Histological processing of un-/cellularized thermosensitive electrospun scaffolds
| Autor: | Gerd Leitinger, Gerit Moser, Amin El-Heliebi, Julia Fuchs, Elisabeth Bock, Birgit Glasmacher, Christine Daxböck, Manuela Stückler, Ingrid Lang, Dagmar Brislinger, Marc Mueller |
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| Rok vydání: | 2018 |
| Předmět: |
Cellularized prosthesis
0301 basic medicine Scaffold Histology food.ingredient Polyesters Immunofluorescence Gelatin Cryofixation Graft Paraffin embedding 03 medical and health sciences chemistry.chemical_compound food medicine Humans Transition Temperature ddc:610 Molecular Biology Acrylic resin Cells Cultured Paraffin Embedding 030102 biochemistry & molecular biology medicine.diagnostic_test Chemistry Keratin-7 technology industry and agriculture Polylactide acid Cell Biology Polycaprolactone Medical Laboratory Technology 030104 developmental biology Antigen retrieval PCL visual_art visual_art.visual_art_medium Dewey Decimal Classification::600 | Technik::610 | Medizin Gesundheit Immunostaining Biomedical engineering |
| Zdroj: | Histochemistry and Cell Biology 18 (2018), Nr. 12 |
| ISSN: | 1432-119X 0948-6143 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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