TP508 is a 23 amino acid peptide derived from human prothrombin. It helps wound healing in both soft tissues and bones. In our previous study, we have demonstrated that TP508 retains chondrocyte in a less mature differentiation state while expanding the cartilage mass, indicating it may partly help bone healing by expand the cartilage template in the endochondral bone formation stage. In our current study, we want to demonstrate that TP508 also blocks chondrocyte apoptosis. We used rat costochondral growth plate chondrocytes as our model. We first established chelerythrine as an apoptogen in chondrocytes. TP508 is able to block apoptosis caused by chelerythrine. Chelerythrine also causes an increase in NO production, which is known to cause both pathological and physiological apoptosis of chondrocyte, and blocking NO production can in turn block apoptosis caused by them. TP508 is also able to block NO production caused by chelerythrine. Therefore, TP508 may partially block chondrocyte apoptosis by blocking NO production. From all above, we conclude that besides decreasing chondrocyte differentiation, TP508 also blocks their apoptosis, so as to conserve the cartilage template in endochondral bone formation

Currently growth plate fractures account for nearly 18.5% of fractures in children and can lead to stunted bone growth or angular deformation. If the body is unable to heal itself a bony bar forms, preventing normal bone growth. Clinical treatment involves removing the bony bar and replacing it with a filler substance, which causes poor results 60% of the time. Using primarily poly(lactic-co-glycolic acid) (PLGA) as the scaffold material, the goal was to develop an implant that would support to the implant site, allow for cell ingrowth, and degrade away over time. Porous scaffolds were fabricated from PLGA microspheres using the salt leaching method. The first part of this work investigated the effect of sintering the microspheres by studying the mechanical properties, degradation and morphology and their potential applications for hard and soft tissue implants. Growth factor or drugs can be encapsulated into PLGA microspheres, which was the second part of this work. Encapsulated insulin-like growth factor I (IGF-I) was able to withstand the scaffold fabrication process without compromising it’s bioactivity and promoted cell proliferation. The next part of this work experimented with the addition of a hydrogel porogen. Porogen particles were made using a quick degrading poly(beta-amino ester) (PBAE) hydrogel and loaded with ketoprofen. The addition of the porogen creates a dual drug-releasing scaffold with a localized delivery system. The final step of this work involved animal studies to determine the effectiveness of the scaffolds in growth plate regeneration and how they compare to the current clinical treatment option. Gross observation, microCT analysis, angular measurement of bone growth and histological methods were employed to evaluate the scaffolds. The goal was to develop a versatile scaffold that could be used for a wide range of tissue engineering applications. The mechanical properties, degradation profiles and drug delivery capabilities can be all tailored to meet the specific needs of an implant site. One specific application was regenerating the native growth plate that can also encourage the endogenous mesenchymal stem cells to follow the desire linage. By regenerating the native growth plate, angular deformation and stunted limb growth were greatly reduced.

