Experimental research on ADSCs-NCSS in wound repair.

New collagen sponge scaffold (NCSS) combined with adipose-derived stem cells (ADSCs) in the repair of full-thickness skin wound in nude mice was investigated. Human ADSCs were extracted via enzyme digestion; NCSS materials were prepared using modified method; the tissue-engineered skin substitute was constructed using ADSCs combined with NCSS. Two 10 mm2 full-thickness skin wounds were designed on the back of 24 female nude mice, respectively. Mice were divided into 4 groups in the experiment: ADSCs-NCSS (group A), simple NCSS (group B), simple ADSCs (group C) and blank control (group D). The wound healing rates were observed at 3, 7, 10 and 14 days after operation, and specimens were taken at 1 and 2 weeks for histological detection and immunohistochemical cluster of differentiation 31 (CD31) vascular density detection, respectively. At 3 and 7 days after construction of new tissue-engineered skin substitute, the infiltration of ADSCs could be seen within NCSS. The wound healing rates at 7, 10 and 14 days after operation in group A were (77.13±1.25%), (89.90±1.08%) and (96.08±0.6%), respectively, which were significantly higher than those in groups B-D; the differences were statistically significant (p<0.05). The detection of regenerated wound tissue thickness at 1 and 2 weeks after operation and CD31 vascular density at 1 week after operation showed that the vascular density in the wound in group A was significantly higher than those in other groups; the differences were statistically significant (p<0.05). After the transplantation of tissue-engineered skin constructed by human ADSCs combined with NCSS, the quality of wound healing in nude mice can be significantly improved, and the wound repair can be promoted.

EGF enhances ADSCs secretion via ERK and JNK pathways.

The objective of this work was to study the effect of epidermal growth factor (EGF) induced secretions of angiogenesis factors in adipose-derived stem cells (ADSCs) and the involvement of mitogen-activated protein kinases (MAPK). ADSCs were cultured and ELISA assays were performed to quantify the vascular endothelial growth factor, the hepatocyte growth factor, and the stromal derived factor-1 in ADSC-conditioned medium before and after EGF treatments and after pharmacological inhibition of MAPKs with PD98059, SB203580, and SP600125. The tube formation assay was used to test the effects of EGF treated and inhibitor treated ADSCs on the human umbilical vein endothelial cells (HUVECs) tube formation. Liposuction was applied and ADSCs were cultured successfully. The ADSCs released a variety of angiogenic factors, with the EGF treatments enhancing secretions and promoting the HUVEC tube formation. The MAPK inhibitors PD98059 and SP600125 increased the paracrine to promote tubular formation, while the SB203580 played an opposite role. In conclusion, (1) the in vitro cultured ADSCs secrete various angiogenic factors and the EGF amplifies the secretion and can enhance the ADSCs on the HUVEC tube formation. (2) ERK1/2 and JNK pathway may be involved in the enhanced secretion capacity of ADSCs while the p38 pathway may exert an opposite effect.

Effect of heat shock protein 47 on collagen synthesis of keloid in vivo.

Keloid is regarded as a fibroproliferative disorder with excessive accumulation of extracellular matrix. However, the molecular mechanism of keloid formation is not well understood and no treatment modality is consistently effective. Heat shock protein 47 (HSP47) is known as a collagen-specific molecular chaperone which plays a critical role in collagen biosynthesis. Results of our previous in vitro experiments demonstrated that HSP47 might be an important reason for excessive collagen accumulation in regard to keloid formation. Our objective is to investigate whether HSP47 has an influence on collagen metabolism in animal keloid models. The constructed plasmids, carrying HSP47-small hairpin RNA (shRNA), were transfected into animal keloid models, in comparison with the control groups. After transfection, the mRNA and protein expression of HSP47 and collage type I were detected by quantitative real-time PCR and Western blot. Both the mRNA and protein levels of HSP47 in animal keloid models were decreased dramatically after transfection of the HSP47- shRNA plasmid, in comparison with the control group. Following the down-regulation of HSP47, we found that the volume of animal keloid models and the major collagen expression were reduced correspondingly. Combining the results of our previous in vitro experiment results, we suggest that overexpression of HSP47 in keloid fibroblast cells could induce excessive collagen accumulation by enhancing collagen synthesis, which not only presents a possible mechanism of keloid formation, but also offers a therapeutic potential of RNA interference to HSP47 for the treatment of keloids and other fibroproliferative disorders.

[The effect of recombinant HSP47siRNA on the pathological scar in nude mice: in vivo study].

OBJECTIVE: To study the significance of HSP47 gene in the development of pathological scar. METHODS: The nude mice were used to reconstruct animal model of pathological scar. 16 days later, the mixture of recombinant HSP47siRNA and liposome was injected into the pathological scar in experimental group. In the control group, 0.25 ml PBS was injected intraperitoneally. 7 days after injection, the specimens were collected for detection of mRNA of HSP47, the collagen and for immunohistochemical study. RESULTS: In the control and experimental group, the collagen content was (91.71 +/- 1.24)% and (82.12 +/- 4.79)%, respectively; the expression of HSP47mRNA was 1 042 862.01 +/- 604 194.36 and 306 123.68 +/- 105 857.08, respectively; the expression of collagen I mRNA was 10 228 614.70 +/- 2 532 879.04 and 6 011 841.97 +/- 2 886 897.17, respectively;the scar volume was (255.60 +/- 21.34) mm3 and (132.99 +/- 24.06) mm3, respectively. All the above results showed significant difference between the two groups (P < 0.05). CONCLUSIONS: The collagen production can be reduced through suppression of the expression of HSP47 gene. It indicates that HSP47 gene enhance the development of keloid and could be used as the target of treatment.

[Replication of pathological scar in nude mice].

OBJECTIVE: To investigate the feasibility of reproduce hypertrophic scar and keloid in nude mice in the study of pathological scars. METHODS: Pieces (0.8 x 0.8 x 0.5 cm) of hypertrophic scars and keloids were implanted into subcutaneous tissue of the nude mice for 16 days, during this period the gross condition of the nude mice and the state of the implants were observed. The implants were extracted after 16 days, and the volume, the microscopic characteristics of the scar, the content of acid mucopolysaccharide, and different types of collagen were determined and compared with that of the original specimens. RESULTS: All mice survived with nice wound healing after the surgery. There was no obvious difference in the acid mucopolysaccharide content in keloid and hyperplastic scar before implantation (3448 +/- 1452, 1940 +/- 509), and after implantation (3237 +/- 1871, 1809 +/- 552, P > 0.05). The implants maintained the collagen pattern, with no signs of cell degeneration and necrosis. CONCLUSION: This experiment showed that the viability and morphology of hypertrophic scars and keloids were maintained after they were implanted in nude mice. Therefore it is feasible to use nude mice as the animal model in the study of hypertrophic scars and keloids.