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Evaluation of purified fractions of human platelet lysate and platelet extracellular vesicles for corneal endothelial regeneration and repair

為了解決Ferrari Leclerc的問題，作者RIFA WIDYANINGRUM 這樣論述：

IntroductionCorneal endothelial cells (CECs) slowly decrease in number with increasing age, which is a clinical issue as these cells have very limited regenerative ability. Certain corneal endothelial disorders, as Fuchs’ endothelial dystrophy, bullous keratopathy, and senescence are associated wit

h oxidative stress. Therapeutic platelet biomaterials have been used in regenerative medicine and cell therapy because of their great safety, cost-effective manufacture, and global availability from collected_ platelet concentrates (PCs). Human platelet lysate (HPL) and platelet extracellular vesicl

es (PEVs) are a complex mixture of potent bioactive molecules instrumental in tissue repair and regeneration. As an alternative to corneal transplantation, we applied heat-treated platelet pellet lysate (HPPL) and PEVs therapy to enhance CECs proliferation and oxidative stress control and ultimately

repair injury.AimTo develop and investigate the suitability of using platelet-derived biomaterials, HPPL and PEVs as the proof concept for implementing innovative regenerative and reconstructive biotherapy for corneal endothelial dysfunction.MethodsTherapeutic grade human PCs were used to isolate H

PPL and PEVs. The protein composition and growth factor content were determined through ELISA assays and proteomics. David biomedical informatic analysis platform was used to further analyze the functional annotation of the proteome. The safety and efficacy of using HPPL in human B4G12 and bovine BC

E C/D- 1b cells were examined by determining cell viability, wound closure rate, flowcytometry, reduced glutathione/oxidized glutathione ratio, liquid chromatography with tandem mass spectrometry (LC-MS/MS), proteomics analysis and western blot. PEVs were isolated from PCs and characterized by atomi

c force microscopy (AFM), nanoparticles tracking analysis (NTA), and dynamic light scattering (DLS). Extracellular vesicles (EVs) and platelet markers were characterized by western blot. PEVs uptake in CECs were observed by confocal microscopy. The safety and efficacy of PEVs were measured by cell v

iability, proliferation markers, wound healing, adhesion assay, immunofluorescence and western blot.ResultsProteomics revealed that HPPL contains multiple growth factors, antioxidants and components involved in many biological processes important for corneal healing. HPPL treatment was found to enha

nce viability, improve wound healing rate, and preserve cell growth and morphology of B4G12 and BCE C/D-1b cells. HPPL effectively protected CECs against TBHP-induced oxidative damage by improving CEC viability, decreasing cell death and reactive oxygen species (ROS) generation, while increasing ant

ioxidant capacity. Proteomics demonstrated that in response to TBHP-induced oxidative stress, HPPL promoted the corneal healing pathway and strengthened the oxidative stress defense mechanism. The PEVs were regular, fairly rounded shape, with an average size of