美国组织工程技术治疗严重尿道狭窄临床应用报告

Articles www.thelancet.com Published online March 8, 2011 DOI:10.1016/S0140-6736(10)62354-9 1 Published Online March 8, 2011 DOI:10.1016/S0140- 6736(10)62354-9 See Online/Comment DOI:10.1016/S0140- 6736(11)60312-7 Wake Forest Institute for Regenerative Medicine and Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA (A Raya-Rivera MD, J J Yoo MD, D R Esquiliano MD, S Soker PhD, Prof A Atala MD); and HIMFG Tissue Engineering Laboratory, Metropolitan Autonomous University and Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, Mexico (A Raya-Rivera, D R Esquiliano, Prof E Lopez-Bayghen PhD) Correspondence to: Prof Anthony Atala, Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston Salem, NC 27157, USA aatala@wfubmc.edu Introduction Complex urethral problems can be caused by injury, which can lead to an inability to void. Catheters might need to be inserted below the abdomen in the suprapubic region for adequate bladder emptying, because often the eff ects of the trauma on the involved tissues need to be minimised. Some patients with urethral strictures repeatedly have infections, experience straining and major discomfort, and have blood in their urine. Urethral functional inadequacy can occur in patients with pelvic fractures or straddle trauma; previous manipulation with indwelling catheters, endoscopy, or surgery; and congenital or acquired disease. Reconstructive techniques depend on the urethral defect location, length, and the surgeon’s preference and previous experience. Short, non-complex urethral defects can be repaired with an end-to-end anastomosis by aligning and joining the normal urethral ends.1 For long defects, surgeons might need to do a pubectomy, to gain better access to the damaged tissue and to help shorten the urethral gap.2 An onlay repair, in which about half the strictured circumference portion of the tubular urethra is replaced with a tissue graft (eg, skin or buccal mucosa), is often used for damaged urethras that have a healthy urethral bed.3,4 Tubularised tissue grafts might be needed for complex or long urethral defects, but have a high proportion of failures (sometimes over 50%).5 Regenerative medicine might help to overcome some of the drawbacks associated with the native tissues that are used for urologic reconstruction.6,7 However, engineered tubularised constructs (eg, urethras or small blood vessels) tend to stricture over time.5,8–10 We aimed to assess whether engineered autologous tubularised urethral tissue could be used as an alternative method for the treatment of complex posterior urethral defects. Methods Patients Five boys with urethral defects were invited to participate in this study of engineered urethras at the Federico Gomez Children’s Hospital in Mexico City, Mexico. Three patients presented with a complete posterior urethral disruption caused by pelvic trauma and had substantial widespread injury and two had previous failed posterior urethral repairs, one with a buccal mucosa graft and one with a skin graft, both of which were tubularised. The study protocol was approved by the hospital’s investigational review board (protocol number 2002055) Tissue-engineered autologous urethras for patients who need reconstruction: an observational study Atlantida Raya-Rivera, Diego R Esquiliano, James J Yoo, Esther Lopez-Bayghen, Shay Soker, Anthony Atala Summary Background Complex urethral problems can occur as a result of injury, disease, or congenital defects and treatment options are often limited. Urethras, similar to other long tubularised tissues, can stricture after reconstruction. We aimed to assess the eff ectiveness of tissue-engineered urethras using patients’ own cells in patients who needed urethral reconstruction. Methods Five boys who had urethral defects were included in the study. A tissue biopsy was taken from each patient, and the muscle and epithelial cells were expanded and seeded onto tubularised polyglycolic acid:poly(lactide-co- glycolide acid) scaff olds. Patients then underwent urethral reconstruction with the tissue-engineered tubularised urethras. We took patient history, asked patients to complete questionnaires from the International Continence Society (ICS), and did urine analyses, cystourethroscopy, cystourethrography, and fl ow measurements at 3, 6, 12, 24, 36, 48, 60, and 72 months after surgery. We did serial endoscopic cup biopsies at 3, 12, and 36 months, each time in a diff erent area of the engineered urethras. Findings Patients had surgery between March 19, 2004, and July 20, 2007. Follow-up was completed by July 31, 2010. Median age was 11 years (range 10–14) at time of surgery and median follow-up was 71 months (range 36–76 months). AE1/AE3, α actin, desmin, and myosin antibodies confi rmed the presence of cells of epithelial and muscle lineages on all cultures. The median