RESUMO
INTRODUCTION: Tissue engineering may become an alternative to current bladder augmentation techniques. Large scaffolds are needed for clinically significant augmentation, but can result in fibrosis and graft shrinkage. The purpose of this study was to investigate the use of multiple scaffolds instead of one large scaffold, to enhance bladder tissue regeneration and bladder capacity. Second, acellular collagen, collagen-heparin, and collagen-heparin scaffolds with growth factors (GFs) were used and the biological activity of the different scaffolds was compared in a large animal model. MATERIALS AND METHODS: Scaffolds were made of bovine type I collagen with or without heparin (Ø = 3.2 cm). Collagen-heparin scaffolds were loaded with GFs, vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and heparin-binding epidermal growth factor (HB-EGF). Three identical scaffolds prepared from collagen (COL-group), collagen with heparin (COLHEP-group), or collagen-heparin with growth factors (COLHEPGF-group) were implanted in one porcine bladder. The outcome was compared with sham-operated animals (Sham-group), in which no scaffold was used. Urodynamic evaluation was performed before surgery and 3 months after bladder reconstruction, together with histological evaluation. RESULTS: Survival rate was 92%, 12 animals completed the study, 3 of every group, 1 animal developed peritonitis due to urine leakage and was sacrificed. The regenerated area was largest in the COLHEP-group, and least in the COL-group (p = 0.002). Histological evaluation revealed a normal urothelial layer and good angiogenesis in all groups, and comparable ingrowth of smooth muscle cells. Urodynamics showed no statistically significant differences in bladder capacity and compliance between groups. Bladder capacity and compliance was very high in this animal model, which made it impossible to study the increase due to augmentation. CONCLUSIONS: Implantation of multiple collagen-heparin scaffolds in one bladder is feasible in a porcine model, resulting in tissue almost indistinguishable from native tissue involving all cell layers of the bladder. Collagen scaffolds with heparin incorporated resulted in a larger area of regenerated tissue. To reach clinically significant augmentation, multiple larger collagen-heparin scaffolds, with or without GFs, need to be tested to study the largest possible diameter of scaffold and number of used scaffolds still resulting in well-vascularized tissue.
Assuntos
Engenharia Tecidual/métodos , Alicerces Teciduais/química , Bexiga Urinária/metabolismo , Animais , Colágeno/química , Feminino , Fator 2 de Crescimento de Fibroblastos/química , Heparina/química , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/química , Suínos , UrodinâmicaRESUMO
PURPOSE: A readily available artificial urinary conduit might be substituted for autologous bowel in standard urinary diversions and minimize bowel associated complications. However, the use of large constructs remains challenging as host cellular ingrowth and/or vascularization is limited. We investigated large, reinforced, collagen based tubular constructs in a urinary diversion porcine model and compared subcutaneously pre-implanted constructs to cell seeded and basic constructs. MATERIALS AND METHODS: Reinforced tubular constructs were prepared from type I collagen and biodegradable Vicryl® meshes through standard freezing, lyophilization and cross-linking techniques. Artificial urinary conduits were created in 17 female Landrace pigs, including 7 with a basic untreated construct, 5 with a construct seeded with autologous urothelial and smooth muscle cells, and 5 with a free graft formed by subcutaneous pre-implantation of a basic construct. All pigs were evaluated after 1 month. RESULTS: The survival rate was 94%. At evaluation 1 basic and 1 cell seeded conduit were occluded. Urinary flow was maintained in all conduits created with pre-implanted constructs. Pre-implantation of the basic construct resulted in a vascularized tissue tube, which could be used as a free graft to create an artificial conduit. The outcome was favorable compared to that of the other conduits. Urinary drainage was better, hydroureteronephrosis was limited and tissue regeneration was improved. CONCLUSIONS: Subcutaneous pre-implantation of a basic reinforced tubular construct resulted in a vascularized autologous tube, which may potentially replace bowel in standard urinary diversions. To our knowledge we introduce a straightforward 2-step procedure to create artificial urinary conduits in a large animal model.
