Fig 1.
Two symmetrical steel frames are connected via screws with a gap nut in between. The observation window (Ø 12 mm) is covered with a coverslip, which is fixed by a circlip. Recesses reduce the weight of the chamber.
Fig 2.
Scanning electron microscopy of the implant materials.
Porous polymer patch (A), unmodified PCL fiber mat (B) and PCL fiber mat modified with CS-g-PCL (C). Scale bars: 200 μm (A), 100 μm (B, C).
Table 1.
Fiber diameter in μm, porosity in %, the maximum pore diameter in μm, the middle pore diameter in μm and the smallest pore diameter in μm of the control group scaffold (control; see Gniesmer et al., 2019), the unmodified PCL fiber mat group (PCL) and the PCL fiber mat modified with CS-g-PCL group.
Table 2.
Venular diameters in μm, volumetric blood flow in pl/s and wall shear rate in s-1 of postcapillary and collecting venules at the border zones of the control group scaffold (control), the unmodified PCL fiber mat group (PCL) and PCL fiber mat modified with CS-g-PCL group immediately (d0) and 3, 6, 10 and 14 days after implantation.
Values are expressed as means ± SEM.
Fig 3.
Results of intravital microscopy.
(A-C) Intravital microscopic overview images of control (A), PCL (B) and CS-g-PCL (C) at day 14. (D) Functional capillary density in cm/cm2 in the border zones of the implants 0, 3, 6, 10 and 14 days after implantation. Means ± SEM; *p < 0.05 vs. PCL and CS-g-PCL; +p < 0.05 vs. PCL on the same day;°p < 0.05 vs. CS-g-PCL on the preceding time point. Scale bars: 400 μm.
Table 3.
Functional capillary density in cm/cm2, number of rolling leukocytes in cells/min and number of adherent leukocytes in cells/mm2 at the periphery of the control group scaffold (control), the unmodified PCL fiber mat group (PCL) and PCL fiber mat modified with CS-g-PCL group immediately (d0) and 3, 6, 10 and 14 days after implantation.
Values are expressed as means ± SEM.
Fig 4.
Leukocyte-endothelium interaction.
Leukocyte-endothelium interaction at the periphery of the implants in post-capillary and collecting venules after implantation. (A) Number of rolling leukocytes 0, 3, 6, 10 and 14 days after implantation shown as number of cells/min. The implantation of different materials did not significantly increase the number of rolling leukocytes. (B) Number of adherent leukocytes 0, 3, 6, 10 and 14 days after implantation as number of cells/mm2. The number of adherent leukocytes at the border zones of the implants decreased over the evaluation period.
Fig 5.
HE staining, van Gieson staining and immunohistochemical detection of CD31.
Representative histological stainings (A-I) 14 days after implantation into the dorsal skinfold chamber of BALB/c mice. (A-C) HE staining (1) implant, (2) skin muscle, (3) subcutaneous tissue. (D-F) Collagen fibers were detected by van Gieson staining (arrowheads). (G-I) The presence of endothelial cells and accordingly enhanced functional capillary density was confirmed by immunohistochemical detection of CD31. (J-L) Negative controls. Areas of the implant are marked (#), arrows denote vascular structures. Scale bars: 100 μm (A—C), 50 μm (D—L).
Fig 6.
Naphtol-AS-D-chloroacetate esterase staining and immunohistochemical detection of CD68.
Representative histological staining of neutrophilic granulocytes (A-C) 14 days after implantation into the dorsal skinfold chamber of BALB/c mice using naphtol-AS-D-chloroacetate-esterase (CAE) staining. Representative histological staining for detection of macrophages and monocytes (D-I) 14 days after implantation into the dorsal skinfold chamber of BALB/c mice. (D-F) Immunohistochemical detection of CD68 for detection of macrophages. (G-I) Negative controls. Areas of the implant are marked (#).
Fig 7.
Immunofluorescence detection of CSF1R, CD86, CD11b and CD3.
Representative histological stainings for detection of macrophages in general, macrophages with predominantly pro-inflammatory actions, monocytes and T-cells (all green fluorescence) (A-C, E-G, I-K, M-O) 14 days after implantation into the dorsal skinfold chamber of BALB/c mice. (A-C) Immunofluorescence detection of CSF1R for detection of macrophages in general. (E-G) Immunofluorescence detection of CD86 for detection of macrophages with predominantly pro-inflammatory actions. (I-K) Immunofluorescence detection of CD11b for detection of monocytes. (M-O) Immunofluorescence detection of CD3 for detection of T-cells. Nuclei were stained using DAPI (blue fluorescence). Scale bars: 50 μm. (D,H,L,P) Quantitative analysis of cell infiltration by immunofluorescent staining for CSF1R (D), CD86 (H), CD11b (L) and CD3 (P). All data are expressed as the fluorescence intensity of the marker of interest normalized to the fluorescence intensity of DAPI staining. Values represent means ± SEM; *p < 0.05 vs. control and PCL; +p < 0.05 vs. control and CS-g-PCL.
Table 4.
Results from quantification of immunofluorescent detection of immune cell markers.