Rita Gobet

Cross-linking of the dermo-epidermal junction of skin regenerating from keratinocyte autografts. Anchoring fibrils are a target for tissue transglutaminase

Since transglutaminases create covalent gamma-glutamyl-epsilon-lysine cross-links between extracellular matrix proteins they are prime candidates for stabilizing tissue during wound healing. Therefore, we studied the temporo-spatial expression of transglutaminase activity in skin regenerating from cultured epithelial autografts in severely burned children by the specific incorporation of monodansylcadaverine into cryostat sections from skin biopsies obtained between 5 d to 17 mo after grafting. The dansyl label was subsequently immunolocalized in the epidermis, dermal connective tissue, and along the basement membrane. Incubation of cryosections of normal and regenerating skin with purified tissue transglutaminase confirmed the dermo-epidermal junction and the papillary dermis as targets for this enzyme and revealed that in regenerating skin transamidation of the basement membrane zone was completed only 4-5 mo after grafting. Immunoelectron microscopy revealed that three distinct regions on the central portion of anchoring fibrils were positive for monodansylcadaverine in normal skin which were negative during the initial phase of de novo formation of anchoring fibrils in regenerating skin. Biochemically, we identified collagen VII as potential substrate for tissue transglutaminase. Thus, tissue transglutaminase appears to play an important role not only in cross-linking of the papillary dermis but also of the dermo-epidermal junction in particular.

Engineering functional bladder tissues

Purpose: End stage bladder disease can seriously affect patient quality of life and often requires surgical reconstruction with bowel tissue, which is associated with numerous complications. Bioengineering of functional bladder tissue using tissue‐engineering techniques could provide new functional tissues for reconstruction. In this review, we discuss the current state of this field and address different approaches to enable physiologic voiding in engineered bladder tissues in the near future. Materials and Methods: In a collaborative effort, we gathered researchers from four institutions to discuss the current state of functional bladder engineering. A MEDLINE® and PubMed® search was conducted for articles related to tissue engineering of the bladder, with special focus on the cells and biomaterials employed as well as the microenvironment, vascularisation and innervation strategies used. Results: Over the last decade, advances in tissue engineering technology have laid the groundwork for the development of a biological substitute for bladder tissue that can support storage of urine and restore physiologic voiding. Although many researchers have been able to demonstrate the formation of engineered tissue with a structure similar to that of native bladder tissue, restoration of physiologic voiding using these constructs has never been demonstrated. The main issues hindering the development of larger contractile tissues that allow physiologic voiding include the development of correct muscle alignment, proper innervation and vascularization. Conclusion: Tissue engineering of a construct that will support the contractile properties that allow physiologic voiding is a complex process. The combination of smart scaffolds with controlled topography, the ability to deliver multiple trophic factors and an optimal cell source will allow for the engineering of functional bladder tissues in the near future. Copyright

99mTechnetium dimercapto-succinic acid renal scintigraphy abnormalities in infants with sterile high grade vesicoureteral reflux

