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AUTOLOGOUS ENGINEERED CARTILAGE RODS FOR PENILE RECONSTRUCTION
0022-5347/99/1623-1119/0
Vol. 162, 1119-1121, September 1999 Printed In U.S.A.
THEJOURNAL OF UROLOGY Copyright 0 1999 by AMERICAN UROLOCICAL ASSOCIATION,INC.
AUTOLOGOUS ENGINEERED CARTILAGE RODS FOR PENILE RECONSTRUCTION JAMES J . YOO, HEUNG JAE PARK, ILWOO LEE
AND
ANTHONY ATALA"
From the Laboratory for Tissue Engineering and Cellular Therapeutics, Department of Urology, Children's Hospital and Harvard Medical School, Boston, Massachusetts
ABSTRACT
Purpose: Conditions such as inadequate and ambiguous genitalia that are caused by rudimentary penis, severe hypospadias or traumatic injury require surgical intervention. Although silicone penile prostheses are an accepted treatment modality, biocompatibility issues may be a problem in select cases. We previously demonstrated that rods composed of cartilage could be created using chondrocytes seeded on biodegradable polymer scaffolds. We showed that the cartilage rods engineered ex situ were readily elastic and withstood high degrees of pressure. We investigated the feasibility of applying the engineered cartilage rods in situ in an animal model. Materials and Methods: Autologous chondrocytes harvested from rabbit ears were grown and expanded in culture. Cells were seeded onto biodegradable poly-L-lactic acid coated polyglycolic acid polymer rods a t a concentration of 50 X lo6 chondrocytes per ~ m .A~total . of 18 chondrocyte polymer scaffolds were implanted into the corporal spaces in 10 rabbits. As controls, 1 corpus in each of 2 rabbits was not implanted. The animals were sacrificed 1, 2, 3 or 6 months after implantation. Histological analysis was performed using hematoxylin and eosin, aldehyde fuschin-alcian blue and toluidine blue staining. Results: All animals tolerated the implants for the duration of the study without any complications. Gross examination after retrieval at 1month showed well formed, milky white cartilage structures within the corpora. All polymers were fully degraded by 2 months. There w a s no evidence of erosion or infection a t any of the implant sites. Histological analysis using alcian blue and toluidine blue staining revealed mature and well formed chondrocytes in the retrieved implants. Conclusions: Autologous chondrocytes seeded on preformed biodegradable polymer structures form cartilage structures within the rabbit corpus cavernosum. This technology appears to be useful for creating autologous penile prostheses. KEYWORDS: penis, prostheses and implants, chondrocytes, cartilage
Reconstructing abnormal male genitalia due to ambiguous genitalia, micropenis or traumatic injury remains a surgical challenge. Achieving functionally and esthetically acceptable genitalia is the prime goal of phalloplasty. Earlier attempts at penile reconstruction involved multiple stages of surgery using autogenous rib cartilage as a stiffener but unsatisfactory functional and cosmetic results due to curvature discouraged its Silicone prostheses have been widely used due t o their unique mechanical proper tie^.^. However, biocompatibility issues may be a problem in select patient^.^.^ A natural prosthesis of autologous chondrocytes may be advantageous due to native biocompatibility. We previously created rods of heterologous cartilage using chondrocytes seeded on biodegradable polymer scaffolds.' We showed that cartilage rods engineered ex situ are readily elastic and withstand high degrees of pressure. In the current study we investigated the feasibility of applying the engineered cartilage rods in situ in the corpora in an animal model (fig. 1). MATERIALS AND METHODS
Polymers. Unwoven sheets of polyglycolic acid polymers with a density of 58 mg./cc were configured into cylindrical rods 2 cm. long and 0.3 cm. in diameter as cell delivery vehicles. The polymer meshes were composed of fibers 15 pm. in diameter with a n interfiber distance of 0 to 200 pm. and
