Significance of an endoscopically injected nodule detected on ultrasound as a predictive factor for the resolution of vesicoureteral reflux.

Endoscopic treatment of vesicoureteral reflux (VUR) is feasible in pediatric patients. As endoscopic injection has a lower success rate than ureteral reimplantation, a postoperative voiding cystourethrogram (VCUG) is usually performed. The present study evaluated whether the presence of a nodule on noninvasive ultrasound could predict the resolution of VUR and replace invasive VCUG. Patients who received an injection of endoscopic bulking agent for VUR from January 2005 to December 2010 were evaluated retrospectively. It was evaluated whether a nodule, an echogenic mass lesion distinguished from the bladder at the ureteral orifice, was present on the ultrasound one month postoperatively. The success of the injection in the group with nodules was compared with that of the group without nodules by VCUG 3 months postoperatively. A total of 149 patients (220 ureters) met the inclusion criteria. The mean age at surgery was 3.5 years (range, 0.6-18 years). The overall success rate was 73.2%. A nodule was present in 152 cases (69.1%). The group with nodules had a higher success rate than the group without nodules (84.2%, 128/152 vs. 48.5%, 33/68, respectively; P<0.001). According to multivariate analysis, injection nodules were a predictive factor for the success of the endoscopic injection (odds ratio, 6.050; P<0.001). The failure rate increased with increasing injection volume. The sensitivity of sonographic injection nodules for predicting success was 79.5% and the specificity was 59.3%. To conclude, the presence of a postoperative nodule can predict the resolution of VUR.

Risk factors for contralateral patent processus vaginalis determined by transinguinal laparoscopic examination.

Concurrent contralateral inguinal exploration in children with unilateral hernia or hydrocele is a subject of debate. The aim of the present study was to investigate the incidence of contralateral patent processus vaginalis (CPPV) using transinguinal laparoscopy (inguinoscopy). In addition, the risk factors of CPPV were evaluated in order to facilitate the selection of appropriate candidates for contralateral examination. A total of 119 patients who presented with unilateral hydrocele, inguinal hernia or cryptorchidism between 2001 and 2008 underwent inguinoscopy during the ipsilateral surgery. All data were collected prospectively. The incidence of CPPV was investigated and the risk factors affecting the presence of CPPV were analyzed. Among these patients, 29 individuals (24.4%) had CPPV confirmed by inguinoscopy. No surgical complications were observed during the inguinoscopy. Cases with suspicious ultrasound findings were at a higher risk of CPPV than cases with normal findings (odds ratio, 13.800; P=0.004). A history of contralateral disease was also found to be a significant risk factor (odds ratio, 4.008; P=0.019). The present study identified that the significant risk factors for CPPV were suspicious findings on ultrasound examination and a history of contralateral disease. Therefore, it is concluded that performing inguinoscopy in children with these risk factors is beneficial.

Urothelial Tumors of the Urinary Bladder in Two Adolescent Patients: Emphasis on Follow-up Methods.

Here we describe two cases of papillary urothelial neoplasm of low malignant potential in adolescent boys. One case was a 16-year-old boy with a polypoid mass beside the right ureteral orifice and the other case was a 13-year-old boy with a papillary mass beside the left ureteral orifice. The initial presentation was hematuria in both cases and the bladder mass was detected by ultrasonography. Complete resection of the bladder tumor was performed by using an 11-Fr pediatric resectoscope. Follow-up has been performed with urine analysis, urine cytology, and bladder ultrasonography or cystoscopy every 3 months with no evidence of recurrence.

Role of Urodynamic Study in Nocturnal Enuresis: Urodynamic Findings and Treatment Outcome Correlation in Children with Pharmacotherapy-resistant Monosymptomatic Nocturnal Enuresis or Severe Non-monosymptomatic Nocturnal Enuresis.

