Biomarcadores ópticos para el diagnóstico de la osteoartritis utilizando la espectroscopía Raman

Programa Oficial de Doutoramento en Biotecnoloxía Avanzada. 5012V01[Resumen] La osteoartritis o artrosis (OA) es la enfermedad reumática más frecuente, caracterizada por la degradación progresiva del cartílago articular, la esclerosis subcondral y la inflamación de la membrana sinovial. Es asintomática en sus primeras fases, y el método de diagnóstico estandarizado en clínica, la radiografía, evalúa cambios en el tejido óseo adyacente al cartílago, limitando el diagnóstico a etapas de la enfermedad de moderadas a severas. La espectroscopía Raman (ER) ha sido descrita como una técnica no destructiva y libre de marcaje para detectar cambios moleculares en tejidos biológicos, generando una huella dactilar molecular del tejido analizado. El objetivo general de esta Tesis Doctoral es evaluar el potencial de la espectroscopía Raman como técnica de diagnóstico de OA, identificando biomarcadores ópticos en un modelo ex vivo de cartílago articular humano. Para ello, se obtuvieron espectros Raman de explantes de cartílago humano procedente de cadera o rodilla, con un láser con longitud de onda (λ) de 785 nm, 50 mW, 120 acumulaciones, y una resolución de 4 cm-1. En base a ratios de las principales señales de los espectros obtenidos se constituyó una propuesta de perfil biomarcador, cuyos parámetros se analizaron cuantitativamente tomando como referencia el grado radiológico de Kellgren-Lawrence (K-L), el Sistema de Graduación Histológico-Histoquímico de Mankin (MS) y la cuantificación bioquímica de glucosaminoglicanos (GAGs) sulfatados y colágeno. Como resultado, se obtuvo un conjunto de parámetros Raman que están directamente relacionados con eventos que ocurren a nivel molecular durante la progresión de la OA, de los cuales se seleccionaron SGAGs (GAGs sulfatados), ColD/F (Colágeno defectivo/funcional) y GMF (Grado de mineralización por fosfato) para constituir modelos predictivos de la severidad de esta enfermedad. Estos parámetros permitieron la diferenciación de 2 perfiles en las muestras analizadas: un perfil de “baja severidad” (K-L 0-II y MS 0-8) y otro de “elevada severidad” (K-L III-IV y MS 9-13). Los modelos predictivos constituidos, tomando como variables respuesta tanto el grado K-L como el MS, fueron buenos, obteniendo valores de área bajo la curva (AUC) superiores a 0,8. Como conclusión general, se demostró que la ER permite diferenciar distintos grados de severidad de OA, mediante el estudio de ratios de señales de la huella molecular del cartílago. Se confirma de esta forma su potencial como herramienta diagnóstica complementaria de la OA.[Resumo] A osteoartrite ou artrose (OA) é a enfermidade reumática máis frecuente, caracterizada pola degradación progresiva da cartilaxe articular, a esclerose subcondral e a inflamación da membrana sinovial. É asintomática bas súas primeiras fases, e o método de diagnóstico estandarizado na clínica (radiografía) avalía cambios no tecido óseo adxacente á cartilaxe, limitando o diagnóstico a etapas da enfermidade dende moderadas a severas. A espectroscopía Raman (ER) é descrita como unha técnica non destructiva e libre de marcaxe para detectar cambios moleculares en tecidos biolóxicos, xerando unha pegada dactilar molecular única do tecido analizado. O obxectivo xeral desta Tese Doutoral é avaliar o potencial da espectroscopía Raman como técnica de diagnóstico de OA, identificando biomardores ópticos nun modelo ex vivo de cartilaxe articular humano. Para isto, obtivéronse os espectros Raman de explantes de cartilaxe humano procedente de cadeira ou nocello en fresco, cun láser de lonxitude de onda (λ) de 785 nm, 50 mW, 120 acumulacións e resolución 4 cm-1. Baseándose nos cocientes das principais sinais dos espectros obtidos, constituíuse unha proposta de perfil biomarcador, do que se analizaron cuantitativamente os diferentes parámetros tomando como referencia o grao radiolóxico de Kellgren-Lawrence (K-L), o Sistema de Graduación Histolóxico-Histoquímico de Mankin (MS) e a cuantificación bioquímica de glucosaminoglicanos (GAGs) sulfatados e coláxeno. Como resultado, obtívose un conxunto de parámetros Raman que están directamente relacionados con eventos que ocorren a nivel molecular durante a OA, dos que se seleccionaron SGAGs (GAGs sulfatados), ColD/F (Coláxeno defectivo/funcional) e GMF (Grao de mineralización por fosfato) para constituir modelos predictivos da severidade desta enfermidade. Estes parámetros seleccionados permitiron a diferenciación de 2 perfiles nas mostras analizadas: un perfil de “baixa severidade” (K-L 0-II e MS 0-8) e outro de “elevada severidade” (K-L III-IV e MS 9-13). Os modelos predictivos constituidos, tomando como variables resposta tanto o grao K-L como o MS, foron bos, obtendo valores de área baixo a curva (AUC) superiores a 0,8. Como conclusión xeral, demostrouse que a ER permite diferenciar distintos graos de severidade de OA, mediante o estudo de ratios de sinais da pegada molecular da cartilaxe. Confírmase desta forma o seu potencial como ferramenta diagnóstica complementaria da OA.[Abstract] Osteoarthritis (OA) is the most common rheumatic disease, characterized by progressive articular cartilage degradation, subchondral sclerosis and inflammation of the synovial membrane. On its early stages, OA is asymptomatic and its current gold-standard diagnosis (X-Rays) mainly evaluates changes in the cartilage adjacent tissue, the bone, limiting the diagnosis to moderate or advanced stages of the disease. Raman spectroscopy (RS) has been recently described as a label-free, non-destructive tool to detect molecular changes in biological tissues, producing a unique fingerprint. The main objective of this Doctoral Thesis is to evaluate Raman Spectroscopy potential as a diagnostic technique, through the identification of optical biomarkers in an ex vivo model of human articular cartilage. To achieve this, Raman spectra of fresh human cartilage explants from hip or knee were obtained, with a NIR laser of wavelength (λ) 785 nm, 50 mW, 120 scans, and a resolution of 4 cm-1. A biomarker profile was proposed based on ratios of the main spectral signals obtained, which were later quantitatively analyzed using as reference the Kellgren-Lawrence (K-L) radiological grade, the Mankin Histological-Histochemical Graduation System (MS) and the biochemical quantification of sulphated glycosaminoglycans (GAGs) and collagen. As a result, a set of Raman parameters that are directly related to events that occur at the molecular level during OA were obtained, from which SGAGs (sulphated GAGs) ColD/F (Defective/functional collagen) and GMF (Phosphate mineralization grade) were selected in order to constitute predictive models of OA severity. Selected parameters allowed the differentiation of 2 profiles in the analysed samples: a “low severity” profile (K-L 0-II y MS 0-8) and another one of “high severity” (K-L III-IV y MS 9-13). The constituted predictive models, taking both K-L grade and MS as response variable, were considered good models, obtaining values of the area under curve (AUC) values greater than 0.8. As a general conclusion, RS allowed the differentiation of OA severity grades, by analysing signals ratios of cartilage molecular fingerprint. This fact confirms its potential as a complementary diagnostic tool for OA

