2 research outputs found
Functionalized CoCr surfaces with adhesive molecules to improve endothelialization
Versi贸 amb diverses seccions encriptades, per drets a'autorCobalt-chromium (CoCr) alloys are widely used as biomaterials for coronary stents due to their excellent mechanical properties, biocompatibility and corrosion resistance. However, these materials are bioinert, retarding the complete endothelialization and resulting in a higher risk of restenosis, narrowing of the artery, and late-stent thrombosis. Therefore, the improvement of implants surface endothelialization has acquired importance in the last years.
Immobilization of cell adhesive biomolecules onto biomaterials surface is a well-known strategy to control cell response. However, the strategy of immobilization, the optimal combination or the appropriate spatial presentation of the bioactive sequences to enhance endothelialization for cardiovascular applications, remains to be elucidated.
The present PhD thesis focused on the development of a new biofunctionalized CoCr alloy surfaces in order to improve the endothelialization. To that end, elastin-like recombinamers (ELR) genetically modified with an REDV (Arg-Glu-Asp-Val) sequence and short synthetized peptides RGDS (Arg-Gly-Asp-Ser), REDV, YIGSR (Tyr-Ile-Gly-Ser-Arg) and their equimolar combination, were attached by physisorption and covalent bonding onto CoCr alloy surfaces and thoroughly characterized physico-chemically and evaluated in vitro with human umbilical vein endothelial cells (HUVECs), coronary artery smooth muscle cells (CASMCs) and platelets from blood donors.
First, biofunctionalized surfaces with ELR were developed and optimized by evaluating different surface activation treatments, oxygen plasma and sodium hydroxide etching, and different binding strategies, physisorption and covalent bonding. The functionalized surfaces demonstrated a higher cell adhesion and spreading of HUVEC cells, this effect is emphasized as increases the amount of immobilized biomolecules and directly related to the immobilization technique: covalent bonding. Nevertheless, the silanization process was not completely effective since a mixture of covalent and physisorption behavior was observed probably due to the use of big molecules that decreased the control of the bonding between the biomolecule and the surface. Secondly, it was synthetized immobilized RGDS, REDV, YIGSR and their equimolar combination peptides onto the different surfaces. Cell studies demonstrated that the covalent functionalization of CoCr surfaces with an equimolar combination of RGDS/YIGSR represented the most powerful strategy to enhance the early stages of HUVECs adhesion, proliferation and migration, indicating a positive synergistic effect between the two peptide motifs. Besides, gene expression and platelet adhesion studies showed that surfaces silanized with the combination RGDS/YIGSR improved anti-thrombogenicity compared to non-modified surfaces.
Finally, cell co-cultures of HUVECs/CASMCs found that functionalization increased the amount of adhered HUVECs onto modified surfaces compared to plain CoCr, independently of the used peptide and the strategy of immobilization. Overall, the present thesis offer a comprehensive view of the effectiveness of immobilizing cell adhesive molecules onto CoCr alloy surfaces to enhance endothelialization while preventing restenosis and thrombosis for cardiovascular applications.Las aleaciones de cobalto-cromo (CoCr) son ampliamente utilizadas como biomateriales para stents coronarios debido a sus excelentes propiedades mec谩nicas, biocompatibilidad y resistencia a la corrosi贸n. Sin embargo, estos materiales son bio-inertales, retardando la completa endotelializaci贸n y resultando en un mayor riesgo de reestenosis, estrechamiento de la arteria y trombosis tard铆a. Por lo tanto, la mejora de la endotelizaci贸n de superficie de los implantes ha adquirido importancia en los 煤ltimos a帽os. La inmovilizaci贸n de biomol茅culas adhesivas celulares sobre la superficie de los biomateriales es una estrategia bien conocida para controlar la respuesta celular. Sin embargo, queda por aclararse la estrategia de inmovilizaci贸n, la combinaci贸n 贸ptima o la presentaci贸n espacial apropiada de las secuencias bioactivas para potenciar la endotelizaci贸n en aplicaciones cardiovasculares. La presente tesis doctoral se centr贸 en el desarrollo de una nueva superficie biofuncionalizada de la aleaci贸n CoCr con el fin de mejorar la endotelializaci贸n. Para ello, los recombinameros tipo elastina (ELR) modificados gen茅ticamente con una secuencia REDV (Arg-Glu-Asp-Val) y p茅ptidos cortos sintetizados RGDS (Arg-Gly-Asp-Ser), REDV, YIGSR (Tyr-Ile-Gly -Ser-Arg) y su combinaci贸n equimolar, fueron anclados por fisisorci贸n y uni贸n covalente a las superficies de la aleaci贸n, se caracterizaron f铆sicamente-qu铆micamente y evalu贸 in vitro con c茅lulas endoteliales de vena umbilical humana (HUVECs), c茅lulas de m煤sculo liso de arteria coronaria (CASMCs) y plaquetas de donantes de sangre. En primer lugar, se desarrollaron y optimizaron las superficies biofuncionalizadas con ELR mediante la evaluaci贸n de