with 95% porosity. The scaffold was designed to degrade via hydrolysis in 6 to 8 weeks. The resulting flexible scaffold was coated with a liquefied copolymer (poly-DL-lactide-coglycolide 50:50, 80 mg./ml. methylene chloride) to achieve adequate mechanical characteristics. The polymers were sterilized in ethylene oxide and placed under sterile conditions until cell delivery. Cell culture and cell seeding. Autologous chondrocytes harvested from rabbit ears were dissected into 2 x 2 mm. fragments. Chondrocytes were harvested under sterile conditions using a previously described t e ~ h n i q u e .lo ~ .Briefly the dissected cartilage fragments were digested in 3% collagenase type I1 solution for 6 to 8 hours. Recovered cells were washed with phosphate buffered saline and plated in culture dishes. The isolated cells were grown in culture in Hamms F-12 media containing 10% fetal calf serum, 5 pg./ml. ascorbic acid, 100 pg./ml. streptomycin and 100 units per ml. penicillin. Cells were incubated at 37C in the presence of 5%carbon dioxide. Chondrocytes were expanded until sufficient cell quantities were available. The cells were trypsinized, collected, washed and counted for seeding. Chondrocytes were seeded onto preformed poly-L-lactic acid coated polyglycolic acid polymer rods a t a concentration of 50 x lo6 chondrocytes per ~ m .The ~ . cell polymer rods were implanted immediately after seeding. Experimental study. A total of 18 chondrocyte polymer scaffolds were implanted in the corporal space in 10 rabbits.
* Requests for reprints: Children's Hospital and Harvard Medical School, 300 Longwood Ave., Boston, Massachusetts 02115. 1119
1120
AUTOLOGOUS ENGINEERED CARTILAGE RODS FOR PENILE RECONSTRUCTION
FIG.1. Autologous chondrocytes were isolated from rabbit ears, expanded and seeded onto preformed polymer rods. Cell polymer scaffolds were implanted intracorporally. Specimens were retrieved for analysis 1, 2, 3 or 6 months after implantation.
FIG.2. Retrieved specimens 1 month after implantation. A, genitalia of control rabbit shows cartilage formation in corpus cavernosum, while control corpus cavernosum appears normal. B , cartilage rod.
Bilateral intracorporal cell polymer scaffolds were implanted in 8 rabbits, while the remaining 2 underwent unilateral implantation, leaving the other corpus cavernosum intact as controls. Three animals were sacrificed 1and 2 months, and 2 were sacrificed 3 and 6 months, respectively, after implantation. The 2 control animals were sacrificed at 1 and 6 months, respectively. The implants were retrieved and analyzed grossly and histologically. Sections (5 p.) of formalin fixed, paraffin embedded tissues were cut and stained with hematoxylin and eosin, aldehyde fuschin-alcian blue and toluidine blue. RESULTS
All animals tolerated the implants for the duration of the study without any noticeable complications. Gross examination after retrieval at 1 month revealed well formed, milky white cartilage structures within the corpora (fig. 2). The retrieved cartilage rod structures remained approximately
the same size as the initial implants within the corporal bodies. The corpora without implantation had normal cavernous tissue. There was no evidence of erosion, inflammation or infection a t any of the implanted cartilage rod sites. Histological analysis with hematoxylin and eosin showed mature and well formed cartilage in all chondrocyte-polymer implants (fig. 3). Polymer fibers were progressively replaced by cartilage with time at 1, 2, 4 and 6 months, respectively. All polymers were fully degraded by 2 months. Aldehyde fuschin-alcian blue and toluidine blue staining demonstrated highly sulfated mucopolysaccharides, which are differentiated products of chondrocytes (fig. 4). DISCUSS I 0N
The management of inadequate and ambiguous genitalia due to rudimentary penis, severe hypospadias or traumatic injury involves phallic reconstruction.'. " Prostheses may be required for genital reconstruction. Natural penile prostheses created from patient cells may be preferable and they may decrease the biocompatibility risks associated with artificial prostheses. Previous studies at our laboratory demonstrated that cartilage rods may be created using chondrocytes seeded on preformed polymer scaffolds.' Stress relaxation studies to measure biomechanical properties were performed on the retrieved tissue engineered structures. Compression, tension and bending studies showed that the cartilage rods are readily elastic and withstand high degrees of pressure. The present study revealed that engineered cartilage tissue composed of autologous chondrocytes seeded on biodegradable polymers may be implanted into corporal bodies in situ.