end maximum urinary fl ow rate was 27·1 mL/s (range 16–28), and serial radiographic and endoscopic studies showed the maintenance of wide urethral calibres without strictures. Urethral biopsies showed that the engineered grafts had developed a normal appearing architecture by 3 months after implantation. Interpretation Tubularised urethras can be engineered and remain functional in a clinical setting for up to 6 years. These engineered urethras can be used in patients who need complex urethral reconstruction. Funding National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. Articles 2 www.thelancet.com Published online March 8, 2011 DOI:10.1016/S0140-6736(10)62354-9 and ethics board. Written informed parental consent was obtained before acquirement of the tissue biopsy and 4–7 weeks before surgery. Procedures All fi ve boys had an engineered urethra created from autologous cells and underwent urethral tubularised posterior urethroplasty by the same surgical method and procedure. Before surgery, the urethral defect length and characteristics such as location and associated pelvic path ology were measured by retrograde urethrography, voiding cystourethrograms, and ureth- roscopy. We took a detailed history from all patients and comp leted symptom question naires from the Inter- national Continence Society (ICS). We took tissue samples from every child and used these to engineer the urethras. All tissue samples were taken by the same person (A Raya-Rivera). A 3 cm suprapubic transverse incision was made and a bladder biopsy (1×1 cm) was taken and sent to the approved manu facturing facility at the Metropolitan Autonomous University, Mexico City, Mexico, where the urethras were engineered under regulatory guidelines (HIM87120BSO) from the Federal Commission on Safety and Health Protection; Investigation, Ethics, and Biosafety Division (COFEPRIS). Primary cultures of smooth muscle and urothelial cells were collected as described previously.6 Briefl y, cells were col lected by explanting pieces of muscle tissue and scraping the uroepithelial tissue onto 10 cm culture plates. The muscle tissue was placed in Dulbecco’s modifi ed Eagle’s medium (Invitrogen; Carlsbad, CA, USA) and the epithelial tissue was placed in keratinocyte growth medium, both with epidermal growth factor. The cells were expanded to a density of 1·0–3·0×10⁷ per cm² for 3–6 weeks before seeding. Muscle and urothelial cells were characterised by histology and immunohistochemistry before seeding onto the scaff olds. 5 μm sections of formalin- fi xed, paraffi n-embedded tissues were processed and stained with haematoxylin and eosin. We used represen- tative sections for immunohistochemistry. Uroepithelial cell layers were identifi ed with broadly reacting monoclonal mouse anti-human cytokeratins AE1/AE3, desmin, and myosin (Dako, CA, USA; product codes M3515, D33, and M3558) antibodies at 1:100, 1:50, and 1:50 dilutions, respectively, for 24 h at 4°C. Smooth muscle fi bres were labelled with monoclonal α smooth muscle anti-actin antibodies (Novocastra, Newcastle, UK) at 1:100 dilution for 24 h at 4°C. Sections were incu- bated with the Dako Real EnVision Peroxidase/DAB detec tion system (Dako). All sections were counter- stained with hematoxylin. Images were taken with a light microscope. We seeded cells onto scaff olds as described previously.7,11,12 Epithelial cells were seeded onto the luminal surface and muscle cells onto the outer surface of the tubular scaff olds. Briefl y, the cells were removed from the culture plate with a 0·05% trypsin solution. Cells were centrifuged, rinsed with medium with no additives, and the pellet was re-suspended to a concentration of 1×10⁷ cells per mL. A biodegradable mesh made of polyglycolic acid (PGA; Sherwood Medical, St Louis, MO, USA) was tubularised with running 5-0 PGA sutures (Ethicon; Piscataway, NJ, USA) and sized for every individual patient, according to measurements of urethral defect from radiography, urethral calibration, and endoscopy. The size of the engineered urethras ranged from 4 to 6 cm (median 5 cm), with a 16 French diameter. The scaff olds were constructed in the same manner as used previously,7,12 and the biomaterials were processed with the same techniques as used previously for engineered bladders implanted in people.12,13 Briefl y, the mesh was sprayed with a 50:50 liquid PGA:poly(lactide-co-glycolide acid) (Sigma; St Louis, MO, USA). The scaff olds were placed in a vacuum desiccator; samples confi rmed scaff old purity.13 The scaff olds were further sterilised with ultraviolet light and sequentially seeded with epithelial cells within the lumen and smooth muscle cells on the outer surface. The seeded scaff olds were incubated in culture for 7 days with Dulbecco’s modifi ed Eagle’s medium. Overall, construction of the neo-urethras took 4–7 weeks. We