OBJECTIVES: This study aimed to determine whether or not a urodynamic study (UDS) is beneficial for management of pediatric nocturnal enuresis (NE), especially in pharmacoresistant monosymptomatic nocturnal enuresis (PRMNE) or severe non-monosymptomatic nocturnal enuresis (NMNE) patients. METHODS: Children with PRMNE or severe NMNE who underwent a UDS for the process of NE treatment were retrospectively reviewed. The UDS findings of patients and treatment outcomes of subsequent tailored therapies according to the UDS findings were analyzed. RESULTS: A total of 80 children (50 boys and 30 girls, mean age 8.4 ± 2.2 years), 19 of which were diagnosed with PRMNE and 61 of which were diagnosed with NMNE, were included in the final analysis. Of the 19 PRMNE children, 12 (63.2%) demonstrated abnormal UDS findings. Ten demonstrated detrusor overactivity (DO) with or without decreased cystometric bladder capacity (CBC); the treatment outcomes markedly improved in all of the children after anticholinergics were added to the initial desmopressin therapy. Biofeedback was found to be helpful for two children with detrusor-sphincter dyssynergia. All of the total 61 children with NMNE demonstrated abnormal urodynamic findings of DO with or without decreased CBC, and 42 (68.9%) achieved more than partial response (> 50% decrease in the number of wet nights) when given a combination therapy of anticholinergics and desmopressin. CONCLUSIONS: The urodynamic findings were helpful for guiding children with PRMNE in the proper choice of further treatment strategies. A routine UDS should not be recommended prior to a first-line combination treatment in children with NMNE.

A novel technique of concealed penis repair.

INTRODUCTION: The correction of concealed penis requires challenging surgical techniques. We describe a novel technique involving inner preputial skin substitution after degloving through a ventral approach that allows sufficient penile skin coverage and evaluated the efficacy and safety of our technique. METHODS: From February 2009 to March 2009, 12 boys with concealed penises underwent correction via our novel technique. We administered a questionnaire about penile size, morphology, and voiding status to parents to evaluate their satisfaction with the results. RESULTS: The mean age of patients at the time of operation was 4.71 ± 4.01 years, and the mean duration of follow-up was 5.11 ± 1.45 months. Parental satisfaction grades (0 to 4) concerning penile size increased from 1.00 ± 1.04 to 2.83 ± 0.94 (p = 0.002) after surgery, penile morphology from 1.00 ± 0.95 to 3.00 ± 0.85 (p = 0.001), and voiding status from 1.08 ± 1.00 to 3.33 ± 0.78 (p < 0.001) at the last follow-up visit. The parental satisfaction rates regarding postoperative changes in size and morphology were 83.3 and 91.7%, respectively. Nine parents (75%) said they would recommend the surgery to others. There were no complications, including postoperative hematoma, infection, and tissue necrosis. CONCLUSION: Our method of correcting concealed penis was demonstrated to be technically feasible and safe. It provides a good cosmetic appearance and excellent parental satisfaction ratings regarding size, morphology, and voiding function.

Factors affecting the outcome of extracorporeal shock wave lithotripsy for unilateral urinary stones in children: a 17-year single-institute experience.

PURPOSE: Extracorporeal shock wave lithotripsy (ESWL) is a first-line treatment for pediatric urinary stone disease. We aimed to determine the factors affecting the outcome of ESWL for unilateral urinary stones in children. MATERIALS AND METHODS: A total of 81 pediatric patients aged 0 to 16 years with urinary stones treated by ESWL from January 1995 through May 2012 were retrospectively reviewed. All patients were required to have unilateral urinary stone disease. Children who underwent other surgical procedures before ESWL were excluded. Outcomes evaluated after ESWL were the stone-free rate at 3 months after ESWL, success within a single session, and success within three sessions. Factors affecting the success within three sessions were also analyzed. RESULTS: The final analysis was for 42 boys and 22 girls (mean age, 9.2±5.2 years). Of these 64 patients, 58 (90.6%) were treated by ESWL without other surgical procedures and 54 (84.4%) were successfully treated within three ESWL sessions. In the multivariate analysis, multiplicity (odds ratio [OR], 0.080; 95% confidence interval [CI], 0.012 to 0.534; p=0.009) and large stone size (>10 mm; OR, 0.112; 95% CI, 0.018 to 0.707; p=0.020) were significant factors that decreased the success rate within three ESWL sessions. CONCLUSIONS: Most of the pediatric urinary stone patients in our study (90.6%) were successfully treated by ESWL alone without additional procedures. If a child has a large urinary stone (>10 mm) or multiplicity, clinicians should consider that several ESWL sessions might be needed for successful stone fragmentation.

