Histological processing of un-/cellularized thermosensitive electrospun scaffolds

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.

TGF–β3 Loaded Electrospun Polycaprolacton Fibre Scaffolds for Rotator Cuff Tear Repair: An in Vivo Study in Rats

“…PCL is biodegradable with slow degradation rates in vivo [23,24] and PCL-based materials for medical devices such as Capronor, a subdermal implant for long-term drug delivery [25] or drug delivery systems for glaucoma treatment [26] are already in clinical use or trial. Studies show that surface modification with fibronectin or blending with polylactide acid (PLA) improve cell attachment of endothelial cells or cell infiltration with trophoblast cells [27,28] as well as osteogenic and tenogenic differentiation of C3H10T1/2 pluripotent stem cells [29]. Furthermore, PCL-PLA and PCL-gelatine fibre mats seeded with mesenchymal stem cells showed high biocompatibility with viabilities higher than 82% after seven days as reported by Gryshkov and coworkers [30].…”

Multi-Layer Nanofibrous PCL Scaffold-Based Colon Cancer Cell Cultures to Mimic Hypoxic Tumor Microenvironment for Bioassay

“…In one previous report, transplanted and harvested tissue-engineered constructs were fixed in formalin and embedded in paraffin using PCL/PLLA fibrous scaffolds, after which tissues were analyzed by IHC [ 29 ]. However, another study reported that histological processing of thermosensitive PCL/PLL scaffolds fails because of the low-melting temperature (T m ≈ 60 °C) characteristic of PCL [ 30 ]. To circumvent this problem, the authors combined PLC with a low-melting-point paraffin-embedding method for histological investigations of thermosensitive specimens [ 30 ].…”

Electrospun PCL/PLA Scaffolds Are More Suitable Carriers of Placental Mesenchymal Stromal Cells Than Collagen/Elastin Scaffolds and Prevent Wound Contraction in a Mouse Model of Wound Healing

“…Recently, we developed electrospun scaffolds consisting of an inner layer of PCL/PLA (100/50 mg/ml) and an outer layer of PCL (200 mg/ml). These scaffolds showed good cell adherence but were thermosensitive and needed a lowtemperature preparation method for histological processing (Fuchs et al, 2019). Fuchs et al showed that the combination of low-melting-point paraffin embedding and pepsin digestion can be used as an antigen retrieval method for histological FIGURE 6 | Cell tracking in hAMSC-treated wounds.…”

“…The fiber morphology of the scaffolds was characterized using scanning electron microscopy (VP-SEM S3400, Hitachi Europe). Six samples were taken from each fiber mat and coated with gold palladium (Sputter Coater SC7620, Emetich) for 45 s. Images were acquired and analyzed according to a standard operating procedure (Fuchs et al, 2019). Fiber diameter distribution and mass of samples were used to calculate the specific surface of the electrospun scaffolds using the following equations:…”