Page 82 - Tailoring Electrospinning Techniques for Regenerative Medicine - Marc Simonet

CHAPTER 4
2-phosphate (0.25 mg/ml; Sigma) that was changed three times a week.
Assessment of cell infiltration
Cell distribution and infiltration were investigated on cryosections of the sca old a er 7 days of culture. The cultured sca olds were washed in PBS, embedded in Tissue-Tek (Sakura) and then snap-frozen in liquid nitrogen. Longitudinal cross-sections of 50 μm were cut by a cryotome, collected on poly-lysine coated coverglasses, and stained with DAPI for 10 minutes. Cells were examined using fluorescent microscopy equipped with a polarizer (Axiovert 200M, Zeiss); pictures were taken using a Zeiss AxioCam HRM camera and Zeiss AxioVision Rel. 4.4 acquisition so ware. The sca old fibers were visualized in cross polarized light and overplayed with the fluorescent DAPI staining image. To assess changes in thickness over time due to compaction by cell traction forces, the sca old thickness was measured with a caliper on the original sca olds and a er 7 days of culture.
4.3.5 Statistical analysis
All data are presented as averages (n=5) and their standard deviations (SD), except the data presented in Figure 4.1. There, each datapoint consists of at least 2 up to 5 spinning sessions, with porosity measured at 3 di erent spots. SD bars are not shown for clarity of the graph. If the SD between spinning sessions was more than 0.5 % the two extremes are shown as datapoints.
Statistical analyses were performed with the t-test (Origin 8.5.1, OriginLab Corporation, USA). Di erences were considered significant for P values <0.05.
4.4 Results and discussion
4.4.1 Influence of polymer deposition and ice crystal formation on 3D sca old porosity
First, we explored how the ratio between polymer deposition rate and growth of the ice crystal layer a ects the final sca old properties. Therefore, we changed the polymer deposition rate by spinning on di erent target areas (i.e. on drums with lengths of 30 mm, 50 mm and 100 mm, respectively) rather than changing process parameters such as flow rate or solution concentration. This allowed us to keep the fiber diameter and the ice crystal growth rate constant. In addition, the spinning time was varied to control the process linearity, such as ice crystal growth and porosity, over time. The ambient conditions were kept between 21 to 26 °C and the relative humidity between 40 to 70 %. The latter has been shown to only have a minor e ect on sca old porosity, if any at all.23,41
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