Assuntos
Bioprótese , Colágeno Tipo I/química , Poliglactina 910 , Engenharia Tecidual/métodos , Derivação Urinária/métodos , Animais , Feminino , Teste de Materiais , Modelos Animais , Suínos , Bexiga Urinária/cirurgiaRESUMO
Tissue engineering may become an alternative to current bladder augmentation techniques. Large scaffolds are needed for clinically significant augmentation, but can result in fibrosis and graft shrinkage. The purpose of this study was to investigate whether smart acellular collagen-heparin scaffolds with growth factors (GFs) VEGF, FGF2, and HB-EGF enhance bladder tissue regeneration and bladder capacity in a large animal model of diseased bladder. Scaffolds of bovine type I collagen with heparin and VEGF, FGF2, and HB-EGF measuring 3.2 cm in diameter were prepared. In 23 fetal sheep, a bladder exstrophy was surgically created at 79 days of gestation. One week after birth (at full term), the bladder was reconstructed by primary closure (PC group) or using a collagen-heparin scaffold with GFs (COLGF group) and compared to a historical group reconstructed with a collagen scaffold without GFs (COL group). Functional (video urodynamics) and histological evaluation was performed 1 and 6 months after bladder repair. The overall survival rate was 57%. Cystograms were normal in all animals, except for low-grade reflux in all groups. Urodynamics showed no statistically significant differences in bladder capacity and compliance between groups. Histological evaluation at 1 month revealed increased urothelium formation, improved angiogenesis, and enhanced ingrowth of smooth muscle cells (SMCs) in the COLGF group compared to the COL group. At 6 months, improved SMC ingrowth was found in the COLGF group compared to the COL group; both scaffold groups showed normal urothelial lining and standard extracellular matrix development. Bladder regeneration using a collagen-heparin scaffold with VEGF, FGF2, and HB-EGF improved bladder tissue regeneration in a large animal model of diseased bladder. Larger GF-loaded constructs need to be tested to reach clinically significant augmentation.
Assuntos
Colágeno , Fator 2 de Crescimento de Fibroblastos , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Regeneração/efeitos dos fármacos , Alicerces Teciduais/química , Bexiga Urinária/fisiologia , Fator A de Crescimento do Endotélio Vascular , Animais , Bovinos , Colágeno/química , Colágeno/farmacologia , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/química , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/farmacologia , Ovinos , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
INTRODUCTION: Cloacal exstrophy is a complex and rare congenital malformation. Because of improvements in medicine, operative techniques, and perioperative management the survival rates are now approaching 100%. Currently, treatment is focused on improving quality of life. Since 1974, we encountered 20 patients with cloacal exstrophy in our hospital. The aim of this study is to evaluate our clinical experience and outcome during the last 39 years. PATIENTS AND METHODS: A retrospective study of the medical records was performed. We evaluated anatomical status, phenotype and genotype at birth, subsequent surgical treatment and current gastrointestinal, urinary, spinal, genital, and gender status and outcome. RESULTS: The records of all 20 patients who were treated in our center were included in this study. Of the 20 patients, 18 were primary patients, 2 referred. Six patients died within the first year of life. Median age of the surviving 14 patients was 25 years (range, 4-39 years) at time of evaluation. At the last follow-up, 11 patients had an endileostomy or endcolostomy. Three patients had a pull-through; two of them were continent for feces. Two patients were continent for urine; one with a sinus urogenitalis and one after urethra reconstruction. Eleven patients are incontinent; six of them had an incontinent urinary deviation (Bricker deviation). Of the other five incontinent patients, two had a urethral reconstruction, one vesicovaginal anastomosis, one perineotomy, and one epispadias bladder. Urinary continence was unknown in one patient. Of the surviving patients, nine have 46 XY karyotype and five 46 XX karyotype. Initially, six XY patients underwent external genital reconstruction with reassignment to female gender; however, one died postoperatively. One declared to feel like a man at age of 15 years. Remaining four XY patients underwent male external genital reconstruction. All XX patients underwent vagina or vulva reconstruction, except for one who still has aplasia vaginae. All patients were born with varying types of spinal dysraphism. DISCUSSION: Despite medical efforts and development in treatment and support, satisfaction in outcome of cloacal exstrophy remains a challenge. Although reconstruction may be very difficult, male genital reconstruction in 46 XY patients has been given preference for more than a decade. A specialized multidisciplinary team should provide lifelong colorectal, urological, sexual, and psychosocial support to these patients, taking into account their age and phase of life.
Assuntos
Cloaca/anormalidades , Procedimentos de Cirurgia Plástica/métodos , Anormalidades Urogenitais/cirurgia , Adolescente , Adulto , Criança , Pré-Escolar , Incontinência Fecal/etiologia , Feminino , Seguimentos , Humanos , Masculino , Procedimentos de Cirurgia Plástica/psicologia , Estudos Retrospectivos , Resultado do Tratamento , Incontinência Urinária/etiologia , Anormalidades Urogenitais/complicações , Anormalidades Urogenitais/psicologia , Adulto JovemRESUMO
OBJECTIVE: To compare the regenerative capacity of diseased bladder in a large animal model of bladder exstrophy with regeneration in healthy bladder using a highly porous collagen scaffold. MATERIALS AND METHODS: Highly porous bovine type I collagen scaffolds with a diameter of 32 mm were prepared. In 12 fetal sheep a bladder exstrophy was surgically created at 79 days' gestation. Lambs were born at full term (140 days' gestation). After 1 week the bladder lesion was reconstructed and augmented with a collagen scaffold (group 1). In nine normal newborn lambs the bladder was augmented with a collagen scaffold 1 week after birth (group 2). Functional (video-urodynamics) and histological evaluation was performed at 1 and 6 months after surgery. RESULTS: The survival rate was 58% in group 1 and 100% in group 2. Cystograms were normal in all lambs, besides low-grade reflux in both groups. Urodynamics showed comparable capacity between both groups and a trend to lower compliance in group 1. Histological evaluation at 1 month revealed a non-confluent urothelial layer, an immature submucosa, and initial ingrowth of smooth muscle cells. At 6 months both groups showed normal urothelial lining, standard extracellular matrix development, and smooth muscle cell ingrowth. CONCLUSIONS: Bladder tissue regeneration with a collagen scaffold in a diseased bladder model and in healthy bladder resulted in comparable functional and histological outcome, with a good quality of regenerated tissue involving all tissue layers. Improvements may still be needed for larger augmentations or more severely diseased bladders.
