Coadministration of platelet-derived growth factor-BB and vascular endothelial growth factor with bladder acellular matrix enhances smooth muscle regeneration and vascularization for bladder augmentation in a rabbit model

Abstract

Tissue-engineering techniques have brought a great hope for bladder repair and reconstruction. The crucial requirements of a tissue-engineered bladder are bladder smooth muscle regeneration and vascularization. In this study, partial rabbit bladder (4×5 cm) was removed and replaced with a porcine bladder acellular matrix (BAM) that was equal in size. BAM was incorporated with platelet-derived growth factor-BB (PDGF-BB) and vascular endothelial growth factor (VEGF) in the experimental group while with no bioactive factors in the control group. The bladder tissue strip contractility in the experimental rabbits was better than that in the control ones postoperation. Histological evaluation revealed that smooth muscle regeneration and vascularization in the experimental group were significantly improved compared with those in the control group (p<0.05), while multilayered urothelium was formed in both groups. Muscle strip contractility of neobladder in the experimental group exhibited significantly better than that in the control (p<0.05) assessed with electrical field stimulation and carbachol interference. The activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 in the native bladder tissue around tissue-engineered neobladder in the experimental group was significantly higher than that in the control (p<0.05). This work suggests that smooth muscle regeneration and vascularization in tissue-engineered neobladder and recovery of bladder function could be enhanced by PDGF-BB and VEGF incorporated within BAM, which promoted the upregulation of the activity of MMP-2 and MMP-9 of native bladder tissue around the tissue-engineered neobladder.

FIG. 1.

The difference of macroscopic findings of tissue engineering neobladder at 6 months after operation between the control group (A, B) and the experimental group (C, D): obvious scar formation and graft shrinkage could be found through the observation of serosal surface in the control group (A). Meanwhile, stone formation could be found on the mucosal surface after opening the regenerated bladder (B). However, the regenerated zone was difficult to distinguish on the serosal surface in the experimental group (C). Scar formation and graft shrinkage were unconspicuous through the observation of mucosal surface. Furthermore, no stone formation can be found (D). Color images available online at www.liebertpub.com/tea

FIG. 2.

Bladder capacity (A) and bladder compliance (B) were evaluated at preoperation time and at 1, 2, 3, and 6 months after surgery in both the control and experimental groups. Bladder capacity and compliance at preoperation time were considered as 100% (*p<0.05).

FIG. 3.

Bladder tissue retrieved at 6 months after surgery in both the experimental and control groups was dissected to muscle strip for electrical field stimulation and carbachol interference. Retrieved bladder tissue was divided into the marginal zone and the central zone for examination, respectively. Normal bladder tissue was retrieved for examination as normal control.

FIG. 4.

Tissue-engineered neobladder urothelium formation was observed by hematoxylin and eosin (HE) staining (100×). Native rabbit bladder (A), central zone (B), and marginal zone (C) of neobladder in the experimental group and the central zone (D), and the marginal zone (E) of neobladder in the control group at 1 month after surgery (scale bars=100 μm). Color images available online at www.liebertpub.com/tea

FIG. 5.

HE staining of marginal zone of the neobladder was performed to observe neovascularity (100×). The functional vessels could be evidenced in the neobladder (scale bars=100 μm). Neobladder at 1 [I(A)], 2 [I(B)], 3 [I(C)], and 6 [I(D)] months in the control group and at 1 [I(E)], 2 [I(F)], 3 [I(G)], and 6 [I(H)] months in the experimental group. Semiquantitative analysis indicated that both the microvessel area [II(A)] and microvessel density [II(B)] in the experimental rabbit neobladder were higher than those in the control group at each time points after surgery (*p<0.05). Color images available online at www.liebertpub.com/tea

FIG. 6.

Immunohistochemical staining of marginal zone of the neobladder with antibody against PCNA in control (A–D) and experimental (E–H) groups at 1 [I (A, E)], 2 [I (B, F)], 3 [I (C, G)], and 6 [I (D, H)] months after surgery. Sections were counterstained with hematoxylin (400×, scale bars=100 μm). Semiquantitative analysis indicated that both the proliferative cell count (PCC) [II(A)] and ratio of proliferative cells (RPC) [II(B)] in the experimental rabbit neobladder were higher than those in the control group at 1 and 3 months after surgery (*p<0.05). At 6 months postoperation, PCC and RPC in the two groups were similar and were also similar with those in the native bladder tissue. PCNA, proliferating cell nuclear antigen. Color images available online at www.liebertpub.com/tea

FIG. 7.

Immunohistochemical staining of the central zone of the neobladder with antibody against PCNA in the control (A–D) and experimental (E–H) groups at 1 [I (A, E)], 2 [I (B, F)], 3 [I (C, G)], and 6 [I (D, H)] months after surgery. Sections were counterstained with hematoxylin (400×, scale bars=100 μm). Semiquantitative analysis indicated that both the PCC [II(A)] and RPC [II(B)] in the experimental rabbit neobladder were higher than those in the control group at 1, 2, and 3 months after surgery (*p<0.05). At 6 months postoperation, PCC and RPC in the two groups were similar and were also similar with those in the native bladder tissue. Color images available online at www.liebertpub.com/tea

FIG. 8.

Immunohistochemical staining of central zone (I) and marginal zone (II) of the neobladder with antibody against SMA in control (A–D) and experimental (E–H) groups at 1 (A, E), 2 (B, F), 3 (C, G), and 6 (D, H) months after surgery (200×, scale bars=100 μm). Semiquantitative analysis indicated that the area of organized smooth muscle both of the central zone [III(A)] and the marginal zone [III(B)] in the experimental rabbit neobladder was higher than those in the control group at each time points after surgery (*p<0.05). SMA, smooth muscle actin. Color images available online at www.liebertpub.com/tea

FIG. 9.

Immunofluorescent double staining of central zone of the neobladder with an antibody against SMA and PCNA in the experimental group at 3 months after surgery (400×, scale bars=100 μm). (A) SMA + Alexa Fluor^® 633, (B) PCNA + Alexa Fluor488, (C) DAPI, and (D) SMA + Alexa Fluor 633 + PCNA + Alexa Fluor 488 + DAPI. DAPI, 4′-6-diamidino-2-phenylindole. Color images available online at www.liebertpub.com/tea

FIG. 10.

Gelatinolytic assay was performed to detect the activity of MMP-2 and MMP-9 of the native bladder tissue around tissue-engineered neobladder. (I) Electrophoresis strip of gelatinolytic assay. N: native bladder tissue of the rabbit preoperation, C: control rabbit, E: experimental rabbit. The strip of MMP-2 and MMP-9 could be found at 62 and 84 kDa. Semiquantitative analysis indicated that the activity of MMP-2 [II(A)] and MMP-9 [II(B)] in the experimental rabbit neobladder was higher than those in the control group at 1 and 2 months after surgery (*p<0.05). The activity of MMP-2 and MMP-9 was declined over time [II(A, B)]. The activity of MMP-2 and MMP-9 in native bladder tissue of the rabbit preoperation was considered as 100%. MMP-2, matrix metalloproteinase-2; MMP-9, matrix metalloproteinase-9.