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Effects of Anti-inflammatory Nanofibers on Urethral Healing
Yvonne Y. Chan, MD1, Matthew I. Bury, MS1, Natalie J. Fuller, BS1, Bonnie Nolan, BS1, Emily M. Yura, MD2, Matthias D. Hofer, MD, PhD2, Arun K. Sharma, PhD3.
1Division of Pediatric Urology, Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA, 2Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA, 3Division of Pediatric Urology, Ann & Robert H. Lurie Children's Hospital of Chicago; Simpson Querrey Institute, Northwestern University; Center for Advanced Regenerative Engineering, Northwestern University, Chicago, IL, USA.

Background: Protracted post-surgical inflammation leading to tissue fibrosis and stricture formation remains a persistent problem in urethral reconstruction. Nanofibers in the form of peptide amphiphiles expressing anti-inflammatory signaling molecules (AIF-PA) have positively modulated local inflammatory responses and promoted proper anatomical and physiological bladder tissue regeneration in a rat bladder augmentation model. Utilizing the same platform to address urethral wound healing, we hypothesize that AIF-PAs will positively modulate post-operative local tissue inflammatory responses.
Methods: Small intestinal submucosa (SIS) was dip-coated with the AIF-PA (2% w/v). Substitution urethroplasty was performed to repair 5 mm ventral surgically-generated urethral defects with: uncoated SIS, SIS+AIF-PA1 (anti-inflammatory), or SIS+AIF-PA6 (control) on 12-week-old male Sprague Dawley rats (n=6/group/timepoint). Animals were euthanized at 14D and 28D post-surgery. Penises were harvested, fixed, and embedded in paraffin followed by H&E and Masson's Trichrome staining on 5μm tissue sections. Immunofluorescence staining was performed using antibodies against myeloperoxidase (MPO; neutrophils), CD68, CD86, and CD206 (macrophages), and pro-inflammatory cytokines TNFα and IL-1β. Images were taken of the native tissue, anastomosis, and regenerated urethral tissue in the wound bed (REGEN) for analysis.
Results: Complete urethra healing, defined as the presence of regenerated urethral tissue at the wound bed, occurred in 3/6 uncoated SIS (50%), 2/6 SIS+AIF-PA6 (33.3%), and 5/6 SIS+AIF-PA1 (83.3%) animals at 14D and all animals at 28D. In all groups, neutrophil, macrophage, and cytokine levels were elevated at the anastomoses and REGEN compared to native tissues. At both 14D and 28D, AIF-PA1 treatment resulted in decreased MPO, CD68, CD86, TNFα and IL-1β levels in anastomoses and REGEN (if present) (0.34-0.58x uncoated SIS 14D, 0.41-0.55x uncoated SIS 28D; Table 1). CD206 levels increased with AIF-PA1 treatment (1.8-2.1x uncoated SIS 14D, 1.7-2.4x uncoated SIS 28D). MPO, CD86, TNFα, and IL-1β levels in the anastomoses and REGEN of uncoated SIS animals decreased from 14D to 28D; at 28D, CD68, CD86, and TNFα had not fallen to the levels of 14D AIF-PA1+SIS animals. Percent vasculature in AIF-PA1 treated animals was 2.2x (anastomoses, 5.44 0.61 vs 2.44 0.16, p<0.01) and 1.8x (REGEN, 3.95 0.38 vs 2.22 0.35, p=0.01) higher than uncoated SIS animals at 28D.
Conclusion: Application of AIF-PA1 to SIS substitution urethroplasty altered the inflammatory microenvironment of post-surgical healing by decreasing neutrophil, CD86+ M1 pro-inflammatory macrophages, TNFα, and IL-1β levels while concurrently increasing levels of CD206+ M2 pro-regenerative/anti-inflammatory macrophages at the anastomoses and REGEN. AIF-PA1 treatment enhanced the healing process and contributed to earlier, complete urethral healing and increased angiogenesis. However, further studies are needed to elucidate the specific mechanism of inflammatory response modulation on angiogenesis and overall urethral healing. AIF-PA1 may have future applications in modulating inflammation and potentially enhancing post-surgical healing in cases of urethral reconstruction.

Table 1. Immune Cell Markers and Cytokine Levels
Tissue Region14D Timepoint
Uncoated SISSIS+AIF-PA6SIS+AIF-PA1SIS+AIF-PA1/uncoated SIS Ratiop-value*
N=6N=6N=6
Native% MPO+1.20.40.80.31.40.5-0.90
% CD68+7.00.96.00.75.60.8-0.38
% CD86+12.31.510.90.89.91.5-0.34
% CD206+5.70.45.00.77.41.0-0.22
% TNFα+9.01.96.71.16.01.1-0.25
% IL-1β+8.11.46.91.05.61.2-0.26
N=6N=6N=6
Anastomosis% MPO+25.01.632.42.414.62.90.580.01
% CD68+48.50.945.02.024.30.50.50<0.01
% CD86+59.61.156.61.726.61.30.45<0.01
% CD206+5.40.86.60.711.50.82.13<0.01
% TNFα+70.61.561.91.326.81.50.38<0.01
% IL-1β+60.91.960.31.723.22.10.38<0.01
N=3N=2N=5
REGEN% MPO+26.11.630.92.111.32.30.43<0.01
% CD68+60.43.157.76.225.71.80.43<0.01
% CD86+63.51.462.91.728.63.30.45<0.01
% CD206+6.61.03.21.112.01.21.820.02
% TNFα+26.11.630.92.111.32.30.43<0.01
% IL-1β+60.43.157.76.225.71.80.43<0.01
28D Timepoint
Uncoated SISSIS+AIF-PA6SIS+AIF-PA1SIS+AIF-PA1/uncoated SIS Ratiop-value*
N=6N=6N=6
Native% MPO+0.10.10.00.00.30.2-0.24
% CD68+2.30.51.30.20.60.20.26x<0.05
% CD86+2.20.82.10.62.50.6-0.91
% CD206+0.60.20.70.20.80.1-0.71
% TNFα+1.80.62.50.72.60.3-0.54
% IL-1β+1.30.31.80.31.20.3-0.99
N=6N=6N=6
Anastomosis% MPO+14.71.713.12.37.91.40.540.04
% CD68+48.70.950.71.619.81.50.41<0.01
% CD86+40.80.847.31.620.70.80.51<0.01
% CD206+8.91.715.61.921.62.42.43<0.01
% TNFα+53.11.054.31.022.22.20.42<0.01
% IL-1β+28.51.631.31.415.82.00.55<0.01
N=6N=6N=6
REGEN% MPO+14.51.315.31.36.80.80.47<0.01
% CD68+50.71.751.51.224.31.20.48<0.01
% CD86+41.61.453.11.420.21.20.49<0.01
% CD206+11.92.112.01.120.41.61.71<0.01
% TNFα+55.30.952.91.025.40.50.46<0.01
% IL-1β+34.42.134.73.316.20.70.47<0.01
*P-values shown for uncoated SIS vs SIS+AIF-PA1

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