measured cell viability on the seeded grafts with an MTT (3-[4, 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide) assay.14 After cells were incubated with 1 mg/mL MTT reagent for about 4 h, an isopropanol:hydrochloric acid (ratio 1:1) solution was added. The samples were read with a GENios (Tecan, Durham, NC, USA) ELISA plate reader (630 nm wavelength). Measure ments from non-seeded grafts were used as controls. We did scanning electron microscopy for seeded and non-seeded scaff olds to assess whether cells had grown on the scaff olds. Random samples were taken on days 3 and 6 after seeding. Fragments were washed with a 0·1 M sodium cacodylate buff er and fi xed for 2 h in a gluta raldehyde (2·5%) solution. Samples were post- fi xed in a 1:100 sodium cacodylate osmium tetroxide solution and dehydrated in a series of ethanol concentrations. We dried the preparations, covered Age (years) Primary diagnosis Previous urethroplasty Defect site Defect length (cm) Follow-up (months) 1 10 Motor vehicle accident No Membranous urethra 5 76 2 14 Straddle trauma Buccal mucosa Membranous urethra 6 73 3 11 Motor vehicle accident No Membranous urethra 4 71 4 11 Motor vehicle accident Foreskin Membranous urethra 4 65 5 12 Straddle trauma No Membranous urethra 5 36* *Patient followed up for 36 months because he was the last patient to enter the study. Table: Characteristics of patients Articles www.thelancet.com Published online March 8, 2011 DOI:10.1016/S0140-6736(10)62354-9 3 them with ionised gold, and examined them with a JEOL JSM-5300 microscope. Once the engineered urethras had been made, the patients underwent surgery. The urethras were catheterised and exposed through a perineal inverted semicircular incision. Defects were identifi ed, the stricture and scar tissue was removed, and the urethral ends were dissected free from surrounding scar tissue. The tubularised engineered urethral constructs were surgically implanted with absorbable sutures. All engineered urethras were trimmed in the overlap region with the native urethras. A urethral catheter was left in place for 2 weeks in the fi rst patient, and for 4 weeks in the other patients. The catheter size ranged from 12 to 14 French. We took patient history, asked patients to complete the ICS questionnaires, and did urine analyses, cystoure throscopy, cystourethrography, and fl ow measurements at 3, 6, 12, 24, 36, 48, 60, and Figure 1: Cellular expression of epithelial and muscular markers, and viability assay and scanning electron microscopy assessments (A) Labelling of smooth muscle and uroepithelial cells in cultures incubated with primary antibodies (positive cells) and without (control cells). All these cell cultures came from patient 3. (B) Assessment of cell viability on the seeded grafts from every patient. Vaginal cells were assessed as positive controls. Bars represent SD. (C) Scanning electron microscopy (15 Ku) of seeded scaff olds 6 days after seeding. Cytokeratins AE1/AE3 α actin Desmin Myosin Positive cells Control cells A C B PGA/PLGA non-seeded grafts PGA/PLGA vaginal cells Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 0 0·2 0·4 0·6 0·8 1·0 1·2 1·4 O pt ica l d en sit y at 5 95 n m Urothelium Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Muscle Scaffold without cells 10 μm 10 μm 10 μm 10 μm 10 μm 10 μm 10 μm 10 μm 10 μm 10 μm 50 μm Articles 4 www.thelancet.com Published online March 8, 2011 DOI:10.1016/S0140-6736(10)62354-9 72 months after surgery. We did serial endoscopic cup biopsies at 3, 12, and 36 months, each time in a diff erent area of the engineered urethras. Role of the funding source The sponsor funded the basic research and its analysis but had no role in the study design (preparing the Figure 2: Neo-urethra implantation and clinical outcome (A) A cell-seeded graft sutured to the normal urethral margins from the fi rst patient. (B) Voiding cystourethrograms of all fi ve patients before surgery (arrows show the abnormal margins), 12 months after surgery (arrows show margins of tissue engineered urethras), and at last follow-up (arrows show margins of tissue engineered urethras). Before surgery 12 months after surgery At last follow-up A B Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Articles www.thelancet.com Published online March 8, 2011 DOI:10.1016/S0140-6736(10)62354-9 5 protocol), data collection, data analy ses, data inter- pretation, or writing of the report. All authors had full access to all the data and had fi nal responsibility for the decision to submit for publication. Results Patients had surgery between March 19, 2004, and July 20, 2007. Follow-up was completed by July 31, 2010. The table shows the characteristics of each patient. Median age was 11 years (range 10–14 years) at the time of surgery and patients were followed up for a median of 71 months (range 36–76 months) after