Efficacy and safety of propiverine in children with overactive bladder.

PURPOSE: Antimuscarinic therapy remains one of the most common forms of therapy for overactive bladder (OAB) in children. However, few clinical studies on the outcomes of antimuscarinics in children with OAB have been published. Therefore, we evaluated the efficacy and safety of propiverine, which is frequently prescribed for the treatment of pediatric OAB. MATERIALS AND METHODS: We retrospectively reviewed children with OAB treated with propiverine within the past 5 years. The response rates were compared between the non-urge incontinence (non-UI) and urge incontinence (UI groups). The cumulative response rate by treatment duration was also compared between the two groups. RESULTS: Among a total of 68 children, 50 children (73.5%) experienced UI. The overall response rate was 86.8%. Functional bladder capacity after treatment was 150 ml, which represented an increase compared with the value (140 ml) before treatment. The voiding frequency per day decreased from 14.0 to 8.5 times. The overall response rate (88.0%) in the non-UI group was not significantly different from that seen in the UI group (83.3%; p>0.05). In non-UI children, the cumulative response rates were 36.0%, 54.0%, 68.0%, 74.0%, 76.0%, and 78.0% at 4, 8, 12, 16, 20, and 24 weeks, respectively. The cumulative response rates in the UI children were 11.1%, 33.3%, 44.4%, 50.0%, 50.0%, and 55.6%, respectively during the same respective time periods. Adverse effects were identified in only two (2.9%) patients, and neither case was severe. CONCLUSIONS: Propiverine is effective and well tolerated as a treatment for children suffering from OAB with or without UI.

A novel WT1 gene mutation in a patient with Wilms' tumor and 46, XY gonadal dysgenesis.

Denys-Drash syndrome (DDS) is a rare genetic disorder featuring the triad of Wilms' tumor, early-onset renal failure, and 46, XY disorder of sex development. DDS is usually caused by heterozygous missense mutations in the zinc-finger region of the WT1 gene. The most frequent constitutional WT1 mutations in DDS patients are missense mutations in exons 8 and 9. We present a new case of variable DDS in a child who was found to have a novel heterozygous missense mutation in exon 7 (c.905G>T) and a splicing mutation in exon 6 (IVS6-1G>T).

The efficacy of enuresis alarm treatment in pharmacotherapy-resistant nocturnal enuresis.

OBJECTIVES: To assess the efficacy of enuresis alarm (EA) treatment in pharmacotherapy-resistant nocturnal enuresis (PRNE). MATERIAL AND METHODS: A retrospective study was performed in children who received EA treatment as a form of combination treatment for PRNE from June 1999 to December 2007. The children included 54 monosymptomatic nocturnal enuresis (MNE) children who had partial response or nonresponse to desmopressin (group 1), 25 nonmonosymptomatic nocturnal enuresis (NMNE) children who had partial response or nonresponse to extended release oxybutynin plus desmopressin (group 2), and 21 MNE or NMNE children who relapsed after responding fully to first-line pharmacotherapy (group 3). EA treatment outcomes were determined as outlined by the International Children's Continence Society Standardisation Committee. RESULTS: Overall, 50% and 53.7%, 40%, and 52.4% of children in groups 1, 2, and 3, respectively, responded fully to EA treatment (no significant differences). In groups 1 and 2, 54.4% (31/57) of the partial responders and 36.4% (8/22) of the nonresponders showed full response (FR) after EA treatment was initiated. Of the children with small bladder capacities, 56.7% showed FR. Of the full responders in each group, 60.7%, 88.9%, and 54.5% of groups 1, 2, and 3, respectively, did not have relapse 6 months after cessation of treatment. CONCLUSIONS: Overall, half of the total population achieved FR, and continued success was observed in more than half of full responders irrespective of the groups. Thus, adding EA treatment to pharmacotherapy is an effective second-line therapeutic strategy for children with PRNE.