diferentes tratamientos de activaci贸n superficial: plasma de ox铆geno y ataque con hidr贸xido de sodio, y diferentes estrategias de uni贸n: fisisorci贸n y uni贸n covalente. Las superficies funcionalizadas demostraron una mayor adhesi贸n celular y propagaci贸n de c茅lulas HUVEC, este efecto se enfatiza a medida que aumenta la cantidad de biomol茅culas inmovilizadas y se relaciona directamente con la t茅cnica de inmovilizaci贸n: la uni贸n covalente. Sin embargo, el proceso de silanizaci贸n no fue completamente efectivo, ya que se observ贸 una mezcla de uniones covalentes y fisisorbidas, probablemente debido al uso de grandes mol茅culas que disminuyeron el control de la uni贸n entre la biomol茅cula y la superficie. En segundo lugar, se inmovilizado los p茅ptidos sintetizados RGDS, REDV, YIGSR y sus combinaciones equimolares sobre las diferentes superficies. Los estudios celulares demostraron que la funcionalizaci贸n covalente de las superficies de CoCr con una combinaci贸n equimolar de RGDS/YIGSR represent贸 la estrategia m谩s potente para potenciar las etapas tempranas de la adhesi贸n, proliferaci贸n y migraci贸n de HUVECs, indicando un efecto sin茅rgico positivo entre los dos motivos pept铆dicos. Adem谩s, la expresi贸n g茅nica y los estudios de adhesi贸n plaquetaria mostraron que las superficies silanizadas con la combinaci贸n RGDS/YIGSR mejoraron la antitrombogenicidad en comparaci贸n con las superficies no modificadas. Finalmente, con los co-cultivos celulares de HUVECs/CASMCs se encontr贸 que la funcionalizaci贸n aumentaba la cantidad de HUVEC adheridas sobre las superficies modificadas en comparaci贸n con CoCr simple, independientemente del p茅ptido usado y la estrategia de inmovilizaci贸n. En general, la presente tesis ofrece una visi贸n completa de la efectividad de la inmovilizaci贸n de mol茅culas adhesivas celulares en la superficie de la aleaci贸n CoCr para mejorar la endotelializaci贸n, mientras que previene la reestenosis y trombosis en aplicaciones cardiovasculares.Postprint (published version
Safety and efficacy of ApTOLL in patients with ischemic stroke undergoing endovascular treatment: a phase 1/2 randomized clinical trial
Importance ApTOLL is a TLR4 antagonist with proven preclinical neuroprotective effect and a safe profile in healthy volunteers. Objective To assess the safety and efficacy of ApTOLL in combination with endovascular treatment (EVT) for patients with ischemic stroke. Design, Setting, and Participants This phase 1b/2a, double-blind, randomized, placebo-controlled study was conducted at 15 sites in Spain and France from 2020 to 2022. Participants included patients aged 18 to 90 years who had ischemic stroke due to large vessel occlusion and were seen within 6 hours after stroke onset; other criteria were an Alberta Stroke Program Early CT Score of 6 to 10, estimated infarct core volume on baseline computed tomography perfusion of 5 to 70 mL, and the intention to undergo EVT. During the study period, 4174 patients underwent EVT. Interventions In phase 1b, 0.025, 0.05, 0.1, or 0.2 mg/kg of ApTOLL or placebo; in phase 2a, 0.05 or 0.2 mg/kg of ApTOLL or placebo; and in both phases, treatment with EVT and intravenous thrombolysis if indicated. Main Outcomes and Measures The primary end point was the safety of ApTOLL based on death, symptomatic intracranial hemorrhage (sICH), malignant stroke, and recurrent stroke. Secondary efficacy end points included final infarct volume (via MRI at 72 hours), NIHSS score at 72 hours, and disability at 90 days (modified Rankin Scale [mRS] score). Results In phase Ib, 32 patients were allocated evenly to the 4 dose groups. After phase 1b was completed with no safety concerns, 2 doses were selected for phase 2a; these 119 patients were randomized to receive ApTOLL, 0.05 mg/kg (n驴=驴36); ApTOLL, 0.2 mg/kg (n驴=驴36), or placebo (n驴=驴47) in a 1:1:v2 ratio. The pooled population of 139 patients had a mean (SD) age of 70 (12) years, 81 patients (58%) were male, and 58 (42%) were female. The primary end point occurred in 16 of 55 patients (29%) receiving placebo (10 deaths [18.2%], 4 sICH [7.3%], 4 malignant strokes [7.3%], and 2 recurrent strokes [3.6%]); in 15 of 42 patients (36%) receiving ApTOLL, 0.05 mg/kg (11 deaths [26.2%], 3 sICH [7.2%], 2 malignant strokes [4.8%], and 2 recurrent strokes [4.8%]); and in 6 of 42 patients (14%) receiving ApTOLL, 0.2 mg/kg (2 deaths [4.8%], 2 sICH [4.8%], and 3 recurrent strokes [7.1%]). ApTOLL, 0.2 mg/kg, was associated with lower NIHSS score at 72 hours (mean difference log-transformed vs placebo, -45%; 95% CI, -67% to -10%), smaller final infarct volume (mean difference log-transformed vs placebo, -42%; 95% CI, -66% to 1%), and lower degrees of disability at 90 days (common odds ratio for a better outcome vs placebo, 2.44; 95% CI, 1.76 to 5.00). Conclusions and Relevance In acute ischemic stroke, 0.2 mg/kg of ApTOLL administered within 6 hours of onset in combination with EVT was safe and associated with a potential meaningful clinical effect, reducing mortality and disability at 90 days compared with placebo. These preliminary findings await confirmation from larger pivotal trials.Peer ReviewedPostprint (author's final draft