FIG. 3. Retrieved cartilage rod specimens 3 months after implantation. A, cartilage tissue has formed within each corpora (C). H & E, reduced from X25. B , mature chondrocytes enclosed within lacunae are adjacent to tunica albuginea (2'). H & E, reduced from x250.
AUTOLOGOUS ENGINEERED CARTILAGE RODS FOR PENILE RECONSTRUCTION
1121
FIG.4. Highly sulfated mucopolysaccharides were detected 6 months after implantation. A, toluidine blue stain, reduced from x 250. B, aldehyde fuchsin-alcian blue stain, reduced from x250.
The architecture of the flexible polymer scaffolds was strengthened by the addition of a liquefied copolymer, polyDL-lactide-co-glycolide, 50:50. The engineered cell polymer scaffolds were snugly implanted within the corpus cavernosum without any technical difficulties. The polymer scaffolds in our study were designed to degrade in 6 to 8 weeks. Seeded cells readily formed mature cartilage tissue in vivo, replacing the degrading polymer fibers during this period. The engineered cartilage remained at the site of initial implantation without any evidence of infection, inflammation or erosion. Histological analysis of the retrieved rods revealed adequate formation of mature cartilage, as evidenced by chondrocytes within lacunae and highly sulfated mucopolysaccharides. Engineered cartilage prostheses may be applicable in the future in patients undergoing penile surgery for congenital or acquired conditions. Autologous cartilage tissue composed of patient cells would be used without corporal tissue for penile reconstruction or intracorporally for erectile dysfunction. However, to engineer cartilage penile rods for clinical use further studies must be performed, including the establishment of functional and biomechanical parameters of the prostheses in vivo as well as use of the rods extracorporally. Studies involving these issues are currently being performed in our laboratory. In conclusion we demonstrated that autologous chondrocytes seeded on preformed biodegradable polymer structures form cartilage rods within the rabbit corpus cavernosum. "his technology may be useful in the future for creating autologous penile prostheses.
REFERENCES
1. Goodwin, W. E. and Scott, W. W.: Phalloplasty. J. Urol., 68 903, 1952. 2. Frumpkin, A. P.: Reconstruction of male genitalia. Amer. Rev. Sov. Med., 2 14,1944. 3. Beheri, G. E.: The problem of impotence solved by a new surgical operation. Kasr. h i . J. Surg., 1: 50,1960. 4. Grabstald, H.: Postradical cystectomy impotences treated by penile silicone implant. New York State J. Med., 7 0 2344, 1970. 5. Lash, H.:Silicone implant for impotence. J. Urol., 100: 709, 1968. 6. Nukui, F., Okamoto, S.,Nagata, M., Kurokawa, J. and Fukui, J.: Complications and reimplantation of penile implants. Int. J. Urol., 4 52, 1997. 7. Kardar, A. and Pettersson, B. A,: Penile gangrene: a complication of penile prosthesis. Scand. J. Urol. Nephrol., 2 9 355, 1995. 8. Yoo, J. J., Lee, I. and Atala, A.: Cartilage rods as a potential material for penile reconstruction. J. Urol., 160 1164,1998. 9. Atala, A,, Cima, L. G., Kim, W., Paige, K. T., Vacanti, J. P., Retik, A. B. and Vacanti, C. A,: Injectable alginate seeded with chondrocytes as a potential treatment for vesicoureteral reflux. J. Urol., 150 745,1993. 10. Atala, A., Kim, W., Paige, K. T., Vacanti, C. A. and Retik, A. B.: Endoscopic treatment of vesicoureteral r e f l w with a chondrocyte-alginate suspension. J. Urol., 152: 641,1994. 11. Horton, C. E. and Dean, J. A.: Reconstruction of traumatically acquired defects of the phallus. World J. Surg., 1 4 757, 1990.
DISCUSSI 0N
Dr. Catherine deVries. I n your model using cartilage what would you propose to use as a model for tunica? Dr. James J . Yoo. When reconstructing the penis, the tunica is important. We recently developed a material that has the physical characteristics of fascia. We plan to use that biomaterial for tunica.
