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Adipose tissue-derived stem cell-seeded small intestinal submucosa for tunica albuginea grafting and reconstruction Limin Maa,b,1, Yijun Yanga,1, Suresh C. Sikkaa,c, Philip J. Kadowitzc, Louis J. Ignarrod, Asim B. Abdel-Mageeda,c,2, and Wayne J. G. Hellstroma,2,3 Departments of aUrology and cPharmacology, Tulane University Health Sciences Center, New Orleans, LA 70112; bDepartment of Urology, Ninth People’s Hospital Affiliated with Medical College of Shanghai, Jiaotong University, Shanghai 200011, China; and dDepartment of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles Center for the Health Sciences, Los Angeles, CA 90095 Edited by Solomon H. Snyder, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved December 13, 2011 (received for review August 29, 2011) Porcine small intestinal submucosa (SIS) has been widely used in cell transplantation has been demonstrated in vascular (6) and car- tunica albuginea (TA) reconstructive surgery. Adipose tissue-derived tilage reconstruction (7) and in restoring immune response and stem cells (ADSCs) can repair damaged tissue, augment cellular hematopoiesis (8). In vivo scaffold-based studies further expanded differentiation, and stimulate release of multiple growth factors. the use of MSCs in new bone formation (9). The aim of this rat study was to assess the feasibility of seeding With the development of tissue engineering, cell-seeded acellu- ADSCs onto SIS grafts for TA reconstruction. Here, we demonstrate lar matrix grafts can theoretically provide off-the-shelf recon- that seeding syngeneic ADSCs onto SIS grafts (SIS-ADSC) resulted in structive materials. Although it has been demonstrated for tissue significant cavernosal tissue preservation and maintained erectile reconstruction in other clinical settings, there are no studies in- fi responses, similar to controls, in a rat model of bilateral incision of vestigating the therapeutic ef cacy of such materials in procedures TA, compared with sham-operated animals and rats grafted with SIS involving surgical intervention of the TA. SIS has been widely used graft (SIS) alone. In addition to increased TGF-β1 and FGF-2 expres- in the reconstruction of various tissues and organs (10). As with sion levels, cross-sectional studies of the rat penis with SIS and SIS- MSCs, ADSCs have the potential to restore damaged tissue and MEDICAL SCIENCES ADSC revealed mild to moderate fibrosis and an increase of 30% prevent some of the above-mentioned postoperative problems. The aim of this study was to assess the feasibility of the use of ADSC- and 40% in mean diameter in flaccid and erectile states, respec- seeded SIS grafts for reconstruction of bilaterally incised TA in a rat tively. SIS grafting induced transcriptional up-regulation of iNOS model. The hypothesis that ADSC seeding onto SIS will maintain and down-regulation of endothelial NOS, neuronal NOS, and VEGF, erectile function was also examined. an effect that was restored by seeding ADCSs on the SIS graft. Taken together, these data show that rats undergoing TA incision Results with autologous SIS-ADSC grafts maintained better erectile function ADSCs Culture, Characterization, and BrdU Labeling. The ADSC compared with animals grafted with SIS alone. This study suggests isolates attached and proliferated within 2–3 d and were pas- that SIS-ADSC grafting can be successfully used for TA reconstruc- saged when they reached 80% confluence. ADSCs became fu- tion procedures and can restore erectile function. siform and exhibited spindle-shaped morphologies (Fig. 1A). Flow cytometric analysis demonstrated that ADSCs (passage 3– Peyronie’s disease | incision and grafting procedures | penile surgery 5) were positive for CD29 (97.62%), CD90 (92.21%), and CD105 (15.87%), but negative for CD45 (4.37%) expression he veno-occlusive mechanism responsible for erection in- (Fig. 1B). To assess engraftment of ADCSs onto SIS, cells were Ttimately depends on the extensibility, rigidity, and compli- efficiently (>95%) labeled with antibromodeoxyuridine (BrdU; ance of the tunica albuginea (TA) (1). Any morphological or Sigma) antibody in vitro (Fig. 1C). functional impairment or defect in the TA can affect compliance of the penile fibro-elastic framework and have profound effects Seeding of ADSCs onto the SIS Grafts. A four-layer porcine SIS was on the hemodynamic and erectile function of the penis (2). TA provided as a soft tissue graft (Surgisis ES) from Cook Medical reconstruction is often used in Peyronie’s disease (PD) with large (Bloomington, IN). Based on histological examination, SIS is an plaques, severe deformities, or short penises that require incision acellular nondirected fibrous material (Fig. 2A). The ADSC- or excision for placement of grafts to augment or lengthen the seeded SIS grafts were cultured in an air fluid