Bioengineered corporal tissue for structuraland functional restoration of the penisKuo-Liang Chen1, Daniel Eberli2, James J. Yoo, and Anthony Atala3

Wake Forest Institute for Regenerative Medicine and Department of Urology, Wake Forest University Health Sciences, Winston-Salem, NC 27157

Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved September 25, 2009 (received for review August 17, 2009)

Various reconstructiveprocedureshavebeenattemptedto restoreacosmetically acceptable phallus that would allow normal reproduc-tive, sexual, and urinary function in patients requiring penile recon-struction. However, these procedures are limited by a shortage ofnative penile tissue. We previously demonstrated that a short seg-ment of the penile corporal body can be replaced using naturallyderived collagenmatriceswith autologous cells. In the current study,weexamined the feasibility ofengineering theentirependularpenilecorporal bodies in a rabbitmodel. Neocorporawere engineered fromcavernosal collagen matrices seeded with autologous cells using amultistep static/dynamic procedure, and these were implanted toreplace the excised corpora. The bioengineered corpora demon-strated structural and functional parameters similar to native tissueand male rabbits receiving the bilateral implants were able to suc-cessfully impregnate females. This study demonstrates that neocor-pora can be engineered for total pendular penile corporal bodyreplacement. This technology has considerable potential for patientsrequiring penile reconstruction.

autologous transplantation | bioengineered corpora |erectile dysfunction | penile reconstruction

Conditions such as congenital anomalies of the genitalia,penile cancer, traumatic penile injury, and some types of

vasculogenic erectile dysfunction often require extensive recon-structive procedures to correct anatomical and functional defi-ciencies of the penis (1–4). Various reconstructive procedureshave been attempted to achieve functional and cosmetic prop-erties, but these are often limited by a shortage of native peniletissue (5–8). In addition, these reconstructive procedures ofteninvolve multiple-stage surgeries, which may include the use ofsilicone penile prostheses or autograft implantation (9), butcorporal tissue function is not restored.The corpus cavernosa, a pair of cylindrical bodies that lie along

the shaft of the penis, make up the body of the penis, and areresponsible for erectile function in males. The corporal bodiesconsist of a sponge-like tissue containing sinusoid bloodfilledspaces lined by endothelium and separated by connective tissuesepta. Under normal conditions, erection is initiated by nitricoxide release from the endothelial cells, which triggers smoothmuscle relaxation and influx of blood into the corporal spaces.Due to the unique tissue structure and complex cellular functionwithin the corpora, reconstruction of functional erectile tissuehas been especially challenging.To address the challenges associated with functional resto-

ration of the corpus cavernosa, cell-based therapies in whichreplacement cavernosal tissue is bioengineered have beenproposed. Previous studies demonstrate that cells derivedfrom the corpus cavernosum are able to reconstitute functionaltissue that is structurally similar to native corpus tissue (10–13). Using this approach, short segments of erectile tissue,approximately one-third of the penile corpora, were previouslyengineered from autologous cells. These cells were seededonto corporal collagen matrices (10). The engineered tissuesegment integrated into native tissue and produced recovery ofapproximately 50% of normal corporal function in terms ofintracorporal pressures. Further recovery was not seen, and

only a limited number of smooth muscle cells could be loadedwithin the sinusoidal spaces of the neocorpora. The collagenmatrices alone, without the cells, contained fibrotic tissue andcalcifications with sparse corporal elements, and there was nofunctionality evident in terms of only scant visualization oncavernosography and a mean maximal intracavernosal pres-sures of only 8% of normal controls.In the present study, we attempted to improve upon the prior

results. Both entire pendular corporal bodies were engineeredand implanted, and a more efficient multistep cell-seedingprocedure was used that resulted in optimal cell density withinthe corporal matrices. Herein, we report the construction andimplantation of functional penile corpora, which resulted insuccessful copulation and impregnation in a rabbit model. Thisis the most complete functional replacement of erectile tissuereported to date (10–13).