Assuntos
Extrofia Vesical/patologia , Colágeno , Matriz Extracelular/patologia , Engenharia Tecidual , Alicerces Teciduais , Bexiga Urinária/patologia , Animais , Animais Recém-Nascidos , Bovinos , Modelos Animais de Doenças , Miócitos de Músculo Liso , Regeneração , Ovinos , UrodinâmicaRESUMO
BACKGROUND/PURPOSE: In fetuses with gastroschisis, toxic products in the amniotic fluid and constriction at the defect of the abdominal wall are considered causative of damage to the eviscerated bowel. The aim of this study was to cover the eviscerated bowel in gastroschisis with a collagen scaffold to protect the bowel and induce cell growth into the scaffold, which could lead to skin or abdominal wall formation replacing the scaffold. METHODS: In 12 fetal lambs gastroschisis was surgically created at 79 days gestation. A dual-layer type I collagen scaffold was sutured into the skin of the abdominal wall around the defect covering the eviscerated bowel. Lambs were examined after caesarean section at 140 days' gestation. RESULTS: Survival was 67%. In 7 of 8 surviving lambs the bowel was found to be covered after birth. One scaffold had ruptured. The bowel was found repositioned in the abdominal cavity in 5 lambs. In 2 lambs it was still partially outside. Only minor adherence of bowel loops and no fibrous peel formation were seen. Connective tissue and skin tissue replaced the scaffold. CONCLUSIONS: Prenatal coverage of the bowel in experimental gastroschisis with a collagen scaffold is feasible in fetal lambs, significantly diminished damage to the bowel wall, and skin and connective tissue replaced the scaffold. This technique may be promising in the care of fetuses with this congenital anomaly.
Assuntos
Colágeno , Feto/cirurgia , Gastrosquise/embriologia , Gastrosquise/cirurgia , Alicerces Teciduais , Animais , Feminino , Masculino , OvinosRESUMO
In spina bifida the neural tube fails to close during the embryonic period and it is thought that prolonged exposure of the unprotected spinal cord to the amniotic fluid during pregnancy causes additional neural damage. Intra-uterine repair might protect the neural tissue from exposure to amniotic fluid and might reduce additional neural damage. Biodegradable collagen scaffolds may be useful in case of fetal therapy for spina bifida, but biochemical properties need to be studied. The aim of this study was to investigate whether biodegradable collagen scaffolds can be used to treat full-thickness fetal skin defects. We hypothesized that the pro-angiogenic growth factors VEGF and FGF2 would enhance vascularization, epidermialization and lead to improved wound healing. To investigate the effect of these two growth factors, a fetal sheep model for skin defects was used. Compared to wounds treated with bare collagen scaffolds, wounds treated with growth factor-loaded scaffolds showed excessive formation of capillaries and less myofibroblasts were present in these wounds, leading to less contraction. This study has demonstrated that collagen scaffolds can be used to treat fetal skin defects and that the combination of collagen scaffolds with VEGF and FGF2 had a beneficial effect on wound healing.
