Fiber density of collagen grafts impacts rabbit urethral regeneration

Abstract

There is a need for efficient and "off-the-shelf" grafts in urethral reconstructive surgery. Currently available surgical techniques require harvesting of grafts from autologous sites, with increased risk of surgical complications and added patient discomfort. Therefore, a cost-effective and cell-free graft with adequate regenerative potential has a great chance to be translated into clinical practice. Tubular cell-free collagen grafts were prepared by varying the collagen density and fiber distribution, thereby creating a polarized low fiber density collagen graft (LD-graft). A uniform, high fiber density collagen graft (HD-graft) was engineered as a control. These two grafts were implanted to bridge a 2 cm long iatrogenic urethral defect in a rabbit model. Histology revealed that rabbits implanted with the LD-graft had a better smooth muscle regeneration compared to the HD-graft. The overall functional outcome assessed by contrast voiding cystourethrography showed patency of the urethra in 90% for the LD-graft and in 66.6% for the HD-graft. Functional regeneration of the rabbit implanted with the LD-graft could further be demonstrated by successful mating, resulting in healthy offspring. In conclusion, cell-free low-density polarized collagen grafts show better urethral regeneration than high-density collagen grafts.

Figure 1

Manufacturing technique of the low-density graft (LD-graft) and high-density graft (HD-graft) (A) Two methods were utilized to manufacture the density controlled collagen grafts by monitoring the water content removed either by rolling compression or air-drying. The polarized collagen fiber distribution characteristics of the LD-graft were achieved by utilizing both a rolling compression followed by air-drying. The uniform collagen fiber distribution characteristics of the HD-graft were achieved by only air-drying the collagen. (B) By weighing and monitoring the collagen gel during production, we could achieve our polarized (compressed and air-dried: LD-graft) and uniform (only air-dried: HD-graft) collagen fiber distribution characteristics. Note: Images in Fig. 1A are Sirius red stained paraffin sections with scale bars of 200 µm.

Figure 2

Description and characterization of manufactured collagen graft with controlled spatial orientation. (A and B) SEM images of LD- and HD-grafts with three different magnifications.

Figure 3

In vivo evaluation of high-density (HD) and low-density (LD) collagen grafts in a rabbit urethral defect model. (A) Functional surgical outcome analyzed by micturating cysto-urethrography of rabbit implanted with LD- and HD-grafts for 1, 3 and 6 months. (B) H&E and MT stained sections of the grafted area of HD-graft 1, 3 and 6 months after implantation. (C) H&E and MT stained sections of the grafted area of low-density graft 1, 3 and 6 months after implantation. (D) Quantification of SMA expression in grafted areas for rabbits implanted with LD- and HD-graft after 1, 3, and 6 months. (E) Immunohistochemistry for compared to the HD-grafts-SMA of LD-graft implanted for 1, 3 and 6 months. (F) Immunohistochemistry for SMA of HD-graft implanted for 1, 3 and 6 months. (G) Immunohistochemistry for Uroplakin-2 (Up2) of LD-graft implanted for 1, 3 and 6 months. (H) Immunohistochemistry for Up2 of HD-graft implanted for 1, 3 and 6 months. Note: areas circled in dashed black line indicating the remaining collagen pieces of the grafts after 1 month (Scale bar H&E and MT 250 µm, Scale bar SMA and Up-2 50 µm). (Error bars represent the standard deviation of four independent samples. **p < 0.01, Student t-test).

Figure 4

Long-term in vivo evaluation of low-density collagen grafts in a rabbit urethral defect model. (A) Functional surgical outcome analyzed by micturating cysto-urethrography of rabbits with an artificially created, circumferential urethral defect that was bridged with a LD-graft (N = 5 rabbits for 9 months, N = 2 rabbits for 11 months). (B,C,E,F, and H,I) H&E and MT stained sections of LD-graft implanted for 9 and 11 months, and a control native rabbit urethra. (D,G and J) Immunohistochemistry for α-SMA of LD-graft implanted for 9 and 11 months, and a control native rabbit urethra. (K) Quantification of SMA expression in LD-graft compared to a control native rabbit urethra. (L) A photo of rabbit offspring from fathers implanted with a LD-graft. Note: green arrow pointing at offspring (Scale bar H&E and MT 250 µm, Scale bar SMA 50 µm). Error bars represent the standard deviation of four independent samples.