ResultsIsolation and Culture of Autologous Cells for Tissue Engineering.Autologous smooth muscle cells (SMC) and endothelial cells(EC) were isolated from corporal biopsies, expanded in vitro,and seeded on the matrices using a multistep procedure (Fig.1A). The expanded cells were characterized. Almost all ofcultured EC were positive for the endothelial cell markerproteins von Willebrand factor (vWF) (Fig. 1B). SMC werecharacterized using antibodies against smooth muscle specificalpha-actin (Fig. 1B).

Production, Seeding, and Implantation of Bioengineered Corpora.Decellularized donor corpora cavernosa were used as collagenscaffolds for producing neocorporal tissue. The corpora collagenmatrices were prepared from donor rabbit phalluses using anestablished decellularization process (10). Matrices were seededwith the autologous SMC and EC using a multistep cell seedingprotocol (14). The cell-seeded matrices were used to replace theentire pendular penile corpora in 12 male New Zealand Whiterabbits. The matrices were seeded with 3.26 ± 0.23 millionEC/mL, and 60.62 ± 0.76 million SMC/mL through the staticmethod, and 117.64 ± 6.60 million SMC/mL through the dy-namic method. The total SMC seeded were 178.26 ± 6.76million/mL. Implantation of unseeded matrices served as scaf-fold alone controls (n = 12) as in our prior studies, and theseshowed no functionality with small segments (10). To producenegative controls, corporal excision without replacement wasperformed (n = 3). Finally, the corpora from the study rabbitsthemselves before surgical intervention served as normal con-

Author contributions: K.-L.C., D.E., J.J.Y., and A.A. designed research; K.-L.C. and D.E.performed research; D.E. and J.J.Y. contributed new reagents/analytic tools; K.-L.C., D.E.,and J.J.Y. analyzed data; and K.-L.C., D.E., J.J.Y., and A.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1Present address: Department of Urology, China Medical University Hospital, No. 2, Yuh-Der Road, Taichung 40447, Taiwan.

2Present address: UniversitatsSpital Zürich, Frauenklinikstrasse 10, DH-8091 Zürich, Swit-zerland.

3To whom correspondence should be addressed. E-mail: aatala@wfubmc.edu.

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trols (n = 16). At 1, 3, and 6 months after surgery, the structureand function of the grafts and themating behavior of the rabbitswere evaluated (n = 4 per time point).

Cavernosometry and Cavernosography. Cavernosometry, whichmeasures vascular pressure in the corpus cavernosum, indicatedthat all rabbits implanted with the cell-seeded neocorporadeveloped adequate intracorporal pressures (ICP). The maximalICP was 257 ± 81 cmH2O in the normal control group (n = 12).The maximal ICP was 244 ± 56, 279 ± 68, and 337 ± 36 cmH2Oin the 1-, 3-, and 6-month cell-seeded experimental groups (n =4 per time point). In contrast, the maximal ICP was 9 ± 0, 9 ±8, and 34 ± 45 cmH2O in the 1-, 3-, and 6-month unseededcontrol groups. The maximal ICP was 0.7 ± 1.2 cmH2O in thenegative control group (n = 3) (Fig. 2A). The differences in ICPmeasurements between the experimental and normal controlswere not significant. However, the measurements between theexperimental and negative controls were statistically significant(P < 0.05).Cavernosography, which demonstrates the corpora cavernosa

and draining veins after injection of contrast medium into thecorpora, indicated that the corpora in the cell-seeded exper-imental group (n = 12) filled with fluid in a homogeneousmanner similar to normal corpora (n = 16) (Fig. 2B). However,multiple filling defects were observed in the unseeded controlgroup (n = 12) and major filling gaps were noted in the animalswhere the corpora were excised (n = 3) (Fig. 2B).