Efficacy of desmopressin and enuresis alarm as first and second line treatment for primary monosymptomatic nocturnal enuresis: prospective randomized crossover study.

PURPOSE: We compared the efficacy of desmopressin and enuresis alarm as first and second line treatment options for monosymptomatic nocturnal enuresis. MATERIALS AND METHODS: A total of 104 children with monosymptomatic nocturnal enuresis were randomly assigned to either desmopressin (54) or enuresis alarm (50) as first line treatment. Following 12 weeks of first line treatment children with a full response were evaluated for relapse 12 weeks after withdrawal of treatment. Children with partial or no response were switched to the alternative treatment and then evaluated after 12 weeks of crossover treatment. Relapse was defined as more than 1 episode of bedwetting monthly. RESULTS: Following first line treatment 77.8% of the desmopressin group and 82% of the enuresis alarm group achieved a successful result, including full response in 37% and 50% of the groups, respectively (p=0.433). Of the children with a full response 50% in the desmopressin group and 12% in the enuresis alarm group experienced a relapse when treatment stopped (p=0.005). Following second line crossover treatment 71.4% of the enuresis alarm-desmopressin group and 67.8% of the desmopressin-enuresis alarm group achieved a successful result, including full response in 47.6% and 45.2% of the groups, respectively (p=0.961). CONCLUSIONS: There was no difference between desmopressin and enuresis alarm during treatment for achieving dryness, but the chance of relapse after treatment stopped was higher following desmopressin. Switching to the alternative treatment following partial or no response provided an additional benefit.

Characteristics and clinical outcomes of renal cell carcinoma in children: a single center experience.

The present study aimed to assess the clinicopathological characteristics and the prognosis of renal cell carcinoma (RCC) in children. We retrospectively reviewed the medical records of 11 Korean children (7 boys and 4 girls) undergoing radical nephrectomy for RCC between September 1997 and January 2008 at our institution. Mean age at diagnosis was 12.7 years. The presenting clinical signs and symptoms included gross hematuria (55%), abdominal mass (45%) and abdominal pain (45%). Two (18%) of the cases were discovered incidentally. Five (45%) were conventional clear cell, four (36%) were papillary, one was a chromophobe and one was a translocation carcinoma. Seven children were T1N0M0, three were T2N0M0 and one child was T1N1M0. While one patient with T2 disease died of disease 89 months postoperatively, the remaining 10 patients, including a patient with regional nodal disease who received adjuvant immunochemotherapy, survived without evidence of recurrence at a mean follow-up of 6.8 years. Our data suggest that the clinical presentation, pathological characteristics and clinical behavior of pediatric RCC are different from those of adult RCC.

An epidemiologic study of voiding and bowel habits in Korean children: a nationwide multicenter study.

