| 摘要: | Introduction
Corneal 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 with 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 vesicles (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.
Aim
To 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.
Methods
Therapeutic grade human PCs were used to isolate HPPL 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 BCE 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 atomic 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 viability, proliferation markers, wound healing, adhesion assay, immunofluorescence and western blot.
Results
Proteomics revealed that HPPL contains multiple growth factors, antioxidants and components involved in many biological processes important for corneal healing. HPPL treatment was found to enhance 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 antioxidant 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 <200 nm and the concentration of particles of EVs approximately 1011 /mL. PEVs express CD41 and CD61 as membrane markers characteristic of platelets, and EVs marker CD9 and CD63. ELISA and proteomics found that the PEVs contained mixtures of growth factors and multiple other functional molecules. Analyses by proteomics bioinformatic analysis identified that PEVs were highly enriched in extracellular exosome with functional activities associated with cell cadherin and adherens pathway. Human cornea endothelial B4G12 cells treated with PEVs had increased viability, enhanced wound healing rate, stronger proliferation markers and improved adhesion rate. PEVs did not exert cellular toxicity as evidenced by the maintenance of cellular morphology and preserved corneal endothelial proteins.
Conclusions
The HPPL and PEVs prepared from clinical-grade PCs are nontoxic and promote CECs proliferation and migration. HPPL exhibits potent antioxidant effects against TBHP – induced oxidative damages. PEVs support corneal endothelial regeneration. These findings clearly support further investigations of HPPL and PEVs platelet-derived biomaterials in preclinical animal models as a new CEC regeneration biotherapy. |