Organ Bath Studies. Bioengineered neocorporal and controltissues were exposed to different pharmacologic agents and toelectrical field stimulation (EFS) over time (n = 4–7 per tissuetype per time point). The response of corporal tissues tocarbachol-induced relaxation was present at 3 months postim-plantation and continued at 6 months. The carbachol inducedrelaxation was significantly higher in the bioengineered cor-poral tissue when compared to the unseeded control group(Fig. 3A). A nitric oxide donor, sodium nitroprusside, couldinduce relaxation of the bioengineered corporal tissues as earlyas 1 month postimplantation, and began to induce relaxationin the unseeded control group at 6 months (Fig. 3B). This

indicated that the bioengineered corpora SMC could respondto nitric oxide induced relaxation as early as 1 month. Incontrast, the tissue strip samples obtained from unseededcorporal tissues remained unresponsive to sodium nitroprus-side with only a mild increase at 6 months. The bioengineeredtissue strips responded to phenylephrine administration asearly as 1 month after implantation and continued to respondin a dose dependent manner over time. The engineeredcorporal tissues exposed to phenylephrine at a concentrationof 3 × 10−5 molar demonstrated contractile forces that were47–63% of those shown by normal corporal tissues (Fig. 3C).Expected maximal contraction (Emax) was 710 mg at 1 month,1,025 mg at 3 months, and 1,301 mg at 6 months. The slopesof the concentration-response curves were 1.346, 0.602, and0.366 at 1, 3, and 6 months, respectively. The Emax and slopesof the bioengineered neocorpora improved with time. EC50(the molar concentration of a drug, which produces 50% of themaximum possible response) was 2.1 × 10−7 molar at 1 month,2.7 × 10−7 molar at 3 months, and 1.8 × 10−7 molar at 6months. In contrast, all implants in the control group re-sponded poorly to phenylephrine administration. Emax wasbetween 103 and 197 mg for control grafts at 1–6 months forunseeded control matrices.EFS-induced contractile responses were obtained on all bioengi-

neered constructs at 80 V with a stimulation frequency of 32 Hz.The bioengineered grafts had a minimal response to EFS at 1month, and a strong contractile response to EFS at 3 and 6 months.However, the control grafts without cells at all time points and theexperimental grafts at 1 month showed minimal response to EFS(Fig. 3D). All response differences measured (phenylephrine, so-dium nitroprusside, carbachol, and electrical stimulation) werestatistically significant (P < 0.05) between experimental and controlgroups at all time points postimplantation.

Fig. 1. Isolation and culture of autologous cells for tissue engineering. (A)Overall study design. (B) Culture expanded endothelial cells (Left) show pos-itive expression of cell specific marker von Willebrand factor protein (vWF),and smooth muscle cells show expression of smooth muscle specific a-actin(Right).

Fig. 2. Cavernosometry and cavernosography. (A) Cavernosometry showsthat all rabbits implanted with the bioengineered corpora after completependular penile corporal excision had sufficient intracorporal pressure (ICP) toattain erection (n = 12). The levels of ICP were comparable to native corpora(n= 12). (B) Cavernosography shows a homogenous appearance of corpora inthe bioengineered group (n = 12), similar to the native corpora (n = 16),numerous filling defects in the unseeded control group (n = 12), and majorfilling gaps in the negative control group (n = 3).

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Histological and Immunohistochemical Analyses. Tissue reorganiza-tion following implantation of bioengineered neocorpora wasobserved at 1 month. Presence of vascular structures was evidentas early as 1 month after implantation. EC, labeled with fluo-rescent membrane bound proteins (PKH26) before implanta-tion, were observed within vascular structures (Fig. 4C). At 3 and6 months, tissue from the bioengineered neocorpora appeared tobe similar to normal controls (Fig. 4 A and B). In addition, the

EC stained positively for vWF and SMC were positive for smoothmuscle alpha-actin in vivo (Fig. 4 D and E).

Mating Assessment. The experimental and control animals wereeach placed with a female rabbit and mating activities wereassessed at 1, 3, and 6 months after implantation. All rabbits withbioengineered neocorpora attempted copulation within 1 min ofintroduction to their female partners, and this occurred as early

Fig. 3. Organ bath studies. (A) The response of corporal tissues to carbachol-induced relaxation at 3months postimplantation. (B) A nitric oxide donor, sodiumnitroprusside, can induce relaxation of the bioengineered corporal tissues as early as 1 month after implantation. (C) Tissue strips obtained from thebioengineered neo-corpora responded to phenylephrine administration as early as 1month after implantation. The responses correlatedwith the concentrationof phenylephrine. (D) The bioengineered grafts at 3 and 6 months showed strong contractile responses to electrical field stimulation (EFS) at 80 V with stimulationfrequency of 32 Hertz. (P < 0.05; compared between experimental and control groups). n = 4–7 per time point. Key: Normal Cont: Normal Rabbits, Exp: Implantswith Cells, Cont: Implants without Cells, Neg Cont: Excision without implants.