Objetivo: Estudar o efeito das diferentes doses (5 J/cm² e 15 J/cm²) do Laser Diodo de 830 nm na cartilagem epifisária de ratos, analisando o comprimento do osso, a espessura e o número de condrócitos de cada camada da cartilagem epifisária. Materiais e Métodos: 30 ratos da linhagem Wistar, machos, pesando em média ± 280 g, com 23 dias de idade, divididos aleatoriamente em 3 grupos: Grupo Controle (GC), Grupo (G5) dose de 5 J/cm², grupo 15 (G15) dose de 15 J/cm². Foram realizadas 10 aplicações do Laser AsGaAl 830 nm em dias alternados. Os animais foram sacrificados 24 horas após a última aplicação, no 21º dia após a primeira aplicação, com dose letal de 200 mg/Kg de pentobarbital sódico. As lâminas foram coradas com H/E (Hematoxilina Eosina). Realizou-se a documentação fotográfica das lâminas em fotomicroscópio ZEISS e, posteriormente, análise histomorfométrica e análise histológica. Para a análise estatística utilizou-se o teste de variância fatorial One-Way ANOVA com nível de significância considerado significativo para < 0,05, seguido pelo teste de Tukey. Resultados: A cartilagem epifisária demonstrou através da análise histológica e pela imagem do RX aumento da espessura da cartilagem epifisária e aumento no número de condrócitos dos grupos G5 e G15. Conclusão: O Laser de AsGaAl 830 nm, dentro dos parâmetros utilizados nesta pesquisa, pode causar alteração na espessura da cartilagem epifisária, aumento no número de condrócitos, porém não é suficiente para causar qualquer alteração no comprimento final do osso. / Objective: To study the effect of an 830-nm GaAlAs diode Laser at two different energy densities (5 J/cm² and 15 J/cm²) on the epiphyseal cartilage of rats, by evaluating the bone length, and the number of chondrocytes and thickness of each zone of the epiphyseal cartilage. Materials and Methods: A total of 30 male Wistar rats with 23 days of age and weighing 280 g on average were randomly divided into 3 groups: control group (CG, no stimulation), G5 group (energy density, 5 J/cm²), and G15 group (energy density, 15 J/cm²). Laser treatment sessions were administered every other day for a total of 10 sessions. The animals were euthanized 24 hours after the last treatment session (21 days after the first application) with a lethal dose of sodium pentobarbitone (200 mg/kg). Histological slides of the epiphyseal cartilage were stained with hematoxylin-eosin (HE), photographed with a Zeiss photomicroscope, and subjected to histometric and histological analyses. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukeys post-hoc test. All statistical tests were performed at a significance level of 0.05. Results: Histological analysis and x-ray radiographs revealed an increase in thickness of the epiphyseal cartilage and in the number of chondrocytes in the G5 and G15 groups. Conclusion: The 830-nm GaAlAs diode Laser, within the parameters used in this study, induced changes in the thickness of the epiphyseal cartilage and increased the number of chondrocytes, but it was not sufficient to induce changes in bone length.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Les protéoglycanes (PGs) jouent un rôle essentiel dans plusieurs processus physiologiques majeurs tels que la signalisation cellulaire, la prolifération et la migration ; ceci grâce aux interactions entre leurs chaînes de glycosaminoglycanes (GAGs) avec des médiateurs solubles et leurs récepteurs. L'initiation de la synthèse des chaînes de GAGs des PGs est catalysée par la xylosyltransferase I (XT-I). Récemment plusieurs études ont montré différentes mutations au niveau du gène de la XT-I associées au syndrome Desbuquois de type II, caractérisée des anomalies ostéoarticulaires. Afin d’étudier le rôle de la XT-I dans le développement ostéoarticulaire, nous avons généré des souris invalidées pour le gène de la XT-I (XT-I KO). L'analyse morphologique des embryons montre que les souris XT-I KO présentent un nanisme prononcé et une hypoplasie frontonasale apparente, indiquant des anomalies du développement ostéoarticulaire. L'évaluation du contenu en PGs a révélé une forte diminution de la synthèse des PGs chez les souris XT-I KO. L'examen des différentes zones chondrocytaires au niveau de la plaque de croissance des os longs a révélé la perte de l’organisation en colonne des chondrocytes prolifératifs et une réduction importante de la zone hypertrophique. Afin d'identifier les mécanismes et les facteurs à l’origine des anomalies squelettiques chez les souris XT-I KO, l'expression de plusieurs gènes impliqués dans le développement du squelette et dans la régulation de la chondrogenèse a