PURPOSE Although vesicoureteral reflux associated with bacteriuria may cause renal scarring, sterile reflux is thought not to cause renal injury. We determined the incidence and associated characteristics of renal abnormalities using 99mtechnetium(Tc) dimercapto-succinic acid (DMSA) renal scintigraphy in infants with high grade vesicoureteral reflux but no history of urinary tract infection. MATERIALS AND METHODS We retrospectively reviewed the results of 99mTc-DMSA renal scintigraphy and renal ultrasonography performed during the first 6 months of life in infants with vesicoureteral reflux detected during the postnatal evaluation of prenatal hydronephrosis or sibling reflux screening. Those with a history of urinary tract infection, or evidence of ureteropelvic junction or bladder outlet obstruction were excluded from study. RESULTS Of the 28 male and 6 female infants who met study criteria vesicoureteral reflux was bilateral in 25 and unilateral in 9. Reflux grade was IV or V, II or III and I in 38, 18 and 3 of the 59 refluxing renal units, respectively. 99mTc-DMSA renal scintigraphy revealed parenchymal abnormalities in 24 refluxing renal units (41%) in 22 patients (65%), of whom 19 (86%) were male and 15 (68%) had bilateral reflux. We noted differential uptake less than 40% with and without cortical defects in 10 and 7 refluxing units, respectively, and cortical defects only in 7. Of the 24 refluxing units with abnormalities 21 were associated with grade IV or V and 3 with grade II or III reflux. Ultrasound showed evidence of renal injury in only 7 of the 17 patients (41%) in whom 99mTc-DMSA scintigraphy was abnormal. CONCLUSIONS In our study the majority of infants with high grade reflux had decreased differential function and/or cortical defects. Parenchymal defects detected by 99mTc-DMSA renal scintigraphy were often not identified by renal ultrasound. Therefore, 99mTc-DMSA renal scintigraphy is especially useful for initially evaluating infants with high grade, sterile vesicoureteral reflux.

Medial clavicular epiphysiolysis in children: the so-called sterno-clavicular dislocation

We retrospectively reviewed six pediatric cases of medial clavicular injury, i.e., epiphyseal separation (Salter/Harris type I or II injury), diagnosed between 1993 and 1997. The clavicular metaphysis was displaced posteriorly in three cases and anteriorly in three. On conventional radiographic views the diagnosis was initially missed in two of three retrosternal dislocations. A special X-ray projection (described by Heinig) or computed tomography (CT) permitted correct diagnosis. Anterior dislocations were immediately and correctly diagnosed. Closed reduction successfully treated retrosternal displacement in two of the three patients. The third patient needed open reduction and internal fixation. Open reduction and internal fixation had to be performed in all three patients with anterior displacement. Follow-up assessment showed perfect functional results in all cases. Direct visualization during open reduction, which was necessary in four of six cases, yielded clear evidence that the so-called sternoclavicular dislocation in children and young adults is, in fact, a fracture of the medial growth plate with posterior or anterior displacement of the metaphysis.

Efficacy of cultured epithelial autografts in pediatric burns and reconstructive surgery

BACKGROUND Cultured epithelial autografts are regularly used in burn patients, but they have not been tested in patients undergoing reconstructive surgery. The aim of this study was to analyze and compare the efficacy of cultured grafts in both burn and reconstructive surgery patients. METHODS In six children with severe and massive burns, full-thickness areas were grafted with cultured grafts. In another six children with hypertrophic or hyperpigmented scars, or both, cultured grafts were used to cover defects resulting from scar excision or deep dermabrasion. RESULTS In burn surgery the final cover rate averaged 60% (range, 0% to 100%). The functional and cosmetic results were good and at least equivalent to results after conventional grafting. Fragility, infection, and, in particular, mechanical instability of cultured grafts during the first weeks after transplantation were the main problems encountered. In reconstructive surgery the final cover rate was 100% in all patients. The functional and cosmetic results were very good and considered better than those obtained by using conventional grafting techniques. No major management problems were encountered. CONCLUSIONS In massively burned children, cultured epithelial autografts represent an effective additional and potentially lifesaving method to conventional grafting. Questions remain regarding the use of this technique to treat less severe burns. For resurfacing-type scar revisions, cultured epithelial autografts yield excellent results that appear to be superior to those of conventional techniques.

