Adipose tissue-derived stem cell-seeded small intestinal submucosa for tunica albuginea grafting and reconstruction

Abstract

Porcine small intestinal submucosa (SIS) has been widely used in tunica albuginea (TA) reconstructive surgery. Adipose tissue-derived stem cells (ADSCs) can repair damaged tissue, augment cellular differentiation, and stimulate release of multiple growth factors. The aim of this rat study was to assess the feasibility of seeding ADSCs onto SIS grafts for TA reconstruction. Here, we demonstrate that seeding syngeneic ADSCs onto SIS grafts (SIS-ADSC) resulted in significant cavernosal tissue preservation and maintained erectile responses, similar to controls, in a rat model of bilateral incision of TA, compared with sham-operated animals and rats grafted with SIS graft (SIS) alone. In addition to increased TGF-β1 and FGF-2 expression levels, cross-sectional studies of the rat penis with SIS and SIS-ADSC revealed mild to moderate fibrosis and an increase of 30% and 40% in mean diameter in flaccid and erectile states, respectively. SIS grafting induced transcriptional up-regulation of iNOS and down-regulation of endothelial NOS, neuronal NOS, and VEGF, an effect that was restored by seeding ADCSs on the SIS graft. Taken together, these data show that rats undergoing TA incision with autologous SIS-ADSC grafts maintained better erectile function compared with animals grafted with SIS alone. This study suggests that SIS-ADSC grafting can be successfully used for TA reconstruction procedures and can restore erectile function.

Fig. 1.

Identification and BrdU labeling of ADSCs. (A) Methyl violet staining of ADSCs. (Scale bar: 100 μm.) (B) Flow cytometric analysis of early passage rat ADSCs with positive expression for CD29 (97.62%), CD90 (92.21%), and CD105 (15.87%) and low expression of CD45 (4.37%). (C) Immunofluorescence to calculate the labeling index (positive rate) of ADSCs incubated with BrdU at 20 μmol/L for 48 h. The nuclei of ADSCs showed green fluorescence, and the labeling rate exceeded 95%.

Fig. 2.

SIS-ADSC grafts and reconstruction surgery of TA. (A) Four-layer SIS showed nondirected fibers, which were completely acellular. (B) H&E staining demonstrated that ADSCs engrafted and formed multiple layers on the SIS scaffolds. (C) Rat penis middle section was chosen as the surgery site. Rats underwent first stage surgery, which included a 5-mm incision (arrows) on both sides of the TA. (D and E) A 10 mm² SIS and stem cell-seeded SIS (arrow) was interpositioned and sutured with 8-0 nylon for the third or fourth groups. (F) All excisions for tunica grafting were performed laterally to protect the dorsal cavernous nerves from injuries.

Fig. 3.

Evaluation of erectile function response to cavernosal nerve electrostimulation. (A) Representative ICP (blue curve) and MAP (red curve) tracing after CNS as described in Materials and Methods. Total erectile response (ICP) was determined by measuring the AUC in millimeter of mercury per second from the beginning of CNS until ICP returned to baseline or prestimulation pressure. The ratio of maximal ICP-to-MAP was determined relative to controls for variations in systemic blood pressure. (B–D) Maximal increase in ICP, ICP/MAP, and total ICP values in response to CNS with different voltage settings. n = 8 individual experiments per condition; Mean ± SE are reported; *P < 0.05; **P < 0.01; and ***P < 0.001.

Fig. 4.

Morphology and histological assessment of penile tissue cross-sections. (A) Compared with the sham group, rats with SIS graft with or without ADSCs seeding revealed ≈38.2–41.0% and 29.5–30% increases in mean penile diameter under flaccid and rigid conditions, respectively. The penile length was not affected by grafting (B). (C) Fluorescent signal from the BrdU-labeled ADSCs (arrows) can be detected on the surface of the graft and in the corpus cavernosum. Penile fibrosis score of SIS graft was significantly high compared with other experimental groups (D). Masson's trichrome staining (40×) in the TA of control (E) and sham-operated rats (F) after 8 wk showed numerous collagen bundles orientated in two directions (inner circular and outer longitudinal) with an abundance of elastic fibers. (G) Moderate fibrosis under the graft and a mild foreign-body reaction around the suture in the SIS group. In the SIS-ADSC group (H), only mild fibrosis was present around the graft, and the elastic fibers of the graft were orientated in two layers, similar to the adjacent tunica albuginea. Orange arrows indicate SIS graft, curved yellow arrows show the suture, solid black arrows depict the normal TA, and lined black arrows denote fibrosis. n = 8 per condition, Mean ± SE are reported. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5.

Immunofluorescence staining of NOS family in the corpus cavernosum of control, sham-operated, SIS, and SIS-ADSC groups. Positive immunostaining was observed in the corpus cavernosum (A, B, and D) and dorsal nerves (C) of the rat penis. Staining of eNOS and nNOS showed diffuse but marked staining in the corpus cavernosum of the control, sham operated, and SIS-ADSC groups, whereas weak staining was detected in the SIS group. Weak nNOS immunoreactivity was detected in the dorsal nerves of the SIS rats compared with the other groups. However, intense immunostaining of iNOS was noticed in corpus cavernosum of the surgical groups, with highest levels detected in the SIS group, compared with controls. Tissue sections processed without primary antibodies served as negative controls.

Fig. 6.

qRT-PCR and immunoblot analysis of NOS isoforms and erectile function related factors. Compared with both the control and sham groups, mRNA levels of the eNOS (A), nNOS (B) and VEGF (D) decreased significantly in the SIS group. However, their expression was restored in the SIS-ADSC group. iNOS (C) and TGF-β1 (E) transcripts increased significantly in the SIS rats, whereas seeding ADSCs on SIS grafts down-regulated their expression. (F) FGF-2 transcripts significantly elevated in the SIS and SIS-ADSC groups in comparison with the controls. (G and H) A representative blot of the respective proteins depicting decreased levels of eNOS and nNOS proteins and an increase in iNOS protein expression in the SIS graft group in comparison with other groups. (I and J) TGFβ1 and FGF2 protein levels were significantly increased whereas VEGF protein expression significantly decreased in the SIS and SIS-ADSC groups in comparison with controls. All experiments were repeated three times. n = 8 per condition; Mean ± SE are reported; *P < 0.05, **P < 0.01, and ***P < 0.001.