été analysée par hybridation in situ à l'aide de la technique RNAscope. Les résultats ont montré une forte expression des marqueurs de l’hypertrophie chondrocytaires suggérant ainsi une maturation précoce des chondrocytes chez les souris XT-I KO. Les embryons XT-I KO montrent également une formation précoce du centre d'ossification secondaire, indiquant une ossification précoce qui participerait aux anomalies de croissance observées chez les souris XT-I KO. L’étude des voies de signalisation impliquées dans la différenciation et la maturation chondrocytaire a révélé une surexpression du récepteur FGFR3 et une activation importante de la signalisation sous-jacente, suggérant ainsi des perturbations de la signalisation du FGF. Compte tenu du rôle important du FGFR3 dans la régulation de la chondrogenèse et de l’ossification endochondrale, ces résultats suggèrent fortement l’implication de la voie de FGF dans le développement des anomalies squelettiques chez les souris XT-I KO et ouvrent la voie pour le développement de de nouvelles thérapeutiques pour le traitement des patients atteints du syndrome Desbuquois de type II. / Proteoglycans (PGs) play an essential role in several major physiological processes such as cell signaling, proliferation and migration; this is mainly due to the interactions between their glycosaminoglycan chains (GAGs) with soluble mediators and their receptors. The initiation of the synthesis of GAG chains of PGs is catalyzed by Xylosyltransferase I (XT-I). Recently several studies have shown that mutations in XT-I gene are associated with Desbuquois syndrome type II which is characterized by skeletal abnormalities. To study the role of XT-I in skeletal development, we generated knockout mice for the XT-I gene (XT-I KO). XT-I KO mice show pronounced dwarfism and apparent frontonasal hypoplasia reflecting abnormalities in skeletal development. Evaluation of PG content revealed a strong decrease in PG synthesis in XT-I KO mice. Analysis of the different chondrocyte zones in the growth plate revealed a loss of columnar organization of proliferative chondrocyte and a significant reduction of the hypertrophic zone. To identify the mechanisms and factors underlying skeletal abnormalities in XT-I KO mice, the expression of several genes involved in skeletal development and in the regulation of chondrogenesis were analyzed by in situ hybridization using RNAscope technique. The results showed a strong expression of markers of chondrocyte hypertrophy thus suggesting early maturation of chondrocytes in XT-I KO mice. The XT-I KO embryos show also a premature formation of the secondary ossification center, indicating a precocious ossification which ultimately leads to the growth abnormalities showed in XT-I KO mice. The study of the signaling pathways involved in differentiation and chondrocyte maturation revealed an overexpression of the FGFR3 receptor and a significant activation of the downstream signaling pathways, thus suggesting disturbances of FGF signaling. Given the important role of FGFR3 in the regulation of chondrogenesis and endochondral ossification, these results strongly suggest the involvement of the FGF pathway in the development of skeletal abnormalities in XT-I KO mice and pave the way for the development of new therapeutics for the treatment of patients with Desbuquois syndrome type II.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Height is a complex physical trait that displays strong heritability. Adult height is related to length of the long bones, which is determined by growth at the epiphyseal growth plate. Longitudinal bone growth occurs via the process of endochondral ossification, where bone forms over the differentiating cartilage template at the growth plate. Estrogen plays a major role in regulating longitudinal bone growth and is responsible for inducing the pubertal growth spurt and fusion of the epiphyseal growth plate. However, the mechanism by which estrogen promotes epiphyseal fusion is poorly understood. It has been hypothesised that estrogen functions to regulate growth plate fusion by stimulating chondrocyte apoptosis, angiogenesis and bone cell invasion in the growth plate. Another theory has suggested that estrogen exposure exhausts the proliferative capacity of growth plate chondrocytes, which accelerates the process of chondrocyte senescence, leading to growth plate fusion. The overall objective of this study was to gain a greater understanding of the molecular mechanisms behind estrogen-mediated growth and height attainment by examining gene regulation in chondrocytes and the role of some of