A B

D EC

Fig. 4. Histological assessment of bioengineered neocorpora. Tissue reorganization following implantation of bioengineered neocorpora was observed at 1month. At 3 and 6months, tissue from the bioengineered neocorpora grafts was similar to normal controls (A and B). Presence of vascular structures was evidentas early as 1 month after implantation. Fluorescent PKH26-labeled EC within vascular structures was also observed (C). EC were positive for immunohistochemicalstain with antibodies detecting vWF and SMC were positive for stain with antibodies against smooth muscle alpha-actin in vivo (D and E).

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as 1 month after implantation. Most control rabbits did notattempt copulation after introduction to their female partners.The intravaginal ejaculation rate was determined using vaginalswabs to detect the presence of sperm after copulation and/orimpregnation. In the experimental group, vaginal swabs con-tained sperm in eight of 12 instances, and four of the 12 femaleswere impregnated, resulting in an intravaginal ejaculation rate of83% (10/12). In the control group without cell seeding, all 12vaginal swabs were negative. A difference between experimentaland control group without cells in the intravaginal ejaculationrates of 75% was noted, with a 95% confidence interval of 36%to 89%. For the negative control group (excision only), allvaginal swabs were negative, and none of the females wereimpregnated (0%).

DiscussionEngineered autologous cartilage rods have been successfullyused as penile prostheses in a rabbit model (9), but these do notrestore corporal function. In this study, we investigated thefeasibility of using autologous cells to engineer the entire lengthof both penile corpora and to restore erectile function in vivo.The entire rabbit pendular penile corpora were engineered usingautologous cells. These bioengineered corpora were functionalin terms of normal erections, adequate copulation, ejaculation,and impregnation.We had previously demonstrated that small segments, approx-

imately one third of the corporal bodies, could be replaced byinterposing autologous engineered tissue in rabbits. However,the maximal pressures within the corporal tissues (ICP) were lessthan 50% of normal, and although the endothelial cell contentwas adequate, the SMC density within the engineered segmentswas reduced (10). The use of decellularized matrices alone,without cells, led to a nonfunctional fibrotic phallus with pres-sures less than 8% of normal. Thus, in this study, we engineeredentire segments of the pendular neocorpora, using autologouscells and maximized the SMC content. A multistep dynamic/static seeding procedure was used (14). With this method, wewere able to seed 5.9 times as many SMC than our priorexperiments. Consistent with our hypothesis, increasing thedensity of SMC cells led to a maximal ICP in the experimentalcell seeded implants that were compatible with normal erectilepressures. We used 3-D cavernosal collagen matrices as scaffoldsfor corporal tissue engineering, which allowed us to engineerneocorpora with a structure nearly identical to native corpora.The 3-D corporal collagen matrices provided an excellent en-vironment for cell survival, attachment, and tissue developmentin vivo.The SMC and EC seeded on the neocorpora began to form

organized tissue with vascular structures as early as 1 month afterimplantation. Grafts retrieved at 3 and 6 months were histolog-ically similar to native tissue. The contractile responses of thesegrafts to phenylephrine in organ bath studies increased at 3months and again at 6 months after implantation. These obser-vations indicate that functional aspects of the engineered cor-pora mature gradually over time.Cavernosography measurements indicated a homogenous ap-

pearance in the bioengineered grafts; whereas, filling defects wereobserved in the negative controls. This suggests that blood is abletoflow smoothly through the engineered corporal tissues. This wasfurther confirmed by cavernosometry, which showed that all rabbitswith the bioengineered corpora developed sufficient ICP to attainerection. In addition, these findings were consistent with the resultsof the mating assessment studies, which tested the ability of thephallus to penetrate through the vaginal vault. The animals thatreceived the engineered corporal tissue were able to copulatenormally, leading to intravaginal ejaculation and/or impregnation.These results indicate that it is possible to use tissue engineered