OBJECTIVES: To evaluate the prevalence of abnormal voiding and bowel habits in healthy children, and to identify possible relationships between personal and familial factors and voiding and/or bowel habits. METHODS: A randomly selected cross-sectional study was conducted in 19,240 children (5-13 years old) nationwide in the Republic of Korea. Parents were asked to complete questionnaires, which included items about nocturnal enuresis, daytime dysfunctional voiding symptoms (DVSs), and abnormal bowel habits (ABHs). Rates and associated factors were investigated. RESULTS: The overall rates of DVS, ABH, and of both were 46.4%, 31.3%, and 18.4%, respectively, and nocturnal enuresis was reported in 919 (5.6%). Daytime dysfunctional voiding symptoms were as follows: increased voiding frequency in 419 (2.5%), decreased voiding frequency in 720 (4.4%), urgency in 2740 (16.6%), daytime incontinence in 1854 (11.2%), urge incontinence in 2775 (16.8%), and holding maneuvers or postponed voiding in 3888 (23.5%). ABHs were as follows: constipation by frequency in 1103 (6.7%), constipation by the Bristol scale in 1941 (11.8%), fecal incontinence in 1293 (7.8%), and difficult or painful defecation in 2609 (15.8%). Delayed stool control, history of urinary tract infection, and a double-income family were positively associated with DVS or ABH or both. Furthermore, fathers with a low level of education and a low income were found to be associated with higher risk of ABH or DVS or both. CONCLUSIONS: Delayed stool control, a history of urinary tract infection, a low paternal level of education, a double-income family, and a lower family income had a negative effect on voiding or bowel habits in children.

Prevalence and associated factors of overactive bladder in Korean children 5-13 years old: a nationwide multicenter study.

OBJECTIVES: To estimate the prevalence of overactive bladder (OAB) in Korean children, 5-13 years of age, and to assess the associated factors for OAB. METHODS: A randomly selected cross-section study was conducted in 26 kindergartens and 27 elementary schools nationwide in Korea. A total of 19 240 children were included; a parent was asked to complete the questionnaires, which included items about OAB and voiding and defecating habits. OAB was defined as urgency with or without urge incontinence, usually with increased daytime frequency and nocturia (International Children's Continence Society, 2006). Its prevalence and associated factors were also investigated. RESULTS: The response rate for the questionnaires was 85.84%. The overall prevalence of OAB was 16.59%. The prevalence of OAB decreased with age from 22.99% to 12.16% (P = .0001). The overall incidence of wet and dry OAB was 26.97% and 73.03%, respectively. Compared with normal children, those with OAB had a greater prevalence of nocturnal enuresis, constipation, fecal incontinence, urinary tract infection, delayed bladder control, and poor toilet facilities (P < .05). The incidence of increased daytime frequency and urge incontinence was 3.69% and 2.31% (P = .009) and 26.97% and 14.78% (P = .0001) in OAB and non-OAB children, respectively. The corresponding prevalence decreased with age from 5.04% to 3.06% and from 45.74% to 18.50% in OAB children (P = .0001). CONCLUSIONS: The overall prevalence of OAB in Korean children, 5-13 years of age, was 16.59% and decreased with age. Nocturnal enuresis, constipation, fecal incontinence, history of urinary tract infection, delayed bladder control, and poor toilet facilities might be factors associated with the development of OAB.

Clinical inconsistency of lower urinary tract symptoms between questionnaire and bladder diary in children with nocturnal enuresis.

PURPOSE: Classification of pediatric enuresis into monosymptomatic nocturnal enuresis and nonmonosymptomatic nocturnal enuresis is crucial for management because these cases differ in many clinical respects. However, correct classification is frequently confused based on questionnaire and/or bladder diary. We tested the consistency of information obtained on lower urinary tract symptoms between our questionnaire and bladder diary. MATERIALS AND METHODS: A total of 108 physically and neurologically normal patients (mean age 7.2 years) with enuresis were included in the study. All patients filled out the questionnaire and 3-day bladder diary completely. Our questionnaire included items on urinary frequency, daytime incontinence and voiding postponement. A total of 93 patients underwent uroflowmetry and residual urine measurements. We analyzed information on lower urinary tract symptoms from the questionnaire and the bladder diary, as well as the uroflowmetry results. RESULTS: The percentage of children with nonmonosymptomatic nocturnal enuresis was 89.8% and 68.5% based on the questionnaire and bladder diary, respectively. In comparisons between the questionnaire and bladder diary we did not observe any significant consistency regarding urinary frequency (p = 0.912), daytime incontinence (p = 0.356) or voiding postponement (p = 0.505). Maximum voided volume, average voided volume and percent maximum voided volume/expected bladder capacity of children with monosymptomatic nocturnal enuresis were significantly higher than in those with nonmonosymptomatic nocturnal enuresis based on the bladder diary (p = 0.006, 0.001 and 0.041, respectively). By contrast, no significant difference was found between children with monosymptomatic and nonmonosymptomatic nocturnal enuresis based on the questionnaire (p = 0.559, 0.597 and 0.947, respectively). CONCLUSIONS: Significant discrepancies in lower urinary tract symptoms were observed between the questionnaire and bladder diary. Therefore, a detailed history and discussion with the patient and family should be performed to correlate with the questionnaire and bladder diary for the proper treatment of children with enuresis. An updated questionnaire containing a detailed scoring system may be needed.