these genes in normal height inheritance. With the heritability of height so well established, the initial hypothesis was that genetic variation in candidate genes associated with longitudinal bone growth would be involved in normal adult height variation. The height-related genes FGFR3, CBFA1, ER and CBFA1 were screened for novel polymorphisms using denaturing HPLC and RFLP analysis. In total, 24 polymorphisms were identified. Two SNPs in ER (rs3757323 C>T and rs1801132 G>C) were strongly associated with adult male height and displayed an 8 cm and 9 cm height difference between homozygous genotypes, respectively. The TC haplotype of these SNPs was associated with a 6 cm decrease in height and remarkably, no homozygous carriers of the TC haplotype were identified in tall subjects. No significant associations with height were found for polymorphisms in the FGFR3, CBFA1 or VDR genes. In the epiphyseal growth plate, chondrocyte proliferation, matrix synthesis and chondrocyte hypertrophy are all major contributors to long bone growth. As estrogen plays such a significant role in both growth and final height attainment, another hypothesis of this study was that estrogen exerted its effects in the growth plate by influencing chondrocyte proliferation and mediating the expression of chondrocyte marker genes. The examination of genes regulated by estrogen in chondrocyte-like cells aimed to identify potential regulators of growth plate fusion, which may further elucidate mechanisms involved in the cessation of linear growth. While estrogen did not dramatically alter the proliferation of the SW1353 cell line, gene expression experiments identified several estrogen regulated genes. Sixteen chondrocyte marker genes were examined in response to estrogen concentrations ranging from 10-12 M to 10-8 M over varying time points. Of the genes analysed, IHH, FGFR3, collagen II and collagen X were not readily detectable and PTHrP, GHR, ER, BMP6, SOX9 and TGF1 mRNAs showed no significant response to estrogen treatments. However, the expression of MMP13, CBFA1, BCL-2 and BAX genes were significantly decreased. Interestingly, the majority of estrogen regulated genes in SW1353 cells are expressed in the hypertrophic zone of the growth plate. Estrogen is also known to regulate systemic GH secretion and local GH action. At the molecular level, estrogen functions to inhibit GH action by negatively regulating GH signalling. GH treated SW1353 cells displayed increases in MMP9 mRNA expression (4.4-fold) and MMP13 mRNA expression (64-fold) in SW1353 cells. Increases were also detected in their respective proteins. Treatment with AG490, an established JAK2 inhibitor, blocked the GH mediated stimulation of both MMP9 and MMP13 mRNA expression. The application of estrogen and GH to SW1353 cells attenuated GH-stimulated MMP13 levels, but did not affect MMP9 levels. Investigation of GH signalling revealed that SW1353 cells have high levels of activated JAK2 and exposure to GH, estrogen, AG490 and other signalling inhibitors did not affect JAK2 phosphorylation. Interestingly, AG490 treatment dramatically decreased ERK2 signalling, although GH did stimulate ERK2 phosphorylation above control levels. AG490 also decreased CBFA1 expression, a transcription factor known to activate MMP9 and MMP13. Finally, GH and estrogen treatment increased expression of SOCS3 mRNA, suggesting that SOCS3 may regulate JAK/STAT signalling in SW1353 cells. The modulation of GH-mediated MMP expression by estrogen in SW1353 cells represents a potentially novel mechanism by which estrogen may regulate longitudinal bone growth. However, further investigation is required in order to elucidate the precise mechanisms behind estrogen and GH regulation of MMP13 expression in SW1353 cells. This study has provided additional evidence that estrogen and the ER gene are major factors in the regulation of growth and the determination of adult height. Newly identified polymorphisms in the ER gene not only contribute to our understanding of the genetic basis of human height, but may also be useful in association studies examining other complex traits. This study also identified several estrogen regulated genes and indicated that estrogen modifies the expression of genes which are primarily expressed in the hypertrophic region of the epiphyseal growth plate. Furthermore, synergistic studies incorporating GH and estrogen have revealed the ability of estrogen to attenuate the effects of GH on MMP13 expression, revealing potential pathways by which estrogen may modulate growth plate fusion, longitudinal bone growth and even arthritis.