Vol. 162, 1119-1121, September 1999 Printed In U.S.A.
THEJOURNAL OF UROLOGY Copyright 0 1999 by AMERICAN UROLOCICAL ASSOCIATION,INC.
AUTOLOGOUS ENGINEERED CARTILAGE RODS FOR PENILE RECONSTRUCTION JAMES J . YOO, HEUNG JAE PARK, ILWOO LEE
AND
ANTHONY ATALA"
From the Laboratory for Tissue Engineering and Cellular Therapeutics, Department of Urology, Children's Hospital and Harvard Medical School, Boston, Massachusetts
ABSTRACT
Purpose: Conditions such as inadequate and ambiguous genitalia that are caused by rudimentary penis, severe hypospadias or traumatic injury require surgical intervention. Although silicone penile prostheses are an accepted treatment modality, biocompatibility issues may be a problem in select cases. We previously demonstrated that rods composed of cartilage could be created using chondrocytes seeded on biodegradable polymer scaffolds. We showed that the cartilage rods engineered ex situ were readily elastic and withstood high degrees of pressure. We investigated the feasibility of applying the engineered cartilage rods in situ in an animal model. Materials and Methods: Autologous chondrocytes harvested from rabbit ears were grown and expanded in culture. Cells were seeded onto biodegradable poly-L-lactic acid coated polyglycolic acid polymer rods a t a concentration of 50 X lo6 chondrocytes per ~ m .A~total . of 18 chondrocyte polymer scaffolds were implanted into the corporal spaces in 10 rabbits. As controls, 1 corpus in each of 2 rabbits was not implanted. The animals were sacrificed 1, 2, 3 or 6 months after implantation. Histological analysis was performed using hematoxylin and eosin, aldehyde fuschin-alcian blue and toluidine blue staining. Results: All animals tolerated the implants for the duration of the study without any complications. Gross examination after retrieval at 1month showed well formed, milky white cartilage structures within the corpora. All polymers were fully degraded by 2 months. There w a s no evidence of erosion or infection a t any of the implant sites. Histological analysis using alcian blue and toluidine blue staining revealed mature and well formed chondrocytes in the retrieved implants. Conclusions: Autologous chondrocytes seeded on preformed biodegradable polymer structures form cartilage structures within the rabbit corpus cavernosum. This technology appears to be useful for creating autologous penile prostheses. KEYWORDS: penis, prostheses and implants, chondrocytes, cartilage
Reconstructing abnormal male genitalia due to ambiguous genitalia, micropenis or traumatic injury remains a surgical challenge. Achieving functionally and esthetically acceptable genitalia is the prime goal of phalloplasty. Earlier attempts at penile reconstruction involved multiple stages of surgery using autogenous rib cartilage as a stiffener but unsatisfactory functional and cosmetic results due to curvature discouraged its Silicone prostheses have been widely used due t o their unique mechanical proper tie^.^. However, biocompatibility issues may be a problem in select patient^.^.^ A natural prosthesis of autologous chondrocytes may be advantageous due to native biocompatibility. We previously created rods of heterologous cartilage using chondrocytes seeded on biodegradable polymer scaffolds.' We showed that cartilage rods engineered ex situ are readily elastic and withstand high degrees of pressure. In the current study we investigated the feasibility of applying the engineered cartilage rods in situ in the corpora in an animal model (fig. 1). MATERIALS AND METHODS
Polymers. Unwoven sheets of polyglycolic acid polymers with a density of 58 mg./cc were configured into cylindrical rods 2 cm. long and 0.3 cm. in diameter as cell delivery vehicles. The polymer meshes were composed of fibers 15 pm. in diameter with a n interfiber distance of 0 to 200 pm. and
with 95% porosity. The scaffold was designed to degrade via hydrolysis in 6 to 8 weeks. The resulting flexible scaffold was coated with a liquefied copolymer (poly-DL-lactide-coglycolide 50:50, 80 mg./ml. methylene chloride) to achieve adequate mechanical characteristics. The polymers were sterilized in ethylene oxide and placed under sterile conditions until cell delivery. Cell culture and cell seeding. Autologous chondrocytes harvested from rabbit ears were dissected into 2 x 2 mm. fragments. Chondrocytes were harvested under sterile conditions using a previously described t e ~ h n i q u e .lo ~ .Briefly the dissected cartilage fragments were digested in 3% collagenase type I1 solution for 6 to 8 hours. Recovered cells were washed with phosphate buffered saline and plated in culture dishes. The isolated cells were grown in culture in Hamms F-12 media containing 10% fetal calf serum, 5 pg./ml. ascorbic acid, 100 pg./ml. streptomycin and 100 units per ml. penicillin. Cells were incubated at 37C in the presence of 5%carbon dioxide. Chondrocytes were expanded until sufficient cell quantities were available. The cells were trypsinized, collected, washed and counted for seeding. Chondrocytes were seeded onto preformed poly-L-lactic acid coated polyglycolic acid polymer rods a t a concentration of 50 x lo6 chondrocytes per ~ m .The ~ . cell polymer rods were implanted immediately after seeding. Experimental study. A total of 18 chondrocyte polymer scaffolds were implanted in the corporal space in 10 rabbits.