A dermal equivalent developed from fibroblast culture alone: effect of EGF and insulin.

We have recently developed a new dermal equivalent without exogenous materials by culturing dermal fibroblasts alone in serum-containing medium treated with several supplements. In this study, we investigated the effects of epidermal growth factor (EGF) and insulin on the formation of a dermal equivalent. After cultured dermal fibroblasts reached a confluence in serum-containing medium, they were treated with EGF or insulin. The combined effects of EGF and insulin were also studied. Macroscopically, in contrast to the culture without supplement, the addition of EGF or insulin produced a fibrous sheet. The combination of EGF and insulin showed a more marked effect than a single factor. Histologically, EGF or insulin alone induced a three-dimensional tissue containing several layers of fibroblasts. The combination of EGF and insulin produced a thicker tissue. It was composed of abundant extracellular matrix containing fibroblasts, suggesting a dermis-like tissue. It revealed collagen fibers by Masson-trichrome staining. Immunohistochemically, the components of dermal extracellular matrix such as type 1 collagen, elastin, and fibrillin-1 were diffusely expressed. Ultrastructurally, a large number of collagen fibrils with cross-striated patterns were found around the fibroblasts. These results showed that a dermal equivalent could be formed by culturing dermal fibroblasts alone postconfluently in serum-containing medium with EGF and insulin. They suggest that the two factors play an important role in the formation of a dermal equivalent.

Nomogram of fetal renal growth expressed in length and parenchymal area derived from ultrasound images.

PURPOSE: We constructed nomograms of fetal renal length and parenchymal area derived from ultrasound images to develop a standard for normal fetal renal growth. MATERIALS AND METHODS: Longitudinal and transverse ultrasound renal images from 216 normal fetuses (16 to 41 weeks of gestation) were evaluated to construct growth charts. We measured the parenchymal area as well as the longitudinal and transverse lengths of each kidney using computer software for image analysis. Data were separately plotted as a mean +/- 2 SD determined by polynomial regression analysis. RESULTS: Nomograms for a renal growth chart were constructed independently for the right and left fetal kidneys. No statistically significant difference was noted between the right and left renal measurements. The polynomial regression equations for the left renal longitudinal length and parenchymal area, respectively, were y = -0.0002x(3) + 0.0139x(2) - 0.2162 x 2.3929 (r(2) = 0.9842), and y = -0.0009x(3) + 0.0724x(2) - 1.5643 x 11.68 (r(2) = 0.9779). The longitudinal and transverse fetal renal growth curves displayed significant growth associated with gestational age (p <0.001). Our data on left longitudinal renal length exhibited an intermediate level compared to 2 published Western growth charts. However, statistical comparisons revealed the differences were partially, but not universally, significant. CONCLUSIONS: We present our nomogram of fetal renal growth expressed in length and parenchymal area. To our knowledge this is the first report of a fetal renal growth chart in Asia that includes the parenchymal area. This nomogram may serve as a valuable tool for evaluation of fetal renal growth.

Histological evidences suggest recommending orchiopexy within the first year of life for children with unilateral inguinal cryptorchid testis.