surgery. AE1/AE3, α actin, desmin, and myosin antibodies confi rmed the presence of cells of epithelial and muscle lineages on all cultures (fi gure 1). There was a substantial increase in cell growth on the seeded scaff olds after 7 days in standard culture conditions. Scanning electron microscopy on day 6 of culture showed all scaff old surfaces were covered with cells. The engineered urethras were implanted without intraoperative complications (fi gure 2). None of the patients had fi stulae or urinary tract infections during follow-up. Patient history and serial symptom questionnaires completed by the patients with their Figure 3: Urofl ow analyses 0 3 6 12 24 36 48 60 72 0 5 10 15 20 25 30 35 Fl ow ra te m L/ s Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Follow-up (months) Figure 4: Histological and immunocytochemical analyses Results of urethral biopsies of engineered segments from every patient at 1 year after surgery and from a control sample of healthy urethral tissue. All scale bars indicate 25 μm. Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Control Haematoxylin and eosin α actin Desmin Mvosin AE1/AE3 Articles 6 www.thelancet.com Published online March 8, 2011 DOI:10.1016/S0140-6736(10)62354-9 parents confi rmed patients’ satisfaction with the results of the operation, as measured by diff erent parameters including the absence of dysuria, voiding frequency, straining, or dribbling. The fi rst patient, who had their catheter removed 2 weeks after surgery, presented with a decreased urinary voiding stream 2 weeks later. Cystourethroscopy showed a narrowing at the proximal superior graft anastomotic site that needed a transurethral incision 4 weeks after surgery. The patient was able to void well thereafter, and no further interventions were needed. Cystourethroscopy confi rmed the radiographic fi ndings of a patent urethra in all patients (fi gure 2). The fourth patient had pelvic disruption that involved the sphincter, and he needed a pubovesical sling after surgery for his daytime stress incontinence. All patients are now continent. Voiding cystourethrograms, which showed the extent of the defects before surgery, showed the maintenance of wide urethral calibres without diverticula 12 months after surgery (fi gure 2). The absence of strictures and diverticula was confi rmed with serial cystourethroscopy. Urofl ow studies showed adequate fl ow rates, with a mean end maximum fl ow rate of 25·1 mL/s, and a median of 27·1 mL/s (range 16–28) at the last follow-up (fi gure 3). Histology of the serial biopsies, confi rmed with immunohistochemistry, showed that the engineered urethras appeared to have a normal architecture by 3 months after implantation, and consisted of distin- guishable layers of epithelia and smooth muscle (fi gure 4). There were no aberrant histological changes over time. Discussion We have successfully constructed engineered urethras with autologous cells and implanted them into patients with urethral defects. All fi ve boys were continent at last follow-up. The results of this study are consistent with the results from experimental studies we did in the early 1990s, when we seeded biodegradable matrices with autologous cells.15 In an experimental model in rabbits, autologous bladder epithelial and smooth muscle cells were grown and seeded onto preconfi gured tubular matrices.11 Entire urethral segments were resected and anterior urethroplasties were done with tubularised matrices, either seeded with cells or without cells. The tubularised matrices that were seeded with autologous cells formed new tissue that was histologically similar to native urethra. There was poor tissue development, fi brosis, and stricture formation from those matrices that were not seeded. The study showed that the use of engineered urethras with matrices of cells was crucial to the success of tubularised repairs in the long term. Although the scaff old alone was not suffi cient for a tubularised repair and there was fi brosis and scarring present, we noted that there was limited tissue regeneration at the edges of the construct. In animal experiments, we noted that the maximum distance at which native cells from the normal urethra migrated onto the scaff old and contributed to adequate tissue regeneration was 0·5 cm.16 This fi nding led us to assess the use of matrices without cells to replace only the top part of the tubular urethra, in an on- lay manner, instead of replacing the entire tubular urethral circumference.17 Thereafter, we showed that if a portion of the circumferential urethral tissue is healthy and can be preserved, off -the-shelf matrices without cells can be used as on-lay grafts with adequate long-term outcomes.18–20 Buccal mucosal cell-seeded matrices have been used as on-lay grafts for urethral disease in patients with lichen sclerosus.21 Lich