研究目的
To evaluate the ability of nanostructured porous boron-doped diamond (BDD) to form an interface with neural cells and the role of molecular functionalization by poly-L-lysine on this interface, assessing biocompatibility, cytotoxicity, and support for neuron adhesion and regeneration.
研究成果
Porous BDD is biocompatible and supports fibroblast proliferation without cytotoxicity. Molecular functionalization with poly-L-lysine is necessary for stable adhesion and physiological responses of newborn neurons on NCD and porous BDD. Porous BDD also supports adult neuron adhesion and network formation even without coating, indicating potential for reduced background noise in neural recordings, though further research is needed on neuronal excitability and glial cell effects.
研究不足
The study did not fully investigate the electrophysiological functionality of adult neurons on uncoated porous BDD, the potential stress responses indicated by F-actin changes, or the long-term effects and in vivo applicability, including glial cell interactions and scar formation. Transparency issues with porous BDD limited direct optical monitoring of neuron growth.
1：Experimental Design and Method Selection:
The study involved preparing planar nanocrystalline diamond (NCD) and porous BDD layers using microwave plasma-enhanced chemical vapor deposition (MW-PECVD) systems. Biocompatibility and neural interface formation were assessed through cell culture experiments with fibroblasts, newborn rat hippocampal neurons, and adult murine neurons, including functionalization with poly-L-lysine.
2：Sample Selection and Data Sources:
Samples included NCD and porous BDD layers on glass or quartz substrates, with controls using glass cover slips. Cell sources were immortalized murine fibroblasts (3T3 cell line), primary newborn rat hippocampal neurons, and primary adult murine neurons from C57BJ/6 mice.
3：List of Experimental Equipment and Materials:
Equipment included MW-LA-PECVD and MW-PECVD systems, SEM (Tescan FERA3), Raman microscope (Renishaw InVia), optical microscopes (Olympus CKX41, FluoView FV-1000), flow cytometer (BD LSR II), and microspectrofluorimetry setup. Materials included diamond nanoparticles, poly-L-lysine, cell culture media, stains (e.g., CellTracker, FURA2-AM), antibodies, and chemicals for stimulation.
4：Experimental Procedures and Operational Workflow:
Diamond layers were prepared, characterized by SEM and Raman spectroscopy, sterilized, and functionalized with poly-L-lysine. Cells were cultured on substrates, stained, and analyzed for proliferation, cytotoxicity, calcium imaging, and immunostaining. Specific protocols included lactate dehydrogenase assay, cell cycle analysis, and calcium imaging with chemical stimulations.
5：Data Analysis Methods:
Data were analyzed using statistical tests (one-way ANOVA in R), software (FlowJo, FluoView, Imaris), and microscopy image analysis to evaluate cell growth, toxicity, and neuronal activity.
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transmission microscope
Olympus CKX41
Olympus
Used for optical microscopy to visualize newborn hippocampal neural cells.
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camera
Olympus E410
Olympus
Attached to the microscope for image acquisition.
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confocal microscopy system
Olympus FluoView FV-1000
Olympus
Used for confocal microscopy to visualize cells in 3D and for image acquisition.
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microspectrofluorimetry setup
IX 81 fluorescence microscope
Olympus
Used for calcium imaging to measure intracellular calcium signals in neurons.
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microwave plasma-enhanced chemical vapor deposition system
MW-LA-PECVD
Used for depositing planar nanocrystalline diamond layers on substrates at low temperatures.
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microwave plasma-enhanced chemical vapor deposition system
ASTeX 5010 reactor
Seki Technotron
Used for depositing porous boron-doped diamond layers on quartz substrates.
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positive tone photoresist
ma-1210
Microresist Technology
Used as part of the porous template for diamond deposition to homogenize and stabilize SiO2 fibers.
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electrospun SiO2 fibers
Elmarco Ltd.
Used in the porous template for diamond deposition, with typical diameters of several hundreds of nm.
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ultrasonic processor
UP400S
Used to ultrasonically chop SiO2 fibers in deionized water for 10 minutes before mixing with polymer.
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poly-L-lysine
M W 30 000–70 000
Sigma
Used for molecular functionalization of diamond surfaces to promote neuron adhesion.
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borosilicate glass cover slips
20 mm diameter, thickness 0.13–0.16 mm
Karl Hecht
Used as control substrates in cell culture experiments.
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scanning electron microscopy tool
Tescan FERA3
Tescan
Used to observe the surface morphology and evaluate thickness of diamond layers.
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Raman microscope
Renishaw InVia
Renishaw
Used for Raman spectroscopy to assess the quality and composition of diamond layers.
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flow cytometer
BD LSR II
BD
Used to measure DNA content for cell cycle analysis.
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