Les polluants environnementaux, en particulier les perturbateurs endocriniens (PE), agissent à très faibles doses sur des cibles multiples. Les effets rapportés portent en majorité sur les organes de reproduction. Très peu d’études ont porté sur le squelette alors que le cartilage et l’os sont sous un puissant contrôle hormonal, depuis le stade fœtal où le système hormonal se met en place jusqu’au vieillissement, en passant par la naissance (hormones thyroïdiennes, hormone de croissance), la puberté et la ménopause chez la femme (stéroïdes sexuels). L’objectif de ce travail est d’étudier les effets de polluants anti-androgènes (vinclozoline, V et métabolite actif M2) ou xenestrogènes (génistéine, G; bisphénol A, BPA), in vivo sur le développement du squelette du rat Wistar et in vitro sur les marqueurs de différenciation chondrogéniques. Les effets in vivo ont été étudiés à des doses inférieures aux “No Observed Adverse Effect Levels ” (NOAEL) fixés par les instances européennes (EFSA) et internationales (US EPA). Des rattes ont été exposées à V, G seuls, combinés (GV) et/ ou associés au BPA (BGV), et ce de la conception des petits jusqu’à leur sevrage (J30) ou leur sacrifice (J30, J110). Les effets ont été recherchés sur des petits de mères et portées différentes, quatre pour chaque traitement, âge et sexe. Les effets in vitro du métabolite M2 de la Vinclozoline, associé ou non avec G et BPA, ont été étudiés sur l’expression de marqueurs chondrogéniques en utilisant : 1) un modèle murin de cellules souches inductibles vers la voie chondrogénique (C1) pour les effets sur la différenciation chondrogénique précoce et 2) des chondrocytes de souriceaux nouveau-nés, différenciés en culture primaire ou dédifférenciés (passages répétés). Comparaison avec les effets du bFGF, facteur de dédifférenciation chondrogénique. Résultats : In vivo, l’exposition à V, seule ou associée à G ou au BPA induit chez les rattes F1 exposées, une cannelure de la queue, discrète mais perceptible à la palpation en regard de chaque articulation intervertébrale. Les xénestrogènes tendent à réduire cet effet de V. Les rats et les animaux F2 ne sont pas atteints. L’examen par micro CT-scan montre une augmentation significative de la largeur des apophyses transverses (ITA) des vertèbres, et une diminution de la hauteur des corps vertébraux chez les rattes F1 exposées en regard des contrôles. Ces modifications anatomiques rappellent certaines pathologies génétiques des collagènes (dysplasies épiphysaires) chez l’homme Elles sont absentes chez les rats F1 et les animaux F2. Elles sont en partie transitoires car présentes à J30 (effets sur ITA et longueur) quand seul l’effet sur l’ITA perdure à J110. L’examen histologique des cartilages de croissance des corps vertébraux montre un déséquilibre entre les zones de prolifération et d’hypertrophie qui évoque une modification de la maturation du cartilage de type estrogénique. Ces effets sont ici aussi transitoires et majoritairement observés chez les rattes. L’effet plus prononcé du BPA lisse toutes les autres activités. Cela suggère que les PE pourraient moduler la différenciation du cartilage de croissance. C’est ce qui a été étudié in vitro. In vitro. Le premier objectif était d’évaluer les effets des PE sur la dynamique d’induction chondrogénique (cellules C1). Nous montrons que l’addition de M2 seul ou avec G ou BPA modifie le processus de maturation du collagène2 sans effet sur les autres marqueurs (SOX9, Agrécane, Col10). M2 prolonge l’expression de COL2A immature et retarde son remplacement par l’isoforme COL2B. Le second objectif était d’étudier les effets des PE sur la régulation de l’expression de COL2A au cours du processus de dé-différenciation des chondrocytes in vitro. L’expression de COL2A augmente avec le degré de dédifférenciation cellulaire (passages successifs) et double en présence de M2, G et BPA. Cet effet dépend des récepteurs aux estrogènes (ER) et des voies p38-MAPK. (...) / Environmental pollutants, particularly Endocrine Disruptors (ED), show effects on multiple targets at very low doses. Mostly known effects target reproductive organs. Very few studies are conducted on skeleton, although cartilage and bone are under