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Feto/patologia , Modelos Animais , Pele/patologia , Engenharia Tecidual/métodos , Útero , Animais , Bovinos , Epitélio/patologia , Feminino , Feto/cirurgia , Fibroblastos/patologia , Microscopia Eletrônica de Varredura , Neovascularização Patológica , Gravidez , Ovinos/cirurgia , Pele/irrigação sanguínea , Alicerces Teciduais , CicatrizaçãoRESUMO
We evaluated the regeneration of the abdominal wall using a dual-layer collagen biomatrix, and the protective effect on the bowel of fetal abdominal wall repair in a fetal sheep model for gastroschisis. In 14 fetal lambs, the abdominal wall was opened at 79 days' gestation, creating a gastroschisis. In group 1, the gastroschisis was left uncovered. In group 2, the bowel was repositioned, and the defect was closed by suturing a collagen biomatrix into the abdominal wall. A cesarean section was performed at 140 days' gestation, and macroscopic and histological evaluation was performed. In the five lambs with a gastroschisis, the eviscerated part of the bowel was coalescent, showed extensive adhesions, and was covered by fibrous peel. In group 2, the abdominal wall had closed, with a firm connection to the native abdominal wall. The biomatrix was largely degraded and replaced by connective tissue with collagen and fibroblasts, neovascularisation, and scattered muscle cells. Minor or no adhesions of the bowel and no peel formation were observed. Abdominal wall tissue replacement using a collagen biomatrix was feasible in fetal lambs, resulting in a closed abdominal wall at birth. Immediate closure of the gastroschisis strongly diminished or prevented bowel adhesions and peel formation.
Assuntos
Parede Abdominal/patologia , Colágeno/metabolismo , Feto/patologia , Gastrosquise/patologia , Cicatrização , Animais , Colágeno/ultraestrutura , Modelos Animais de Doenças , Matriz Extracelular/ultraestrutura , Feminino , Gravidez , Carneiro DomésticoRESUMO
OBJECTIVES: To evaluate histological changes in an animal model for bladder exstrophy and fetal repair of the bladder defect with a molecular-defined dual-layer collagen biomatrix to induce fetal bladder wall regeneration. METHODS: In 12 fetal lambs the abdominal wall and bladder were opened by a midline incision at 79 days' gestation. In 6 of these lambs an uncorrected bladder exstrophy was created by suturing the edges of the opened bladder to the abdominal wall (group 1). The other 6 lambs served as a repair group, where a dual-layer collagen biomatrix was sutured into the bladder wall and the abdominal wall was closed (group 2). A caesarean section was performed at 140 days' gestation, followed by macroscopic and histological examination. RESULTS: Group 1 showed inflammatory and maturational changes in the mucosa, submucosa and detrusor muscle of all the bladders. In group 2, bladder regeneration was observed, with urothelial coverage, ingrowth of fibroblasts and smooth muscle cells, deposition of collagen, neovascularization and nerve fibre formation. This tissue replaced the collagen biomatrix. No structural changes of the bladder were seen in group 2. CONCLUSIONS: The animal model, as in group 1, for bladder exstrophy shows remarkable histological resemblance with the naturally occurring anomaly in humans. This model can be used to develop new methods to salvage or regenerate bladder tissue in bladder exstrophy patients. Fetal bladder wall regeneration with a collagen biomatrix is feasible in this model, resulting in renewed formation of urothelium, blood vessels, nerve fibres, ingrowth of smooth muscle cells and salvage of the native bladder.
Assuntos
Materiais Biocompatíveis/uso terapêutico , Extrofia Vesical/cirurgia , Colágeno/uso terapêutico , Doenças Fetais/cirurgia , Feto/cirurgia , Regeneração Tecidual Guiada , Animais , Extrofia Vesical/embriologia , Extrofia Vesical/patologia , Modelos Animais de Doenças , Doenças Fetais/patologia , Feto/patologia , Ovinos/embriologia , Alicerces TeciduaisRESUMO
Despite being one of the most common congenital defects in boys, the etiology of hypospadias remains largely unknown. In this case-referent study, we evaluated a wide spectrum of potential risk factors for hypospadias. Cases were identified from the hospital information system, and referents were recruited through the parents of the cases. Both parents of cases and referents completed written questionnaires that they received through the mail. Logistic regression analyses were used to assess the independent contribution of different factors to the risk of hypospadias. The final database included 583 cases and 251 referents. Hypospadias more often occurred in children whose father had hypospadias (OR=9.7; 95%CI: 1.3-74.0) and in children with a low birth weight (OR=2.3; 95%CI: 1.2-4.2). Indications for elevated risks were found when mothers were DES-daughters (OR=3.5; 95%CI: 0.8-15.6), fathers were subfertile (OR=1.8; 95%CI: 0.7-4.5), the parents had undergone fertility treatment (OR=2.3; 95%CI: 0.9-5.8), and in twin or triplet pregnancies (OR=2.0; 95%CI: 0.8-5.1). Maternal use of iron supplements (OR=2.2; 95%CI: 0.8-6.0), maternal smoking (OR=1.5; 95%CI: 1.0-2.4), paternal prescriptive drug use (OR=2.6; 95%CI: 1.1-6.6), and paternal exposure to pesticides (OR=2.1; 95%CI: 0.6-7.1) during the 3 months immediately prior to conception or in the first trimester of pregnancy also appeared to increase the risk of hypospadias. The associations found in this study support the hypothesis that genetic predisposition, placental insufficiency, and substances that interfere with natural hormones play a role in the etiology of hypospadias.