Medizinische Universität Graz - Research portal

Logo MUG Resarch Portal

Selected Publication:

SHR Neuro Cancer Cardio Lipid

Fuchs, J; Mueller, M; Daxböck, C; Stückler, M; Lang, I; Leitinger, G; Bock, E; El-Heliebi, A; Moser, G; Glasmacher, B; Brislinger, D.
Histological processing of un-/cellularized thermosensitive electrospun scaffolds.
Histochem Cell Biol. 2019; 151(4):343-356 [OPEN ACCESS]
Web of Science PubMed PUBMED Central FullText FullText_MUG

 

Authors Med Uni Graz:
Bock Elisabeth
Brislinger Dagmar
Daxboeck Christine
El-Heliebi Amin
Fuchs Julia
Lang-Olip Ingrid
Leitinger Gerd
Moser Gerit
Stückler Manuela
Altmetrics:

Dimensions Citations:

Plum Analytics:
Number of Figures: 8
| | | | | | | |
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.

Find related publications in this database (Keywords)
PCL
Cellularized prosthesis
Polycaprolactone
Polylactide acid
Graft
Paraffin embedding
© Meduni GrazImprint