The Penile Perception Score: An Instrument Enabling Evaluation by Surgeons and Patient Self-Assessment After Hypospadias Repair

PURPOSE Studies of the outcome of hypospadias repair must document quality, including assessment of complications and appraisal of appearance. To our knowledge the Pediatric Penile Perception Score is the first validated instrument for the outcome assessment of hypospadias repair in prepubertal males by surgeons and patients. We validated the instrument for adult genitalia. MATERIALS AND METHODS Standardized photographic documentation was prepared for 19 men after hypospadias repair and 3 with normal genitalia after circumcision. This was sent to 21 urologists, who rated the outcome with a questionnaire comprising items on the penile meatus, glans, shaft skin and general appearance. Each item was rated with a 4-point Likert scale. The Penile Perception Score is a sum score of all items. Patients were asked to provide a self-assessment with the same instrument. RESULTS When calculated with the ICC and the rank correlation using Kendall W, concordance among urologist scores was fair and good (0.46 and 0.64, respectively, p <0.001). Instrument stability was 0.78, indicating good reproducibility. Using the Spearman rank correlation coefficient general appearance correlated well with single items, including the meatus (r = 0.93, p = 0.000), glans (r = 0.92, p = 0.000) and shaft skin (r = 0.89, p = 0.000). No significant differences were found between patient and urologist Penile Perception Scores. CONCLUSIONS The Penile Perception Score is a reliable instrument for urologist assessment and self-assessment of postpubertal genitalia after hypospadias repair. The instrument can be recommended for all age groups because it was previously validated for the pediatric population.

Is there an ideal age for hypospadias repair? A pilot study.

OBJECTIVE To assess whether repairing hypospadias before or after 18 months affects psychological adjustment, health-related quality of life (HRQoL) and surgical outcome. PATIENTS AND METHODS Seventy-seven boys aged 6-17 years were assigned to one of two groups, according to whether they had a hypospadias repair before or after the age of 18 months. The surgical outcome was assessed using the pediatric penile perception score by non-involved urologists. A psychologist interviewed the patients to assess HRQoL and gender-role behavior. The childs psychological adjustment was assessed with a questionnaire for parents. RESULTS The surgical outcome and complication rate were not significantly different between groups. A covariance analysis of HRQoL, gender-role behavior and psychological adjustment as a function of age at the last operation with current age as covariant was performed, but differences did not reach significance. CONCLUSION This study does not provide evidence to support recommendations concerning the ideal age for hypospadias repair. In the absence of evidence of a benefit of early surgery, anesthesia-related risk factors must be considered when operating in very early infancy. Large, prospective studies, measuring surgical and psychological outcome with similar instruments to those presented may reveal whether there is a true ideal age for hypospadias surgery.

Long-term follow up (37–69 years) of patients with bladder exstrophy treated with ureterosigmoidostomy: Uro-nephrological outcome

OBJECTIVE To describe the urological and nephrological long-term outcome of patients born with classical bladder exstrophy treated with bilateral ureterosigmoidostomies in early childhood. PATIENTS AND METHOD Out of 42 patients born with bladder exstrophy in Switzerland between 1937 and 1968, 25 participated in this study; seven had died, seven were lost to follow up and three refused consent. Assessment included chart review, clinical examination, and assessment of renal function and morphology. RESULTS After a follow-up period of 37-69 years ((mean 50 years), 13 of the 25 participants (52%) had their ureterosigmoidostomy still in place. All others had different forms of urinary diversions. Fifteen (60%) patients had normal renal function or mild chronic kidney disease as assessed by estimated glomerular filtration rate. Three patients were on renal replacement therapy. MRI (n=16) showed 10 morphologically normal kidneys. One patient suffered from adenocarcinoma of the colon, five had benign colonic polyps, one urethral papillary carcinoma and 18 no evidence of tumor. CONCLUSION The majority of our patients have normal or mildly impaired renal function and a well functioning ureterosigmoidostomy. This is remarkable, given the fact that ureterosigmoidostomies are considered to be refluxing high-pressure reservoirs at risk of renal injury and malignancy.

Fetal partial urethral obstruction causes renal fibrosis and is associated with proteolytic imbalance.