Síntese de nanopartículas con propiedades adsorbentes mediante métodos de química sustentable

[Resumen] La nanotecnología es un campo que está creciendo en estos últimos años debido a que las nanopartículas de algunos materiales ven incrementadas u optimizadas sus propiedades o aplicaciones en esa escala de tamaño. Al mismo tiempo, la problemática ambiental mundial actual hace cada vez más necesario el desarrollo de procesos de química sostenible y la obtención de nuevos materiales mediante métodos de síntesis verde. En este trabajo se han sintetizado nanopartículas metálicas de hierro mediante un método respetuoso con el medioambiente, una alternativa frente a los procesos convencionales basada en la capacidad antioxidante de extractos vegetales naturales que reducen metales en disolución. Los extractos se preparan con biomasa procedente de especies vegetales autóctonas gallegas, y su caracterización se realiza evaluando su capacidad antioxidante mediante diversos métodos y determinando otros parámetros, como el potencial redox y la DQO (Demanda Química de Oxígeno). La estimación del tamaño de las nanopartículas sintetizadas con dichos extractos se lleva a cabo mediante imágenes de TEM (Transmission Electron Microscopy). Una vez escogido el extracto considerado apropiado, se realiza un estudio de la capacidad de eliminación de cromo hexavalente de las correspondientes nanopartículas de hierro sintetizadas en función del pH, realizando su inmovilización en varios soportes.[Abstract] Nanotecnology is a field rising these last years due to the fact that properties or applications of nanoparticles of some materials are increased or optimized in this size scale. At the same time, current global environmental problems required the development of sustainable chemical processes and new materials obtained by green synthesis methods. In this essay, metallic nanoparticles of iron have been synthesised by an environmental friendly method, an alternative to conventional processes based in the antioxidant capacity of vegetal extracts to reduce metals in solution. Extracts were prepared with biomass from galician native vegetal especies and their characterisation was made evaluating their antioxidant capacity using various methods, and determining other parameters such as redox potential and COD (Chemistry Oxigen Demand). Estimated size of synthesised nanoparticles with each of these extracts was performed by TEM (Transmission Electron Microscopy) images. Once the appropriate extract is selected, a study of de removal of hexavalent chromium at differents values of pH was made with corresponding synthesised iron nanoparticles, making their inmobilization in various supports.[Resumo] A nanotecnoloxía é un campo que está crecendo nestes últimos anos debido a que as nanopartículas dalgúns materiais ven incrementadas ou optimizadas as súas propiedades ou aplicacións nesa escala de tamaño. Ao mesmo tempo, a problemática ambiental mundial actual fai cada vez máis necesario o desenvolvemento de procesos de química sostible e a obtención de novos materiais mediante métodos de síntesis verde. Neste traballo sintetizáronse nanopartículas metálicas de ferro mediante un método respetuoso co medioambiente, unha alternativa aos procesos convencionais baseada na capacidade antioxidante de extractos vexetais naturais que reducen metais en disolución. Os extractos prepáranse con biomasa procedente de especies vexetais autóctonas galegas, e a súa caracterización realizase evaluando a súa capacidade antioxidante mediante diversos métodos e determinando outros parámetros como o potencial redox e a DQO (Demanda Química de Osíxeno). A estimación do tamaño das nanopartículas sintetizadas cos extractos lévase a cabo mediante imaxes de TEM (Transmission Electron Microscopy). Unha vez escollido o extracto considerado apropiado, realízase un estudio da capacidade de eliminación de cromo hexavalente das correspondentes nanopartículas de ferro sintetizadas en función do pH, realizando a súa inmobilización en varios soportes.Traballo fin de grao (UDC.CIE). Química. Curso 2014/201

Molecular analysis of the destruction of articular joint tissues by Raman spectroscopy

Review[Abstract] Introduction: Osteoarthritis (OA) is a highly heterogenous disease influenced by different molecular, anatomic, and physiologic imbalances. Some of the bottlenecks for enhanced diagnosis and therapeutic assessment are the lack of validated biomarkers and early diagnosis tools. In this narrative review, we analyze the potential of Raman spectroscopy (RS) as a label-free optical tool for the characterization of articular joint tissues and its application as a diagnosis tool for OA. Areas covered: Raman spectra produce a unique 'molecular fingerprint' providing rotational and vibrational molecular information, allowing the identification and follow-up of molecular changes associated with OA pathological mechanisms. Focusing on multiple joint tissues (cartilage, synovium, bone, tendons, ligaments, and meniscus) and their contribution in disease incidence and progression, this review highlights the current knowledge on the application of RS in the characterization of organic and inorganic molecules present at these tissues and alterations that occur in the onset of OA. Expert opinion: Vibrational spectroscopy techniques, such as RS, are low cost, rapid and minimally invasive approaches that offer high specificity in the assessment of the molecular composition of complex tissues. Combined with multivariate statistical methods, RS offers great potential for optical biomarkers discovery or disease diagnosis applications, and we hereby discuss clinical translational progresses on the field

Optical Biomarkers for the Diagnosis of Osteoarthritis through Raman Spectroscopy: Radiological and Biochemical Validation Using Ex Vivo Human Cartilage Samples

[Abstract] Osteoarthritis (OA) is the most common rheumatic disease, characterized by progressive articular cartilage degradation. Raman spectroscopy (RS) has been recently proposed as a label-free tool to detect molecular changes in musculoskeletal tissues. We used cartilage samples derived from human femoral heads to perform an ex vivo study of different Raman signals and ratios, related to major and minor molecular components of articular cartilage, hereby proposed as candidate optical biomarkers for OA. Validation was performed against the radiological Kellgren–Lawrence (K-L) grading system, as a gold standard, and cross-validated against sulfated glycosaminoglycans (sGAGs) and total collagens (Hyp) biochemical contents. Our results showed a significant decrease in sGAGs (SGAGs, A1063 cm−1/A1004 cm−1) and proteoglycans (PGs, A1375 cm−1/A1004 cm−1) and a significant increase in collagen disorganization (ColD/F, A1245 cm−1/A1270 cm−1), with OA severity. These were correlated with sGAGs or Hyp contents, respectively. Moreover, the SGAGs/HA ratio (A1063 cm−1/A960 cm−1), representing a functional matrix, rich in proteoglycans, to a mineralized matrix-hydroxyapatite (HA), was significantly lower in OA cartilage (K-L I vs. III–IV, p < 0.05), whilst the mineralized to collagenous matrix ratio (HA/Col, A960 cm−1/A920 cm−1) increased, being correlated with K-L. OA samples showed signs of tissue mineralization, supported by the presence of calcium crystals-related signals, such as phosphate, carbonate, and calcium pyrophosphate dihydrate (MGP, A960 cm−1/A1004 cm−1, MGC, A1070 cm−1/A1004 cm−1 and A1050 cm−1/A1004 cm−1). Finally, we observed an increase in lipids ratio (IL, A1450 cm−1/A1670 cm−1) with OA severity. As a conclusion, we have described the molecular fingerprint of hip cartilage, validating a panel of optical biomarkers and the potential of RS as a complementary diagnostic tool for OA.Xunta de Galicia; ED431E 2018/03Xunta de Galicia; IN607A2017/1