potent hormonal control, from fetal stage, where hormonal system takes place, until aging, through birth stage (thyroid hormones, growth hormones), puberty and menopause for women (steroid hormones). The aim of this work is to study effects of anti-androgenic pollutants (vinclozolin, V, and its active metabolite M2) or xenoestrogens (genistein, G; bisphenol A, BPA), in vivo on Wistar rat skeletal development and in vitro on chondrogenic differenciation markers.In vivo effects were studied at doses below the “No Observed Adverse Effect Levels” (NOAEL) established by European and American agencies (EFSA and US EPA respectively). Female Wistar rats were exposed to V and G alone, in combination (GV) and/or associated to BPA (BGV), from pups conception until weaning (d30) or sacrifice (d30, d110). Effects were investigated on offsprings from different mothers and litters, on four animals by treatment, age and gender. In vitro effects of M2 metabolite of Vinclozolin, combined or not to G and BPA, were studied on chondrogenic markers expression using : 1) inducible murine stem-cells model towards chondrogenesis (C1) to sudy effects on early chondrogenic differentiation and 2) post-natal mouse differentiated chondrocytes, in primary culture or dedifferenciated chondrocytes by successive passages. Comparison with bFGF, a dedifferentiation factor for chondrocytes.Results : In vivo, exposure to V, alone or combined to G or BPA, induce discrete but palpable annealing in F1 treated female rat tails, in front of each intervertebral articulation. Xenoestrogens tend to decrease V effect. Male rats and F2 offsprings were not affected. Micro-CT Scan analysis shows significative increase of vertebrae inter transverse apophyses (ITA) distance, and decrease of vertebral body height in F1 female rats comparing to control animals. Anatomical modifications recall human collagen genetic diseases (epiphyseal dysplasias). They are absent in F1 male rats and F2 offsprings. Furthemore they are partly transient, ITA and height effects being present at d30 whereas ITA effect alone remains until d110. Histological analysis of vertebral body growth plate shows unbalance between proliferative and hypertrophic zones, which evokes estrogenic acceleration of cartilage maturation. Those effects are still transient and mainly observed in female rats. BPA activity is dominant above G and V effects. This result suggests ED can modulate growth plate cartilage differentiation, which was studied in vitro. In vitro : First, we aimed to evaluate eventual role of ED on the dynamic of the chondrogenic differentiation process. We show that M2 addition, alone or in combination with G or BPA, modifies collagen 2 maturation process without any effect on other markers (SOX9, Agrecan, COL10). M2 addition extends immature isoform COL2A expression and delays its replacement by mature isoform COL2B. Second, we studied the effects of ED on the regulation of COLA expression through the dedifferentiating process of chondrocytes in vitro. COL2A expression increases with cell dedifferenciation degree (successive passages) and double with M2, G and BPA. No other chondrogenic marker was modified. This effect depends on estrogen receptors (ER) and p38-MAPK pathway. (...)

Magnetresonanztomographische Studie zur altersabhängigen Abbildung der Wachstumsknorpel des distalen Radius des Pferdes unter besonderer Berücksichtigung des Epiphysenfugenknorpels. Es wurden magnetresonanztomographische Untersuchungen von 28 Gliedmaßenabschnitten des distalen Radius im Alter von zwei Tagen bis 17 Jahren durchgeführt. Die Studie wurde an einem 1,5 Tesla Magnetom “Symphony“ (Siemens) in vier unterschiedlichen Sequenzen (T1-gewichtet T1w, T2-gewichtet T2w, Protonendichte PD, T2 Double Echo in Steady State T2-dess) und zwei Schnittebenen (dorsal, sagittal) durchgeführt. Die Darstellung der knorpeligen Wachstumsregionen des distalen Radius mit besonderem Hinblick auf seine Epiphysenfuge wurde deskriptiv erfasst und altersbedingte Unterschiede definiert. Die durchschnittliche Dicke des sich darstellenden Epiphysenfugenknorpels wurde in zwei Sequenzen (T1w und T2-dess) vermessen und in Bezug zu dem ansteigenden Alter des Probenmaterials