Histological processing of un-/cellularized thermosensitive electrospun scaffolds

Julia Fuchs,Gerd Leitinger,Gerit Moser,Elisabeth Bock,Dagmar Brislinger,Marc Mueller,Birgit Glasmacher,Manuela Stückler,Amin El-Heliebi,Christine Daxböck,Ingrid Lang

doi:10.1007/s00418-018-1757-7

Julia Fuchs, Gerd Leitinger + Show 9 more

Open Access

https://doi.org/10.1007/s00418-018-1757-7

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Abstract

Histological processing of thermosensitive electrospun poly(ε-caprolactone)/poly(l-lactide) (PCL/PLA) scaffolds fails, as poly(ε-caprolactone) (PCL) is characterized by its low-melting temperature (Tm = 60 °C). Here, we present an optimized low-temperature preparation method for the histological processing of un-/cellularized thermosensitive PCL/PLA scaffolds.Our study is aimed at the establishment of an optimized dehydration and low-melting-point paraffin-embedding method of electrospun PCL/PLA scaffolds (un-/cellularized). Furthermore, we compared this method with (a) automatized dehydration and standard paraffin embedding, (b) gelatin embedding followed by automatized dehydration and standard paraffin embedding, (c) cryofixation, and (d) acrylic resin embedding methods. We investigated pepsin and proteinase K antigen retrieval for their efficiency in epitope demasking at low temperatures and evaluated protocols for immunohistochemistry and immunofluorescence for cytokeratin 7 (CK7) and in situ padlock probe technology for beta actin (ACTB). Optimized dehydration and low-melting-point paraffin embedding preserved the PCL/PLA scaffold, as the diameter and structure of its fibers were unchanged. Cells attached to the PCL/PLA scaffolds showed limited alterations in size and morphology compared to control. Epitope demasking by enzymatic pepsin digestion and immunostaining of CK7 displayed an invasion of attached cells into the scaffold. Expression of ACTB and CK7 was shown by a combination of mRNA-based in situ padlock probe technology and immunofluorescence. In contrast, gelatin stabilization followed by standard paraffin embedding led to an overall shrinkage and melting of fibers, and therefore, no further analysis was possible. Acrylic resin embedding and cyrofixation caused fiber structures that were nearly unchanged in size and diameter. However, acrylic resin-embedded scaffolds are limited to 3 µm sections, whereas cyrofixation led to a reduction of the cell size by 14% compared to low-melting paraffin embedding. The combination of low-melting-point paraffin embedding and pepsin digestion as an antigen retrieval method offers a successful opportunity for histological investigations in thermosensitive specimens.

Highlights

Poly(ε-caprolactone)/poly(l-lactide) (PCL/PLA) scaffolds are a focus of tissue engineering as a promising substitute for expanded polytetrafluoroethylene scaffolds (Siddiqui et al 2018; Pfeiffer et al 2014)
Acrylic resin embedding of un‐/cellularized PCL/PLA scaffolds
Attached cells appeared reduced in their size compared to the cells attached on PCL/PLA scaffolds processed by low-melting-point paraffin embedding (Fig. 7 b, d, e)

Summary

Introduction

Poly(ε-caprolactone)/poly(l-lactide) (PCL/PLA) scaffolds are a focus of tissue engineering as a promising substitute for expanded polytetrafluoroethylene (ePTFE) scaffolds (Siddiqui et al 2018; Pfeiffer et al 2014). PCL/PLA scaffolds act as a three-dimensional (3D) microenvironment for cell adhesion, proliferation, differentiation, and extracellular matrix (ECM) formation and, mimic the conditions that allow recreating the native tissue (Dhandayuthapani et al 2011). Various types of PCL/PLA scaffolds and applications are under investigation; e.g., electrospun PCL/PLA blend nanofibrous scaffolds to facilitate new bone formation, small-diameter fibrous vascular grafts, and nerve tissue regeneration (Yao et al 2017; Prabhakaran et al 2008). Immunocytochemistry in combination with laser scanning electron microscopy and scanning electron microscopy (SEM) is usually the methods of choice to prove that there are cellular adhesion, proliferation, and differentiation on the PCL/PLA scaffolds. Histological examinations of in vivo systems using implanted PCL/PLA scaffolds showed that cells intruded into the PCL/PLA scaffolds seem to increase the thermostability of the material (Shao et al 2006; Gredes et al 2017). There is an obvious lack of appropriate histological processing methods for thermosensitive materials such as PCL/PLA scaffolds used for in vitro experiments

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