Impact of prevalence ratios of chondroitin sulfate (CS)- 4 and -6 isomers derived from marine sources in cell proliferation and chondrogenic differentiation processes

[Abstract] Osteoarthritis is the most prevalent rheumatic disease. During disease progression, differences have been described in the prevalence of chondroitin sulfate (CS) isomers. Marine derived-CS present a higher proportion of the 6S isomer, offering therapeutic potential. Accordingly, we evaluated the effect of exogenous supplementation of CS, derived from the small spotted catshark (Scyliorhinus canicula), blue shark (Prionace glauca), thornback skate (Raja clavata) and bovine CS (reference), on the proliferation of osteochondral cell lines (MG-63 and T/C-28a2) and the chondrogenic differentiation of mesenchymal stromal cells (MSCs). MG-G3 proliferation was comparable between R. clavata (CS-6 intermediate ratio) and bovine CS (CS-4 enrichment), for concentrations below 0.5 mg/mL, defined as a toxicity threshold. T/C-28a2 proliferation was significantly improved by intermediate ratios of CS-6 and -4 isomers (S. canicula and R. clavata). A dose-dependent response was observed for S. canicula (200 µg/mL vs 50 and 10 µg/mL) and bovine CS (200 and 100 µg/mL vs 10 µg/mL). CS sulfation patterns discretely affected MSCs chondrogenesis; even though S. canicula and R. clavata CS up-regulated chondrogenic markers expression (aggrecan and collagen type II) these were not statistically significant. We demonstrate that intermediate values of CS-4 and -6 isomers improve cell proliferation and offer potential for chondrogenic promotion, although more studies are needed to elucidate its mechanism of action.Xunta de Galicia; IN607A 2017/11Xunta de Galicia; IN607B 2018/19European Commission; 0245_IBEROS_1_

Impact of prevalence ratios of chondroitin sulfate (CS)- 4 and -6 isomers derived from marine sources in cell proliferation and chondrogenic differentiation processes

Osteoarthritis is the most prevalent rheumatic disease. During disease progression, differences have been described in the prevalence of chondroitin sulfate (CS) isomers. Marine derived-CS present a higher proportion of the 6S isomer, offering therapeutic potential. Accordingly, we evaluated the effect of exogenous supplementation of CS, derived from the small spotted catshark (Scyliorhinus canicula), blue shark (Prionace glauca), thornback skate (Raja clavata) and bovine CS (reference), on the proliferation of osteochondral cell lines (MG-63 and T/C-28a2) and the chondrogenic differentiation of mesenchymal stromal cells (MSCs). MG-G3 proliferation was comparable between R. clavata (CS-6 intermediate ratio) and bovine CS (CS-4 enrichment), for concentrations below 0.5 mg/mL, defined as a toxicity threshold. T/C-28a2 proliferation was significantly improved by intermediate ratios of CS-6 and -4 isomers (S. canicula and R. clavata). A dose-dependent response was observed for S. canicula (200 µg/mL vs 50 and 10 µg/mL) and bovine CS (200 and 100 µg/mL vs 10 µg/mL). CS sulfation patterns discretely affected MSCs chondrogenesis; even though S. canicula and R. clavata CS up-regulated chondrogenic markers expression (aggrecan and collagen type II) these were not statistically significant. We demonstrate that intermediate values of CS-4 and -6 isomers improve cell proliferation and offer potential for chondrogenic promotion, although more studies are needed to elucidate its mechanism of action.Xunta de Galicia | Ref. AGRUP 2015/05 (CICA-INIBIC)Xunta de Galicia | Ref. Grant IN607A 2017/11Xunta de Galicia | Ref. Grupos de Potencial Crecimiento IN607B 2018/19European Commission | Ref. 0245_IBEROS_1_