A Modified Suture Technique to Improve Scar Appearance in Wounds Under High Tension.

ABSTRACT: The purpose of this study was to introduce a modified suture technique and to compare its effects on skin scar formation with 2 traditional suture methods: simple interrupted suture (SIS) and vertical mattress suture (VMS). Three groups of healthy adult female Sprague-Dawley rats were selected (6 replicates in each group), and the full-thickness skin of 5 cm × 0.2 cm was cut off on the back of the rats after anesthesia. The wounds were then sutured using 1 of the 3 methods for each group: SIS, VMS, and a newly introduced modified vertical mattress suture (M-VMS) technique with the needle reinsertion at the exit point. A traction device was installed on the back of the rats to achieve high tension wounds. The tensile distance was increased by 1 mm every day for 20 days. After 20 days of healing, the hematoxylin-eosin staining method was used for observation of scar morphology. The collagen production rate was measured by Masson staining, and the type I collagen and type III collagen were detected by the immunofluorescence method. Immunohistochemical staining was used to detect the expression of myofibroblast marker α-smooth muscle actin, and real-time quantitative polymerase chain reaction and Western blot techniques were used to detect the expressions of transforming growth factors TGFß1, TGFß2, and TGFß3 to understand the mechanisms of scar formation. Results showed that the quantity and density of collagen fibers were both lower in the M-VMS group than in the other 2 groups. Immunofluorescence results showed that type I collagen was significantly lower, whereas type III collagen was significantly higher in the M-VMS group than in the other 2 groups. The expressions of α-smooth muscle actin and TGFß1 both were lower in the M-VMS group than in the other 2 groups. The expression of TGFß2 and TGFß3 had no obvious difference among the 3 groups. For wounds under high tension, compared with SIS and VMS methods, the M-VMS technique we proposed can reduce scar formation due to the reduction of collagen formation, myofibroblast expression, and TGFß1 expression.

[Effects of different ways in repairing scrotum of pigs with full-thickness burn on apoptosis and bcl-2 protein expression of spermatogenic cells].

OBJECTIVE: To observe the effects of different ways in repairing scrotum of pigs with full-thickness burn on spermatogenesis of testis. METHODS: Twenty male Guizhou miniature pigs were divided into normal control (NC), natural-healing (NH), flap-repairing (FR), and skin-grafting (SG) groups according to the random number table, with 5 pigs in each group. Pigs in NC group were not subjected to any injury. Scrotum of pigs in the latter three groups were inflicted with full-thickness burn. Wounds in NH group healed naturally. Wounds in FR group were repaired with inguinal region flap, and those in SG group with full-thickness skin from lower abdomen. Appearance of scrotum in the latter three groups was observed right after injury, and three months post injury or surgery (PIM or PSM). Specimens of testes of pigs in the latter three groups were obtained in PIM or PSM 3 to detect apoptosis of spermatogenic cells with TUNEL, and bcl-2 protein expression with immunohistochemistry. The same indexes were observed and determined in pigs of NC group. Data were processed with one-way analysis of variance and LSD test. RESULTS: (1) Scrotum of pigs in NC group had skin folds with contraction function. Scrotum of pigs became hard with a leathery appearance right after burn in the other three groups. In PIM or PSM 3, wounds of pigs in NH group healed with scar, and the testes were squeezed into inguinal region. Scrotal skin of pigs in FR group was thick with testes in the scrotum, and that of pigs in SG group was thin with testes in the scrotum. (2) Spermatogenic cells in each level in NC group were arranged regularly, with few apoptotic spermatocytes and spermatoblasts. In NH, FR, and SG groups, seminiferous epithelium was thinner with most of the spermatogenic cells showing apoptosis, and they were mainly spermatogonia and spermatocytes. Apoptotic index of spermatogenic cells in NH, FR, SG, and NC groups was respectively (46.3 ± 3.3)%, (40.9 ± 3.5)%, (20.6 ± 2.3)%, (7.5 ± 1.9)%, and the difference among them was statistically significant (F = 405.65, P < 0.01). There were significant statistical differences among the former three groups (with P values below 0.01). (3) bcl-2 protein expression in NH, FR, SG, and NC groups was respectively (52 ± 5)%, (53 ± 4)%, (64 ± 5)%, (75 ± 5)%, and the difference among them was statistically significant (F = 56.63, P < 0.01). There was no significant statistical difference in bcl-2 expression between NH group and FR group (P = 0.66), and it was lower in both groups as compared with SG group (with P values below 0.01). CONCLUSIONS: Either scar healing, flap transplantation, or SG in repairing scrotum with full-thickness burn in pigs inhibits spermatogenesis, but repair with SG produces less deleterious effect on the testis.

[Influence of temperature on spermatogenesis-related genes in spermatogonia cultured in vitro].