gesetzt. Die Proben konnte man fünf Gruppen zuordnen. In Gruppe 1 konnten sowohl der Wachstumsknorpel der distalen Ossifikationszentren als auch die knorpeligen Anteile der Epi- und Apophysenfugen dargestellt werden. In der Gruppen 2 ließen sich die Apo- und Epiphysenfugen darstellen, in Gruppe 3 nur die Epiphysenfugen. Gruppe 4 beschrieb partiell geschlossene Epiphysenfugen und in Gruppe 5 stellten sich nur 88 noch Fugennarben dar. Eine alterskorrelierende Abnahme der mittleren Knorpeldicke der Epiphyse konnte mittels Vermessungen der Knorpelschichten nachgewiesen werden. Der hyaline Knorpel war mit den gewählten Sequenzen sehr gut beurteilbar. Der Wachstumsknorpel der Epi- und Apophysen stellte sich in den T1w und PD mit hell-intermediärer und in den T2w mit intermediärer Signalintensität dar. Die knorpeligen Anteile der Epi- und Apophysenfuge wurden in T1w intermediär bis hellintermediär, in T2w hell-intermediär und in der PD hell-intermediär bis hyperintens dargestellt. Angrenzende Strukturen wie die subchondrale Knochenplatte und die Mineralisationszone der Epiphysenfuge konnten in dunkel-intermediären bis hypointensen Signalen abgebildet werden. Die Magnetresonanztomographie (MRT) hat sich als geeignetes Verfahren erwiesen, die knorpeligen Strukturen der Wachstumsregion des distalen Radius des Pferdes bildlich wiederzugeben. Altersabhängige strukturelle Unterschiede konnten im MRT dargestellt werden. Damit leistet die vorgestellte Studie einen wichtigen Beitrag über die anatomischen Verhältnisse und deren physiologische Darstellung.

Tesis por compendio / [ES] El cartílago hialino es un tejido conectivo denso con poca capacidad de auto regeneración cuando es afectado por patologías degenerativas. Por lo tanto, la estimulación eléctrica se ha propuesto como una terapia alternativa no invasiva para mejorar la reparación del cartílago hialino. De acuerdo con esto, este trabajo presenta un enfoque computacional y experimental combinado para entender mejor la biología del cartílago hialino y su respuesta a la estimulación eléctrica usando diferentes modelos in vitro. En primer lugar, se ha desarrollado un modelo mecanobiológico para simular el proceso de osificación endocondral. Por otro lado, se ha evaluado el efecto de la estimulación eléctrica sobre el cartílago hialino en tres escenarios diferentes. Inicialmente se ha analizado la proliferación celular y la síntesis de glicosaminoglicanos de condrocitos cultivados en monocapa y estimulados con campos eléctricos. Luego, se ha realizado un análisis histomorfométrico a explantes de condroepífisis que fueron estimulados eléctricamente. Por último, se ha evaluado el efecto de los campos eléctricos sobre la diferenciación condrogénica de células madre mesenquimales cultivadas en hidrogeles. Los resultados indican que la estimulación eléctrica es un estímulo biofísico prometedor, ya que este tipo de estimulación mejora la viabilidad y la proliferación celular, induce cambios morfológicos en los condrocitos, y estimula la síntesis de las principales moléculas que componen el cartílago hialino, tales como SOX-9, glicosaminoglicanos y agrecan. Además, este proyecto es el primer paso hacia la implementación de un estímulo biofísico alternativo que modifica la dinámica celular de los condrocitos de la placa de crecimiento en condiciones ex vivo. Adicionalmente, este estudio resalta el efecto potencial de los campos eléctricos para inducir el proceso de condrogénesis de células madre mesenquimales cultivadas en condiciones basales. En general, la evaluación de la estimulación eléctrica sobre condrocitos, tejidos y andamios es una herramienta útil que puede contribuir al conocimiento actual de las terapias regenerativas enfocadas en la regeneración del cartílago hialino. / [CA] El cartílag hialí és un teixit connectiu dens amb poca capacitat d'auto regeneració quan es veu afectat per patologies degeneratives. Per tant, l'estimulació elèctrica s'ha proposat com una teràpia alternativa no invasiva per millorar la reparació del cartílag articular. D'acord amb això, aquest treball presenta un enfoc computacional i experimental combinat per entendre millor la biologia del cartílag hialí i la seva resposta a l'estimulació