Speaker
Description
Introduction
Cell-seeded scaffolds made from natural or synthetic materials are widely tested in animal models as biodegradable implants for treatment of joint disease (1). One of the most commonly used material for scaffold production is polycaprolactone (PCL), known for its biocompatibility and mechanical properties close to human cartilage. To validate cell distribution within scaffold and its biocompatibility, histological assessment is usually performed. However, processing of PCL polymers is associated with challenges related to temperature sensitivity and solubility in histological reagents (2). Therefore, to overcome them formalin-fixed paraffin-embedded (FFPE) and frozen samples histological techniques were optimised.
Methods
3D fibrous scaffolds featuring interconnected porous networks with gradient design were fabricated using a 3D fiber printer, which integrates melt electrospinning and fused deposition modeling techniques. Surface hydrophilicity was enhanced via non-thermal plasma (NTP) treatment. Modified protocols of standard FFPE and frozen samples histology were used. Cross-sections of PCL scaffolds were acquired with microtome and cryomicrotome, respectively.
Results
Standard FFPE histology caused shrinkage of both NTP-treated and -nontreated PCL scaffolds and disrupted their fiber network. Intact sections were obtained when replacing xylene with D-limonene-based clearing agent ROTI®Histol (Carl Roth) in combination with low-melting paraffin. Paraffin with 51-53°C melting point was applied to nontreated scaffolds, however, for NTP-treated scaffolds 42-44°C melting point paraffin was required, as these scaffolds had lower melting point. The best quality of sections was achieved by processing PCL scaffolds with automated tissue processor LOGOS (Milestone Medical), which employs vacuum to infiltrate paraffin, while avoiding clearing reagents at all.
Furthermore, prolonged incubation in cryoprotective reagents were found to be required to achieve optimal cryosections. To prevent PCL fiber disruption PCL scaffolds were sequentially incubated in 5 and 20 % glucose, mixture of 20% glucose and cryogel, and finally in pure cryogel.
Discussion
In this work we optimised several histological techniques of PCL scaffolds. The best results were achieved by using automated tissue processor with low-melting paraffin for FFPE and prolonged incubation in cryoprotectants for frozen samples. These optimizations extend applicability of histological evaluation to hydrophilic PCL scaffolds.
References
1. Chen R. et al. Multiphasic scaffolds for the repair of osteochondral defects: Outcomes of preclinical studies. Bioact Mater. 2023 Apr 28;27:505-545. doi: 10.1016/j.bioactmat.2023.04.016.
2. Dębski T. et al. Modified Histopathological Protocol for Poly-ɛ-Caprolactone Scaffolds Preserving Their Trabecular, Honeycomb-like Structure. Materials (Basel). 2022 Feb 25;15(5):1732. doi: 10.3390/ma15051732.
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