OBJECTIVE: To investigate the influence of temperature on the expressions of c-kit and PI3K in spermatogonia cultured in vitro at 32 degrees C and 37 degrees C, and provide basic scientific data for the mechanism of spermatogenic impairment due to body temperature (37 degrees C). METHODS: Isolated spermatogenic cells were cultured in vitro at 32 degrees C and 37 degrees C, and their adherence, proliferation and morphologic changes were observed and recorded under the inverted phase contrast microscope. At 8 days, the spermatogonia were separated by Percoll density gradient centrifugation and the differential adhesion method. The expressions of c-kit and PI3K mRNA and proteins in the separated cells were detected by real time polymerase chain reaction and Western blot, respectively. The c-kit gene was sequenced to identify the occurrence of mutations. RESULTS: Adherence, division and proliferation of the cells were observed in both the 32 degrees C and 37 degrees C groups. The expressions of c-kit and PI3K mRNA and proteins in the spermatogonia were significantly higher in the 32 degrees C group than in the 37 degrees C group (P < 0.05). The 32 degrees C group showed no mutation of c-kit in exon 9, 11 and 13; the 37 degrees C group exhibited no mutation in exon 11 and 13, but possible insertion or deletion mutations in exon 9. CONCLUSION: Culturing in vitro at 37 degrees C could inhibit the expression of proliferation- and differentiation-related genes in spermatogenic cells and lead to the mutation of the c-kit gene.

[Impact of the reconstruction of the scrotum with third-degree burns on spermatogenic function: experimental study of three different methods].

OBJECTIVE: To explore the effects of different methods of scrotal reconstruction on the apoptosis of spermatogenic cells and expression of the bcl-2 protein in patients with third-degree scrotal burns. METHODS: Forty male and 24 female 2-month-old Guizhou mini-pigs were used in this study, the former equally randomized to groups I (normal control), II (natural healing), III (skin grafting) and IV (skin flap grafting). Ten months after the establishment of the model of third-degree burns, 6 male pigs from each group were paired with the female pigs and fed for 3 weeks. Then the female pigs were fed for another 4 months, followed by observation of their reproductivity. At 12 months, the bilateral testes were taken from the male pigs for detection of the apoptosis index of spermatogenic cells by TUNEL and determination of the expression of the bcl-2 protein by immunohistochemistry. The data obtained were subjected to single factor analysis of variance. RESULTS: The apoptosis indexes of spermatogenic cells were (7.07 +/- 3.5), (40.34 +/- 4.85), (15.14 +/- 1.36) and (39.29 +/- 5.73)% in groups I , II, III and IV, respectively, significantly higher in groups II , III and IV than in I (P<0.05), with statistically significant differences between group III and groups II and IV (P<0.05) but not between II and IV (P>0.05). The expression rates of the bcl-2 protein were (75.07 +/- 3.74), (54.93 +/- 4.03), (66.85 +/- 3.06) and (53.33 +/- 5.22)% in groups I, II, III, and IV, respectively, remarkably higher in I than in the other three (P<0.05), with significant differences between group III and groups II and IV (P<0.05) but not between II and IV (P>0.05). Pregnancies were found in all the female pigs of group I with 10.0 +/- 1.18 newborns and in 4 of group III with 9.92 +/- 1.31 newborns, but in none of groups II and IV, with significant differences between group I and the other three (P<0.05) as well as between group III and groups II and IV (P<0.05), but not between II and IV (P>0.05). CONCLUSION: All the three methods of reconstruction for the scrotum with third-degree burns can suppress spermatogenic function, increase the apoptosis of spermatogenic cells and decrease the expression of the bcl-2 protein, among which, skin grafting least affects spermatogenic function.

[Identification of biological characteristics of human keloid-derived stem cells].

OBJECTIVE: To investigate the biological characteristics of human keloid-derived stem cells (KDSC) in order to further research its role in keloid pathogenesis. METHODS: Human keloid specimens were harvested to isolate and select KDSC by enzyme digestion and subculturing. Primary and (or) the third generation of KDSC were collected for identification of biological characteristics as follows. (1) After addition of mouse anti-human monoclonal fluorescent antibodies (CD29-PE, CD34-PE, CD44-FITC, CD90-FITC, CD45-PerCP), the expression of cell surface antigen phenotype (CD29, CD34, CD44, CD45, CD90) as well as cell cycle was analyzed by flow cytometry. (2) After addition of mouse anti-human cell keratin (CK19) monoclonal antibody and mouse anti-human vimentin monoclonal antibody, the expression level of CK19 and vimentin was respectively determined with immunocytochemical method. RT-PCR was used to detect the expression of Oct4. The multipotent differentiation capacity of the first generation KDSC was observed with osteogenic, chondrogenic and adipogenic nutrient media. RESULTS: After being subcultured, the sizes of cells were similar, and the majority of them were spindle-shaped with disorderly arrangement. The cells highly expressed typical surface markers of mesenchymal stem cells (such as CD29, CD44, and CD90, etc.) with low expression of hematopoietic stem cell surface markers (such as CD34, CD45, etc.). 67.66% of cells were in G0/G1 phase, 26.24% of cells were in G2/M phase, and 6.11% of cells were in S phase. Vimentin was positively expressed in KDSC while CK19 was negatively expressed. The expression of Oct4 was also positive. After being cultured in inducing differentiation media, the cells could differentiate into osteoblasts, chondrocytes, and adipocytes. CONCLUSIONS: Stem cells existing in human keloid, which are similar to mesenchymal stem cells, may play an important role in keloid pathogenesis.