corporal tissue in reconstructive procedures where restoration oferectile function is necessary.Further, organ bath studies showed that the bioengineered

corporal tissues had contractile responses to low concentra-tions of phenylephrine at 3 and 6 months. In addition, thebioengineered corporal tissue responded to other pharmaco-logical agents including carbachol and sodium nitroprusside,which confirmed that the endothelial and smooth muscle cellsin the grafts were functionally active. Finally, the engineeredtissue showed strong contraction in response to EFS, anindicator of innervation. These data indicate that both ade-quate cell seeding and time for tissue maturation are essentialelements for engineered corpora to become organized andfunction appropriately.We used SMC and EC from primary cultures of autologous

corporal tissues in this study. These cells are derived fromtissue that produces penile erectile function and are best-suited to recapitulate that function after being seeded onto thecollagen matrices. This study shows that the entire pendularpenile corpora can be engineered using autologous cells. Theengineered tissues are able to maintain normal intracorporalpressures and are able to contract and relax in response toelectrical field and pharmacological stimulation. We also showthat animals implanted with neocorpora in most instances areable to penetrate, ejaculate, and impregnate the female part-ners that go on to deliver healthy pups. While further studiesare required, these results are encouraging. Patients withcongenital anomalies, penile cancer, traumatic penile injury,and some types of organic erectile dysfunction could benefitfrom this technology in the future.

Materials and MethodsMatrix Preparation. Corporal tissues were obtained through two longitudinaldorsal incisions on the tunica albuginea of each donor rabbit phallus (up to 3cm in length). All animal experiments were approved by the Animal Care andUse Committee (ACUC) at the Wake Forest University School of Medicine.Decellularization (removal of cellular components) was performed by vigor-ously stirring sequentially with distilled water, 1% Triton X-100 (Sigma-Aldrich) with 0.1% ammonium hydroxide (Fisher Scientific), and PBS solutionfor a total of 33 days (10). Samples of tissue fragments were processed forhistology and stained with standard hematoxylin and eosin staining every 14days until no cellular remains were detected. The decellularized corporalscaffolds were lyophilized, sterilized with low temperature ethylene oxide,and stored in a dessicator until use.

Smooth Muscle and Endothelial Cell Culture. Corporal tissue biopsies wereharvested from experimental rabbits. Corporal tissue specimens wereminced into 1 mm2 pieces. EC were explanted in a gelatin coated dish withEndothelial Cell Basal Medium-2 (BM-2; Cambrex) supplemented withEndothelial Cell Growth Medium-2 (GM-2; Cambrex). SMC were explantedin Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) supplemented with10% FBS (Gemini) and 1% penicillin-streptomycin (Gibco). EC were char-acterized by immunohistochemical staining with antibodies against vonWillebrand factor protein (vWF) (Santa Cruz Biotechnology). SMC werecharacterized by immunohistochemical staining with antibodies againstsmooth muscle actin (DakoCytomation). SMC and EC were expanded untilsufficient cell numbers were available for seeding the acellular matrices.The EC were expanded between 3 and 7 passages, and SMC between 3 and17 passages. Cells subcultured for less than 8 passages were used in thisstudy.

Static and Dynamic Cell Seeding. A multistep static/dynamic seeding methodwas used to improve cell attachment and homogeneity on the matrices(14). On the first seeding day, approximately 60 × 106 SMC/mL wereinjected into the decellularized corporal tissue matrix at multiple sitesusing a 22-gauge needle, and then the construct was placed in culture for24 h in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin.On the second seeding day, approximately 3 × 106 EC/mL were injectedonto the corporal matrices with a 22-gauge needle, and the construct wascultured for 24 h in BM-2 supplemented with GM-2. On the third day,approximately 120 × 106 SMC/mL were additionally seeded and the cor-

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poral tissue matrix was placed in a spinner flask containing 120 mL ofDMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Theneocorpora constructs were suspended in the SMC medium with a mag-netic stir bar spinning at 40 RPM for another 48 h.

