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. 2022 Feb 25;15(5):1732.
doi: 10.3390/ma15051732.

Modified Histopathological Protocol for Poly-ɛ-Caprolactone Scaffolds Preserving Their Trabecular, Honeycomb-like Structure

Tomasz Dębski  1 , Juliusz Wysocki  1 , Katarzyna Siennicka  1 , Jakub Jaroszewicz  2 , Karol Szlązak  2 , Wojciech Święszkowski  2 , Zygmunt Pojda  1
Affiliations

Modified Histopathological Protocol for Poly-ɛ-Caprolactone Scaffolds Preserving Their Trabecular, Honeycomb-like Structure

Tomasz Dębski et al. Materials (Basel). .

Abstract

Poly-ɛ-caprolactone (PCL) is now widely studied in relation to the engineering of bone, cartilage, tendons, and other tissues. Standard histological protocols can destroy the carefully created trabecular and honeycomb-like architecture of PCL scaffolds, and could lead to scaffold fibers swelling, resulting in the displacement or compression of tissues inside the scaffold. The aim of this study was to modify a standard histopathological protocol for PCL scaffold preparation and evaluate it on porous cylindrical PCL scaffolds in a rat model. In 16 inbred Wag rats, 2 PCL scaffolds were implanted subcutaneously to both inguinal areas. Two months after implantation, harvested scaffolds were first subjected to μCT imaging, and then to histopathological analysis with standard (left inguinal area) and modified histopathological protocols (right inguinal area). To standardize the results, soft tissue percentages (STPs) were calculated on scaffold cross-sections obtained from both histopathological protocols and compared with corresponding µCT cross-sections. The modified protocol enabled the assessment of almost 10× more soft tissues on the scaffold cross-section than the standard procedure. Moreover, STP was only 1.5% lower than in the corresponding µCT cross-sections assessed before the histopathological procedure. The presented modification of the histopathological protocol is cheap, reproducible, and allows for a comprehensive evaluation of PCL scaffolds while maintaining their trabecular, honeycomb-like structure on cross-sections.

Keywords: PCL; full cross-section of scaffold; histopathological protocol; scaffold; scaffold architecture preservation.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Scaffold design: (a) scaffold dimensions; (b) the fiber pattern repeated every five layers during scaffold printing.
Figure 2
Figure 2
Scaffold implantation: (a) incision marking after the epilation of inguinal areas; (b) implanted scaffolds; (c) closed wound after implantation.
Figure 3
Figure 3
Scaffold µCT imaging: (a) 3D scaffold reconstruction; (b) µCT images of scaffold longitudinally (red frame) and cross-sectionally (green frame).
Figure 4
Figure 4
Microscopical images of the scaffold pores before (a) and after (b–f) xylene addition at a temperature of 23 °C. Representative images are shown at 10× magnification. Images were taken in 20-s intervals. Scale bars represent 100 μm.
Figure 5
Figure 5
Microscopical images of scaffold pores before (a) and after bathing in water at a temperature of 57 °C for 10 min. Representative images are shown at 10× magnification. Scale bars represent 100 μm.
Figure 6
Figure 6
Scaffold harvesting after 2 months of follow-up (a,b).
Figure 7
Figure 7
HE-stained sections prepared according to the standard (a) and modified histopathological protocol (b).
Figure 8
Figure 8
μCT imaging of the scaffolds before (a) and after the standard (b) and modified histopathological protocol ((c,d), accordingly).
Figure 9
Figure 9
Scaffold cross-section surface (S1, blue) and soft tissue area inside the scaffold (S2, orange) on sections obtained after the standard histopathological (a,b) and modified histopathological protocols (c,d).
Figure 10
Figure 10
Scaffold cross-section surface (S1, blue) and soft tissue area inside the scaffold (S2, orange) on μCT images obtained before the standard histopathological (a,b) and modified histopathological protocols (c,d).
Figure 10
Figure 10
Scaffold cross-section surface (S1, blue) and soft tissue area inside the scaffold (S2, orange) on μCT images obtained before the standard histopathological (a,b) and modified histopathological protocols (c,d).
Figure 11
Figure 11
Scaffold cross-section surface (S1, blue) and soft tissue area inside the scaffold (S2, orange) on μCT images obtained after the standard histopathological (a,b) and modified histopathological protocols (c,d).
Figure 12
Figure 12
STP calculated on: (a) H–E sections after standard and modified histopathological protocols. Significant difference: p < 0.05 (*); (b) μCT images obtained before and after the standard and modified histopathological protocols. Significant difference: p < 0.05 (*), p > 0.05 (ns).
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