escala de contagem de Lysholm e escala de avaliação para a articulação fêmoro-patelar) e eletromiografia (EMG) dos músculos vasto medial (VM), vasto lateral (VL), bíceps femoral (BF) e gastrocnêmio porção lateral (GT) durante contrações isométricas de flexão e extensão da perna. Posteriormente, os pacientes foram divididos em quatro grupos de tratamento: o Grupo 1 (G1) realizou alongamento dos músculos da cadeia posterior pela técnica de reeducação postural global, o Grupo 2 (G2) realizou alongamento segmentar dos músculos isquiotibiais e gastrocnêmio, o Grupo 3 (G3) fortaleceu o músculo quadríceps femoral em cadeia cinética aberta e o Grupo 4 (G4) fortaleceu o mesmo músculo em cadeia cinética fechada, ambos com aumento progressivo da carga. Antes e após cada sessão de tratamento, as pacientes preenchiam a intensidade da dor no joelho numa escala visual analógica. O tratamento durou oito semanas com freqüência de duas vezes semanais. As variáveis obtidas antes e após o tratamento para cada grupo foram analisadas pelo teste de Wilcoxon e entre os grupos pela Anova e teste de Duncan ou Anova de Friedman ou Manova (&#945; < 0,05). Comparando as variáveis antes e após o tratamento, os resultados mostram que a intensidade da dor apresentou melhora significante no G1 e G3 e o ângulo Q diminuiu no G1 e G2. Para a atividade EMG, o músculo VM não sofreu alteração após o tratamento, o músculo VL aumentou no G2 e G3, o músculo BF aumentou apenas no G3 e o músculo GT aumentou no G3 e G4. Em todos os grupos, as escalas mostram melhora da capacidade funcional, diminuição do encurtamento dos músculos isquiotibiais e aumento da flexibilidade. Entre os grupos, o G1 tendeu a apresentar maior flexibilidade, o G4 maior atividade EMG do músculo BF, enquanto o G1 e G3 mostraram maior ativação do músculo GT. Os dados sugerem que os exercícios de alongamento muscular, em especial o global, também devem ser indicados no tratamento de pacientes com SFP, principalmente nas fases iniciais, onde se objetiva uma redução efetiva da intensidade da dor. Além disso, também possibilitou maior realinhamento do joelho (redução do ângulo Q) e aumento da flexibilidade, o que pode facilitar o fortalecimento muscular. / The patellofemoral syndrome (PFS) affects young adults and is characterized by knee pain during functional activities. PFS etiology includes bigger Q angle and muscular imbalances. For treatment, quadriceps femoris strengthening are normally performed, but we were unable to locate documentation about the application of stretching exercises in the patellofemoral joint rehabilitation. The aim of this study was to compare the efficacy of quadriceps femoris muscle strengthening and posterior chain stretching exercises in the functional recovery of PFS patients. Forty female nonathletes aged between 18 and 32 years old were recruited. Before treatment, it was measured: flexibility, hamstring tightness, Q angle, functional injury level of the knee by the application of questionnaires and electromyography (EMG) of the vastus medialis (VM), vastus lateralis (VL), biceps femoris (BF) and lateral gastrocnemius(LG) muscles during isometric contractions of leg flexion and extension. After this initial evaluation, patients were divided into four treatment groups: Group 1 (G1) performed posterior chain global stretching exercises, Group 2 (G2) segmentary stretching exercises of hamstring and gastrocnemius muscles, Group 3 (G3) quadriceps femoris strengthening exercises in open kinetic chain and Group 4 (G4) quadriceps femoris strengthening exercises in closed kinetic chain, with progressively resistance increases. This treatment lasted eight weeks, twice a week. Before and after each treatment session, the visual analogue scale accessed pain intensity. The data obtained before and after treatment for all groups were analyzed by Wilcoxon test, and the data between groups by Anova and Duncan test or Friedman Anova or Manova (&#945; < 0.05). Comparing the data before and after treatment, the results showed a significant improvement in pain intensity in G1 and G3 and a decreased Q angle in G1 and G2. For the EMG activity, VM muscle activity was not modified after treatment, VL activity increased in G2 and G3, BF activity increased only in G3 and LG activity increased in G3 and G4. Between groups, G1 showed greatest flexibility, G4 greatest EMG activity of BF muscle, while G1 and G3 showed greatest activity of LG muscle. Based on these results, we suggest that stretching exercises, specifically in global modality, can be prescribed in PFS treatment, especially in its initial phases, for an effective reduction of pain intensity. Moreover, this treatment caused a knee alignment (by reducing Q angle)and flexibility improvement, which may facilitate muscular strengthening.