Histological processing of un-/cellularized thermosensitive electrospun scaffolds

Area of Science:

• Biomaterials Science
• Histotechnology
• Tissue Engineering

Background:

• Thermosensitive electrospun scaffolds made of poly(ε-caprolactone)/poly(L-lactide) (PCL/PLA) are difficult to process histologically due to PCL's low melting point (60°C).
• Standard histological techniques often fail, leading to scaffold degradation and inaccurate analysis of cellular infiltration and gene expression.

Purpose of the Study:

• To establish an optimized low-temperature preparation method for histological processing of thermosensitive PCL/PLA scaffolds.
• To compare the efficacy of this method against automated dehydration, standard paraffin embedding, gelatin embedding, cryofixation, and acrylic resin embedding.

Main Methods:

• Development and optimization of a low-temperature dehydration and low-melting-point paraffin embedding protocol for PCL/PLA scaffolds.
• Comparative analysis with standard paraffin embedding, gelatin embedding, cryofixation, and acrylic resin embedding.
• Evaluation of pepsin and proteinase K for antigen retrieval, and application of immunohistochemistry, immunofluorescence, and in situ padlock probe technology for CK7 and ACTB.

Main Results:

• The optimized low-melting-point paraffin embedding method successfully preserved the PCL/PLA scaffold structure and fiber integrity.
• Cells on the scaffolds exhibited minimal size and morphological changes compared to controls.
• Pepsin digestion enabled efficient antigen retrieval for CK7, revealing cell invasion, and combined methods confirmed ACTB and CK7 expression.

Conclusions:

• Optimized low-temperature paraffin embedding combined with pepsin digestion is a viable method for histological analysis of thermosensitive PCL/PLA scaffolds.
• This technique preserves scaffold architecture and cellular details, facilitating the study of cell-scaffold interactions and molecular expression.
• Alternative methods like gelatin embedding proved unsuitable, while acrylic resin and cryofixation presented limitations in sectioning or cell size preservation.