TY - JOUR

T1 - Histological processing of un-/cellularized thermosensitive electrospun scaffolds

AU - Fuchs, Julia

AU - Müller, Marc

AU - Daxböck, Christine

AU - Stückler, Manuela

AU - Lang, Ingrid

AU - Leitinger, Gerd

AU - Bock, Elisabeth

AU - El-Heliebi, Amin

AU - Moser, Gerit

AU - Glasmacher, Birgit

AU - Brislinger, Dagmar

N1 - Funding Information: Open access funding provided by Medical University of Graz. This work was supported by the Austrian Science Fund (FWF) and Christian Doppler Research Association (CDG): PIR7-B28 and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (EXC 62/1).

PY - 2019/4/8

Y1 - 2019/4/8

N2 - 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.

AB - 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.

KW - Cellularized prosthesis

KW - Graft

KW - Paraffin embedding

KW - PCL

KW - Polycaprolactone

KW - Polylactide acid

UR - http://www.scopus.com/inward/record.url?scp=85058676697&partnerID=8YFLogxK

U2 - 10.1007/s00418-018-1757-7

DO - 10.1007/s00418-018-1757-7

M3 - Article

C2 - 30560287

AN - SCOPUS:85058676697

VL - 151

SP - 343

EP - 356

JO - Histochemistry and Cell Biology

JF - Histochemistry and Cell Biology

SN - 0948-6143

IS - 4

ER -