* Requests for reprints: Children's Hospital and Harvard Medical School, 300 Longwood Ave., Boston, Massachusetts 02115. 1119
1120
AUTOLOGOUS ENGINEERED CARTILAGE RODS FOR PENILE RECONSTRUCTION
FIG.1. Autologous chondrocytes were isolated from rabbit ears, expanded and seeded onto preformed polymer rods. Cell polymer scaffolds were implanted intracorporally. Specimens were retrieved for analysis 1, 2, 3 or 6 months after implantation.
FIG.2. Retrieved specimens 1 month after implantation. A, genitalia of control rabbit shows cartilage formation in corpus cavernosum, while control corpus cavernosum appears normal. B , cartilage rod.
Bilateral intracorporal cell polymer scaffolds were implanted in 8 rabbits, while the remaining 2 underwent unilateral implantation, leaving the other corpus cavernosum intact as controls. Three animals were sacrificed 1and 2 months, and 2 were sacrificed 3 and 6 months, respectively, after implantation. The 2 control animals were sacrificed at 1 and 6 months, respectively. The implants were retrieved and analyzed grossly and histologically. Sections (5 p.) of formalin fixed, paraffin embedded tissues were cut and stained with hematoxylin and eosin, aldehyde fuschin-alcian blue and toluidine blue. RESULTS
All animals tolerated the implants for the duration of the study without any noticeable complications. Gross examination after retrieval at 1 month revealed well formed, milky white cartilage structures within the corpora (fig. 2). The retrieved cartilage rod structures remained approximately
the same size as the initial implants within the corporal bodies. The corpora without implantation had normal cavernous tissue. There was no evidence of erosion, inflammation or infection a t any of the implanted cartilage rod sites. Histological analysis with hematoxylin and eosin showed mature and well formed cartilage in all chondrocyte-polymer implants (fig. 3). Polymer fibers were progressively replaced by cartilage with time at 1, 2, 4 and 6 months, respectively. All polymers were fully degraded by 2 months. Aldehyde fuschin-alcian blue and toluidine blue staining demonstrated highly sulfated mucopolysaccharides, which are differentiated products of chondrocytes (fig. 4). DISCUSS I 0N
The management of inadequate and ambiguous genitalia due to rudimentary penis, severe hypospadias or traumatic injury involves phallic reconstruction.'. " Prostheses may be required for genital reconstruction. Natural penile prostheses created from patient cells may be preferable and they may decrease the biocompatibility risks associated with artificial prostheses. Previous studies at our laboratory demonstrated that cartilage rods may be created using chondrocytes seeded on preformed polymer scaffolds.' Stress relaxation studies to measure biomechanical properties were performed on the retrieved tissue engineered structures. Compression, tension and bending studies showed that the cartilage rods are readily elastic and withstand high degrees of pressure. The present study revealed that engineered cartilage tissue composed of autologous chondrocytes seeded on biodegradable polymers may be implanted into corporal bodies in situ.