PURPOSE We determine whether fetal bladder outlet obstruction induces renal fibrosis, and is associated with an alteration in the regulation of connective tissue degradation and the presence of fibrogenic interstitial cells. MATERIALS AND METHODS Partial bladder outlet obstruction was surgically induced in 33 fetal sheep at 95 days of gestation. These animals and 24 normal age matched controls were sacrificed at 109, 116 and 135 (term) days of gestation, and the kidneys were rapidly retrieved, drained and weighed. Representative whole kidney samples were snap frozen for assessment of deoxyribonucleic acid, protein and collagen content. Morphometric analysis and alpha-smooth muscle actin immunohistochemistry were performed on histological specimens from formalin fixed kidneys. Tissue extract from fresh kidney specimens were analyzed for metalloproteinase and tissue inhibitor of metalloproteinase activity. Urine samples obtained at the time of sacrifice were analyzed for electrolyte, creatinine and N-acetyl glucosaminidase excretion. RESULTS All obstructed kidneys were hydronephrotic and larger than age matched controls. Obstructed kidneys at term showed interstitial fibrosis, as measured by increased extracellular matrix volume fraction (45% in male obstructed kidneys versus 2.5% in normal male kidneys, p = 0.0004), increased total collagen content (120 mg./kidney in male obstructed versus 20 mg. in normal male animals, p = 0.016) and collagen/deoxyribonucleic acid content per kidney (2.78 versus 0.53 mg./mg., p = 0.016). Metalloproteinase-1 activity was significantly lower in obstructed kidneys (210 versus 380 U./mg. protein in normal kidneys). Tissue inhibitor of metalloproteinase activity was undetectable in both groups. The presence of an increased population of myofibroblasts often associated with fibrotic processes was seen by alpha-smooth muscle actin staining which was localized to interstitial cells throughout the cortex in obstructed kidneys. CONCLUSIONS Fetal partial bladder outlet obstruction induces renal interstitial fibrosis as early as 2 weeks after obstruction. A possible mechanism for this process is a shift in proteolytic activity to reduce matrix degradation in obstructed kidneys. These changes might be mediated by the increased number of fibrogenic interstitial cells. The observations suggest several potential approaches to developing an understanding of congenital obstructive uropathy.

EXPERIMENTAL VESICOURETERAL REFLUX IN THE FETUS DEPENDS ON BLADDER FUNCTION AND CAUSES RENAL FIBROSIS

PURPOSE Prenatal diagnosis allows the early detection of vesicoureteral reflux in an increasing number of newborns, some of whom are born with impaired kidney function. This situation challenges our current understanding of the pathophysiology, natural history and, therefore, treatment of reflux. We created a fetal sheep model of vesicoureteral reflux to study the mechanisms of fetal reflux nephropathy. MATERIALS AND METHODS Vesicoureteral reflux was induced in fetal sheep at 95 days of gestation (term 140 days) by open bladder incision of the intravesical ureteral tunnel. All animals underwent urachal ligation and in female subjects mild bladder outlet obstruction was created with a gold ring. RESULTS At term reflux was detected in 18 of 28 renal units by filling cystography. Refluxing kidneys were hydronephrotic and larger than normal. At term mean kidney weight was 21.1 gm. (range 12.2 to 35.0) in male subjects with reflux compared to 8.5 gm. (range 6.5 to 11.3) in normal male subjects (p <0.001) and 11.5 gm. (range 8.5 to 15.8) in male subjects with urachal ligation only (p = 0.035). In female subjects there was no change in renal weight. Renal histology revealed a thin, structurally normal cortex with small subcortical cysts and a hypoplastic medulla with mesenchymal tissue replacing normal ducts. Total mean renal collagen content was significantly increased to 51.7 mg. (range 35 to 81) in the refluxing kidneys of male animals, while it was 23.8 mg. (range 12.1 to 38.4) in normal male animals (p = 0.03). The fractional excretion of sodium was elevated in refluxing kidneys based on sodium-to-creatinine ratios in bladder urine. CONCLUSIONS In a novel model of fetal vesicoureteral reflux we showed that prenatal reflux nephropathy is characterized by altered renal growth regulation, structural maldevelopment without overt dysplasia, excess matrix deposition and impaired excretory function.