elèctrica usant diferents models in vitro. En primer lloc, s'ha desenvolupat un model mecanobiològic per simular el procés d'ossificació endocondral. D'altra banda, s'ha avaluat l'efecte de l'estimulació elèctrica sobre el cartílag hialí en tres escenaris diferents. Inicialment s'ha analitzat la proliferació cel·lular i la síntesi de glicosaminoglicans de condròcits cultivats en monocapa i estimulats amb camps elèctrics. Després, s'ha realitzat una anàlisi histomorfomètrica a explants de condroepífisis que van ser estimulats elèctricament. Finalment, s'ha avaluat l'efecte dels camps elèctrics sobre la diferenciació condrogénica de cèl·lules mare mesenquimals cultivades en hidrogels. Els resultats indiquen que l'estimulació elèctrica és un estímul biofîsic prometedor, ja que aquest tipus d'estimulació millora la viabilitat i la proliferació cel·lular, indueix canvis morfològics en els condròcits, i estimula la síntesi de les principals molècules que componen el cartílag hialí, com ara SOX-9, glicosaminoglicans i agrecan. A més, aquest projecte és el primer pas cap a la implementació d'un estímul biofísic alternatiu que modifica la dinàmica cel·lular dels condròcits de la placa de creixement en condicions ex vivo. Addicionalment, aquest estudi ressalta l'efecte potencial dels camps elèctrics per induir el procés de condrogènesi de cèl·lules mare mesenquimals cultivades en condicions basals. En general, l'avaluació de l'estimulació elèctrica sobre condròcits, teixits i scaffolds és una eina útil que pot contribuir al coneixement actual de les teràpies regeneratives enfocades a la regeneració del cartílag hialí. / [EN] Hyaline cartilage is a dense connective tissue with low self-healing capacity when is affected by degenerative pathologies. Therefore, electrical stimulation has been proposed as a possible non-invasive alternative therapy to enhance the restoration of the cartilaginous tissue. Accordingly, this work presents a combined computational and experimental approach to understand better the hyaline cartilage biology and its response to electrical stimulation using different in vitro models. On the one hand, a mechanobiological model was developed to simulate the endochondral ossification process. On the other hand, the electrical stimulation on hyaline cartilage was evaluated in three different scenarios. Initially, cell proliferation and glycosaminoglycans synthesis of chondrocytes, cultured in monolayer and stimulated with electric fields, was analyzed. Then, a histomorphometric analysis was performed to chondroepiphysis explants that were electrically stimulated. Finally, the effects of the electric fields on chondrogenic differentiation of mesenchymal stem cells cultured in hydrogels was assessed. The results indicated that electrical stimulation is a promising biophysical stimulus, due to the fact that this type of stimulation enhances the viability and the proliferation of cells, induces morphological changes in the chondrocytes, and stimulates the synthesis of the main molecules that compose the hyaline cartilage, such as SOX-9, glycosaminoglycans and aggrecan. Moreover, this project is the first step towards the implementation of an alternative biophysical stimulus that modifies the cellular dynamics of growth plate chondrocytes in ex vivo conditions. Additionally, this study highlights the potential effect of electric fields to induce the chondrogenesis process of mesenchymal stem cells cultured in basal conditions. Overall, the assessment of electrical stimulation on chondrocytes, tissues and scaffolds is a useful tool that may contribute to the current knowledge of regenerative therapies focused on hyaline cartilage healing. / To the financial support from COLCIENCIAS – COLFUTURO through the fellowship No. 647 for national doctorates. To the financial support from COLCIENCIAS through the research grant 712-2015 No. 50457. To the financial support from the Spanish Ministry of Economy and Competitiveness through the MAT2016-76039-C4-1-R project. / Vaca González, JJ. (2019). The effect of electric fields on hyaline cartilage: an in vitro and in silico study [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/120023 / TESIS / Compendio