Corporal Implantation. General anesthesia was induced with intramuscularketamine (35 mg/kg) and xylazine (5 mg/kg), and then maintained withinhaled isoflurane (2%–5%). After surgical preparation, a longitudinal inci-sion was made on the dorsal surface of penis and the tunica albuginea wasexposed. Careful dissection was performed to preserve the dorsal neurovas-cular bundles. The tunica albuginea was opened longitudinally along themidline. The pendular corporal tissue size of 2.5–3 cm was removed, andreplaced with the bioengineered neocorpora (n = 12) or unseeded controlmatrices (n = 12). A negative control group (n = 3) received corporal tissueexcision without implantation. The wound was closed with interrupted 4–0and 3–0 absorbable sutures in layers.

Cavernosometry and Cavernosography. Each rabbit underwent cavernosom-etry and cavernosography before any surgical procedure was performed todetermine its own baseline values as normal control. These values werecompared with those obtained during examinations performed at 1, 3, and 6months after surgery. Two 23-gauge needles were inserted into the corporacavernosa. One served as a pathway for normal saline infusion, and the otheras a port tomeasure intracorporal pressure (ICP). ICP was recorded as erectionwas induced by infusion of normal saline at a rate of 1 mL/min as well asinjection of papaverine (5 mg), which is a vasodilator. Then, contrast mediumwas infused into the corpora cavernosa via one of the 23-gauge needles underfluoroscopy for cavernosography.

Mating Assessment. Each male rabbit was coupled with a female adult rabbitat 1, 3, and 6 months after surgery to evaluate erection of the neophallus,vaginal penetration, ejaculation, and reproduction. The rabbits were ob-served to determine the time to first mating attempt. Vaginal swabs weretaken after mating to confirm vaginal penetration and intravaginal ejacula-tion. The presence of sperm was confirmed by light microscopy after hemo-toxylin and eosin staining. Female rabbits were observed for 28–35 days toconfirm conception, gestation, and delivery.

Organ Bath Studies. Corporal tissue was harvested at 1, 3, and 6 months aftersurgery and placed in DMEM at 4 °C. Longitudinal tissue strips were attachedto a tissue support hook at one end and an isometric force transducer at theother end. The specimens were mounted in isolated baths containing Krebs’Ringer solution and a 95% oxygen/5% carbon dioxide mixture at 37 °C. SMCfunction was tested by treating the tissue with 10−10 to 3 × 10−5 molarphenylephrine, 5 × 10−6 molar sodium nitroprusside, and electrical stimula-tion at 80 V/32 Hz. EC function was tested by treatment with 5 × 10−6 molarcarbachol and 6 × 10−2 molar potassium chloride. Peak contractions wererecorded for each individual strip of tissue exposed to these stimuli andtransducer signals were transmitted to a recorder. The organ bath studyincluded 4–7 samples per time point.

Histological and Immunohistochemical Analyses. The retrieved corporal tissueswereprocessed for standardhistological analysis. Paraffin-embedded sectionswere stainedwith hemotoxylin and eosin, and immunohistochemical stainingwas performed using frozen sections. EC were characterized using antibodiesagainst vWF protein and SMC were characterized using antibodies againstSMA. Biotin/Avidin was used for immunolabeling and the peroxidase sub-strate 3,3′-diaminobenzidine (DAB) or Vector VIP substrate (Vector) were usedas chromagens. Nuclear counterstaining by Gill’s hematoxylin was used withDAB, and methyl green with Vector VIP substrate.

Statistical Analysis. Data were presented as mean ± standard deviation.One-way analysis of variance (ANOVA) with Dunnett’s post test was per-formed using GraphPad Prism version 3.00 for Windows (GraphPad Soft-ware) to compare the data from the cell-seeded (experimental) and controlgroups. Concentration-response curves were fitted with non-linear curvesusing the least squares method to calculate 50% effective concentration(EC50, the molar concentration of a drug, which produced 50% of themaximum possible response for that drug). P values <0.05 were consideredsignificant.

ACKNOWLEDGMENTS. We thank Drs. Luiz Freitas Filho and Du-Geon Moonfor technical support and Drs. Colin Bishop, Belinda Wagner, and JenniferOlson for editorial assistance with this manuscript.

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