OBJECTIVE: To determine the optimal timing for orchiopexy, we evaluated the histological parameters of the cryptorchid testis. METHODS: We prospectively performed testicular biopsy in a total of 65 consecutive children with palpable unilateral inguinal cryptorchid testes. For controls, we used testicular histological slides from 15 age-matched children with testicular tumor. To investigate the fertility potential, we analyzed the parameters including mean tubular diameter (MTD), mean tubular fertility index (MTFI), germ cell count/tubule (GCC), Sertoli cell index (SCI) and interstitial fibrosis index (IFI). RESULTS: The MTFI and GCC in children < or =1 years of age were significantly higher than those of other older age groups. The MTFI, GCC and IFI were significantly better in patients < or =2 years of age when compared to those of > 2 years. Compared to the controls, the MTFI and GCC in the patients were significantly worse in those aged > 2 years at surgical repair. In the < or =2-year age group, the MTFI and GCC of the cryptorchid testis showed a decreasing tendency with age, which were contrasting with the ascending curves in the control and the curves crossed at 1-2 years of age in each parameter. CONCLUSIONS: To protect fertility potential, we recommend, orchiopexy should be performed within the first year of life, and no later than 2 years of age in patients with palpable inguinal cryptorchid testes.

Clinical and genetic analysis of HLXB9 gene in Korean patients with Currarino syndrome.

Currarino syndrome (CS) is a rare autosomal dominant disease that has been described as a triad of partial sacral agenesis, anorectal anomalies, and a presacral mass. Mutations in the HLXB9 gene have been suggested to be the genetic background of CS. In this study, sequence analysis of the HLXB9 gene was performed in two familial and two sporadic Korean patients showing the clinical features of CS, and two mutations in the HLXB9 gene were identified only in the two familial cases. One mutation (R295W) has been reported previously, and the other (H260_Q261delinsLELLELE) is novel. Consistent with previous observations, the phenotypic expression of the mutation carriers in the CS families varies from mild to severe, including the complete triad. This study confirms that familial CS patients in Korea have the same genetic background as other ethnicities and reaffirms the phenotype variability among CS patients with the same mutation.

A new dermal equivalent: the use of dermal fibroblast culture alone without exogenous materials.

BACKGROUND: During the past decade, several kinds of skin equivalents have been developed. However, the dermal equivalents have all contained exogenous materials, which can be difficult to obtain and a source of infections. OBJECTIVES: The aim of this study was to develop a new dermal equivalent by culturing dermal fibroblasts alone without exogenous materials and to evaluate its applicability in vitro and in vivo. METHODS: The postconfulent cultures of dermal fibroblasts in serum containing medium, that was supplemented with epidermal growth factor, insulin, hydrocortisone, transferrin and triiodothyronine for 3 weeks, produced a fibrous sheet that was visible macroscopically. To construct a skin equivalent, epidermal keratinocytes were cultured on the top of the fibrous sheet at the air-liquid interface. To evaluate its fate in vivo, the fibrous sheet was grafted into a nude mouse. RESULTS: Histologically, the fibrous sheet showed dermis-like tissue that consisted of an extracellular matrix around dermal fibroblasts, and revealed collagen fibers by Masson-trichrome staining. The components of dermal matrix such as type I collagen, type III collagen, elastin, fibrillin-1 and fibronectin were diffusely expressed. Some collagen fibrils were found by electron microscopy. In the skin equivalent, a multilayered epidermis with a horny layer was formed. Some differentiation markers (keratin 1 and 10, and involucrin) and the components of basement membrane (beta4 integrin chain, type IV and VII collagens) were expressed in a similar fashion to those in normal skin in vivo. Ultrastructurally, basement membrane zone such as hemidesmosomes, lamina lucida and lamina densa was found, although it was still incomplete. When the fibrous sheet was grafted in vivo, it revealed blood vessels that were derived from the nude mouse, and persisted for 4 weeks. CONCLUSION: These findings demonstrated that a new dermal equivalent, closely resembling a dermis in vivo, could be constructed by culturing dermal fibroblasts alone in a special culture medium. In addition, the dermal equivalent may be useful for experimental and clinical purposes, such as the reconstruction of a skin equivalent in vitro and grafting in vivo.