media for 1 wk. penis (3). There are myriad different autologous graft tissues Random H&E staining revealed that ADSCs grew well, attached harvested from the patient’s body, such as dermis, temporalis onto the SIS, exhibited a similar cellular morphology, and fascia, tunica vaginalis, penile skin, and saphenous vein, as well formed multilayer epithelium on the SIS scaffolds (Fig. 2B). as cadaveric tissues, such as dermis, fascia, pericardium, and porcine small intestine submucosa. A number of synthetic Measurement of Erectile Responses. The SIS and SIS-ADSC were C–F materials such as Gore-Tex, Dacron, and silastic mesh have been surgically implanted into the rat penis (Fig. 2 ). Erectile used for grafting procedures (4). Erectile dysfunction (ED), re- currence of penile curvature, and penile shortening are recog- nized complications from such grafting procedures (4). Author contributions: L.M., Y.Y., A.B.A.-M., and W.J.G.H. designed research; L.M. and Y.Y. Mesenchymal stem cells (MSCs) are a well-characterized pop- performed research; S.C.S., P.J.K., and L.J.I. analyzed data; and S.C.S., P.J.K., A.B.A.-M., and ulation of adult stem cells capable of self-renewal. Based on their W.J.G.H. wrote the paper. fl plasticity, several lines of evidence demonstrate that MSCs have The authors declare no con ict of interest. the potential to engraft and differentiate into multiple lineages This article is a PNAS Direct Submission. both in vitro and in vivo (5). The growing interest in the use of MSCs 1L.M. and Y.Y. contributed equally to this work. in regenerative medicine and tissue reconstruction is explained, in 2A.B.A.-M. and W.J.G.H. contributed equally to this work. part, by the relative ease of their isolation from several adult human 3To whom correspondence should be addressed. E-mail: [email protected]. tissues, propagation in culture, and their differentiation capacity. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Using tissue-engineering techniques, the clinical utility of stem 1073/pnas.1113810109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1113810109 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 A C Bright Field BrdU Merged 100˩m 100˩m CD29 B CD45 CD90 CD105 Fig. 1. Identification and BrdU labeling of ADSCs. (A) Methyl violet staining of ADSCs. (Scale bar: 100 μm.) (B) Flow cyto- metric analysis of early passage rat ADSCs with positive ex- pression for CD29 (97.62%), CD90 (92.21%), and CD105 (15.87%) and low expression of CD45 (4.37%). (C) Immuno- fluorescence 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%. responses were examined 8 wk after surgical intervention. difference in all groups in mean penile length under either Fig. 3A shows maximum increase in intracavernosal pressure flaccid or erectile conditions (Fig. 4B). (ICP), ICP/mean arterial pressure (MAP), and total ICP values In the BrdU immunostained frozen penile sections grafted in response to cavernous nerve stimulation (CNS) for 1 min in SIS-ADSC, a weak signal on the surface of the graft and slight control groups in a voltage-dependent manner. Conversely, sporadic reactivity on the corpus cavernosum were detected (Fig. fi erectile responses in rats with the SIS were signi cantly lower 4B). Masson’s trichrome staining of the TA of control (Fig. 4C) (P < 0.05) (Fig. 3 B–D). Similar to controls, seeding ADSCs on D fi and sham-operated rats (Fig. 4 ) revealed numerous collagen the SIS grafts resulted in a signi cant restoration of the erectile bundles oriented in two directions with an abundance of elastic function compared with SIS group (P < 0.05) (Fig. 3 B–D). fibers. In the SIS group (Fig. 4E) there was moderate fibrosis under the graft and a mild foreign-body reaction around the Penile Measurements and Histological Assessment. We measured F fi the diameter and length of the rat penis in the four surgical suture. In the SIS-ADSC group (Fig. 4 ), only mild brosis was groups to illustrate the properties of the SIS. Compared with the observed around the graft, and the elastic fibers of the graft were sham group, rats with SIS-ADSC and SIS revealed 40% and 30% orientated in two layers, similar to the adjacent TA. The assessed increase in mean diameter under flaccid and erectile states, re- penile fibrosis score of the SIS group was significantly greater spectively (P < 0.001) (Fig. 4A). We also observed no significant compared with the other three groups (Fig. 4G). Fig. 2. SIS-ADSC grafts and reconstruction surgery of TA. (A) Four-layer SIS showed non- directed fibers, which were completely acel- lular. (B) H&E staining demonstrated that ADSCs engrafted and formed multiple layers on the SIS scaffolds. (C) Rat penis middle sec- tion was chosen as the surgery site. Rats un- derwent first stage surgery, which included a 5-mm incision (arrows) on both sides of the TA.

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