FIG. 3. Retrieved cartilage rod specimens 3 months after implantation. A, cartilage tissue has formed within each corpora (C). H & E, reduced from X25. B , mature chondrocytes enclosed within lacunae are adjacent to tunica albuginea (2'). H & E, reduced from x250.
AUTOLOGOUS ENGINEERED CARTILAGE RODS FOR PENILE RECONSTRUCTION
1121
FIG.4. Highly sulfated mucopolysaccharides were detected 6 months after implantation. A, toluidine blue stain, reduced from x 250. B, aldehyde fuchsin-alcian blue stain, reduced from x250.
The architecture of the flexible polymer scaffolds was strengthened by the addition of a liquefied copolymer, polyDL-lactide-co-glycolide, 50:50. The engineered cell polymer scaffolds were snugly implanted within the corpus cavernosum without any technical difficulties. The polymer scaffolds in our study were designed to degrade in 6 to 8 weeks. Seeded cells readily formed mature cartilage tissue in vivo, replacing the degrading polymer fibers during this period. The engineered cartilage remained at the site of initial implantation without any evidence of infection, inflammation or erosion. Histological analysis of the retrieved rods revealed adequate formation of mature cartilage, as evidenced by chondrocytes within lacunae and highly sulfated mucopolysaccharides. Engineered cartilage prostheses may be applicable in the future in patients undergoing penile surgery for congenital or acquired conditions. Autologous cartilage tissue composed of patient cells would be used without corporal tissue for penile reconstruction or intracorporally for erectile dysfunction. However, to engineer cartilage penile rods for clinical use further studies must be performed, including the establishment of functional and biomechanical parameters of the prostheses in vivo as well as use of the rods extracorporally. Studies involving these issues are currently being performed in our laboratory. In conclusion we demonstrated that autologous chondrocytes seeded on preformed biodegradable polymer structures form cartilage rods within the rabbit corpus cavernosum. "his technology may be useful in the future for creating autologous penile prostheses.
REFERENCES
1. Goodwin, W. E. and Scott, W. W.: Phalloplasty. J. Urol., 68 903, 1952. 2. Frumpkin, A. P.: Reconstruction of male genitalia. Amer. Rev. Sov. Med., 2 14,1944. 3. Beheri, G. E.: The problem of impotence solved by a new surgical operation. Kasr. h i . J. Surg., 1: 50,1960. 4. Grabstald, H.: Postradical cystectomy impotences treated by penile silicone implant. New York State J. Med., 7 0 2344, 1970. 5. Lash, H.:Silicone implant for impotence. J. Urol., 100: 709, 1968. 6. Nukui, F., Okamoto, S.,Nagata, M., Kurokawa, J. and Fukui, J.: Complications and reimplantation of penile implants. Int. J. Urol., 4 52, 1997. 7. Kardar, A. and Pettersson, B. A,: Penile gangrene: a complication of penile prosthesis. Scand. J. Urol. Nephrol., 2 9 355, 1995. 8. Yoo, J. J., Lee, I. and Atala, A.: Cartilage rods as a potential material for penile reconstruction. J. Urol., 160 1164,1998. 9. Atala, A,, Cima, L. G., Kim, W., Paige, K. T., Vacanti, J. P., Retik, A. B. and Vacanti, C. A,: Injectable alginate seeded with chondrocytes as a potential treatment for vesicoureteral reflux. J. Urol., 150 745,1993. 10. Atala, A., Kim, W., Paige, K. T., Vacanti, C. A. and Retik, A. B.: Endoscopic treatment of vesicoureteral r e f l w with a chondrocyte-alginate suspension. J. Urol., 152: 641,1994. 11. Horton, C. E. and Dean, J. A.: Reconstruction of traumatically acquired defects of the phallus. World J. Surg., 1 4 757, 1990.
DISCUSSI 0N
Dr. Catherine deVries. I n your model using cartilage what would you propose to use as a model for tunica? Dr. James J . Yoo. When reconstructing the penis, the tunica is important. We recently developed a material that has the physical characteristics of fascia. We plan to use that biomaterial for tunica.