Prolonged ethanol replacement by CO2 increases splits on articular cartilage surface after critical point drying

Area of Science:

• Biomaterials Science
• Microscopy Techniques
• Tissue Engineering

Background:

• Preservation of articular cartilage for scanning electron microscopy (SEM) is crucial for understanding joint health.
• Critical point drying (CPD) is a common method for preparing biological specimens, but the preceding ethanol dehydration step requires careful optimization.
• Ethanol replacement using carbon dioxide (CO2) is an alternative to traditional air-drying, aiming for better structural preservation.

Purpose of the Study:

• To investigate the effects of ethanol replacement by CO2 on the surface ultrastructure of rabbit articular cartilage prior to critical point drying (CPD).
• To assess the efficacy of monitoring ethanol removal using an alcometer during the CO2 exchange process.
• To compare the surface preservation achieved by CO2 exchange followed by CPD versus air-drying.

Main Methods:

• Rabbit articular cartilage specimens were fixed with glutaraldehyde and dehydrated with ethanol.
• Ethanol was replaced by CO2, with monitoring of ethanol removal using a gas chromatograph and an alcometer at different time intervals (2 h and 16 h).
• Specimen surface structure was analyzed using scanning electron microscopy (SEM) and a semiquantitative scoring method. Air-dried specimens served as a control.

Main Results:

• Short-term CO2 treatment (2 h) resulted in a smooth articular surface with minor striations and superficial splits.
• Prolonged CO2 treatment (16 h) completely removed ethanol but led to increased superficial splitting of the cartilage surface after CPD.
• Air-drying resulted in inferior preservation, characterized by pitted and leafy surfaces, although without superficial splits.

Conclusions:

• Prolonged ethanol replacement by CO2 prior to CPD can degrade the surface structure of articular cartilage, necessitating careful experimental design.
• Alcometer monitoring provides a convenient and effective method for tracking ethanol removal during CO2 exchange for CPD.
• Optimizing the duration of CO2 treatment is critical for achieving high-quality SEM imaging of cartilage ultrastructure.