[Comparative study on repair of full-thickness burn wound with different artificial dermal stent in pigs].

OBJECTIVE: To compare differences of angiogenesis among collagen- chitosan, collagen-sulfonated carboxymethyl chitosan porous scaffolds and acellular dermal matrix after these three different scaffolds with silicone membrane were transplanted on the wounds of full thickness burn, and the wound repair of different scaffolds with epidermis grafting on. METHODS: Angiogenesis in different dermal scaffolds, the wound surface and epidermis survival were observed in 1, 2, and 3 weeks after the three different scaffolds were respectively transplanted on wounds of full thickness burn with debridement in 6 Bama miniature pigs (total 18 pigs in 3 groups). At the same time, CD34 positive signals (neo-forming microvessels) were detected by immunohistochemical staining. The wounds without any scaffold transplantation were studied as the control. RESULTS: Angiogenesis had been fundamentally finished in 2 weeks after implantation of collagen- sulfonated carboxymethyl chitosan porous scaffold. And fundamental angiogenesis in collagen- chitosan porous scaffolds and acellular dermal matrix needed at least 3 weeks. Neo-forming micro-vessels perpendicular to wound beds with these three different scaffolds were more than those in the control wounds without scaffold. CD34 positive signals (neo-forming micro-vessels) were significantly higher in wounds at the second week than those in wounds at the first week. And those in wounds at the third week were significantly higher than those in wounds at the second week in all wounds with different scaffold transplantations and the control wounds. CD34 positive signals in the group of sulfonated carboxymethyl chitosan porous scaffold on the 1st, 2nd and 3rd week after the scaffold transplantation were significantly higher than those corresponding signals in the other three groups. Epidermis on the sulfonated carboxymethyl chitosan porous scaffold which had been transplanted on burn wound for 1 week could survive perfectly, however, epidermis on the collagen- chitosan porous scaffold or acellular dermal matrix could not survive until these two scaffolds had been transplanted on the burn wounds for at least 2 weeks. CONCLUSIONS: These three different scaffolds could repair the full thickness skin defects caused by burn, and angiogenesis of sulfonated carboxymethyl chitosan porous scaffold is the best.

[Differences of wound contraction and apoptosis in full-thickness burn wounds repaired with different artificial dermal stent in pigs].

OBJECTIVE: To investigate the roles and differences of angiogenesis of different dermal scaffolds on wound contraction and apoptosis during full-thickness burn wound repair. METHODS: Wounds were observed at different time after the collagen-sulfonated carboxymethyl chitosan porous scaffold or collagen-chitosan porous scaffold or acellular dermal matrix were respectively transplanted on wounds of full thickness burn with debridement in Bama miniature pigs. At the same time, vessels and myo-fibroblasts expressing α-smooth muscle action(α-SMA) and apoptosis in wounds of different time were detected in situ by immunohistochemical staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling. The burn wounds without any scaffold transplantation were studied as the control. RESULTS: Wounds with different scaffolds transplantation were different from granulation wounds. Vessels expressing α-SMA had been increasing continuously in the wounds from 1 to 3 weeks after different scaffolds transplantation and decreased in wounds after epidermis had been grafted for 2 weeks on surface of the scaffolds transplanted on wounds for 2 weeks. Vessels expressing α-SMA were the most in the wounds with collagen-sulfonated carboxymethyl chitosan porous scaffold transplantation and the least in the control wounds without dermal scaffold at different time. Myo-fibroblasts expressing α-SMA was the least in the wounds with collagen-sulfonated carboxymethyl chitosan porous scaffold transplantation and the peak of expressions was on the 2nd week, however, the peak in the wounds with the other two scaffolds transplantation and in the control wound without dermal scaffold was on the 3rd week. Myo-fibroblasts expressing α-SMA was the most in the control wounds. Apoptosis had been increasing continuously in the transplantation wounds from 2 to 4 weeks after different scaffolds transplantation, however, apoptosis had begun to increase continuously from 3 to 4 weeks in the control wounds. Apoptosis was the most in the wounds with collagen-sulfonated carboxymethyl chitosan porous scaffold transplantation and the least in the control wounds without dermal scaffold from 3 to 4 weeks. CONCLUSION: Sulfonated carboxymethyl chitosan can promote migration of reparative cells and angiogenesis, and it can repair full-thickness burn wound fast and well.

[Proliferation of the mesenchymal stem cells in a delayed fat flap: an experimental study in rabbits].

OBJECTIVE: To investigate the possibility to enhance the proliferation of adipose-derived stem cells (ASCs) in a delayed fat flap in rabbits. METHODS: A delayed fat flap was formed in one side of inguinal region of a rabbit. 21 days after operation, the fat tissues at the delayed flaps and at the unoperated side were harvested and digested with 0.25% collagenase and sieved. The cell suspensions were centrifuged. The cells were obtained from tissue precipitate after centrifugation. The expression rates of the surface marker (CD29, CD44, CD14 and CD45) were measured by FCM and compared between the experimental and control groups. RESULTS: Expression rates of CD29 and CD44 were higher in the delayed fat flap (74.06% and 90.74%) than in the contralateral fat tissue (62.88% and 77.54%, P < 0.05), while those of CD14 and CD45 were lower in the delayed fat flap (57.66% and 4.84%) than in the contralateral fat tissue (72.10% and 75.82%, P < 0.05 and P < 0.01). CONCLUSIONS: Tissue hypoxic ischemia such as fat tissue in a delayed fat flap can promote proliferation of ASCs. It indicates that tissue in the delayed flap may be transplanted with better survival rate. The ischemia pretreatment of fat tissue may become a new method for fat transplantation.

[Mechanisms and effects of biosynthesis and apoptosis in repair of full-thickness skin defect with collagen-chitosan dermal stent].

OBJECTIVE: To investigate biosynthetic and apoptotic mechanisms in repair of full thickness skin defect with collagen-chitosan porous scaffold transplantation, and to determinate differences between wound repair with the scaffold transplantation and scar healing without the scaffold transplantation. METHODS: The full thickness skin defects were made on 10 Bama miniature pigs and the bilayer dermal equivalent (BDE) composed of collagen-chitosan porous scaffold and silicone membrane was transplanted on wounds. Surfaces of wounds were observed at 1, 2, and 3 weeks after the BDE transplantation, and so were done the wound repairs after epidermis had been grafted for 2 weeks on surface of the scaffold which had been transplanted on skin defect wounds for 2 weeks. At the same time, TGF-beta1 expressions, apoptosis and self collagen replacement of scaffolds in wounds were detected in situ by immunohistochemical staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) and picrosirius red polarized light. Wounds without scaffold transplantation were studied as control. RESULTS: 1) Wounds with the scaffold transplantation were different from granulation tissue. 2) The peak of TGF-beta1 expression in the scaffold wounds was from 1 to 2 weeks after BDE transplantation, and TGF-beta1 expressions decreased continuously from 3 to 4 weeks. TGF-beta1 expressions increased continuously in the control wounds from 1 to 3 weeks and decreased on 4 weeks. TGF-beta1 expressions in the scaffold wounds on 1st and 2nd week were significantly higher than those in the corresponding control wounds, whereas, TGF-beta1 expressions in the scaffold wounds on 3rd and 4th week were significantly lower than those in the corresponding control wounds. 3) Apoptosis increased continuously in the scaffold wounds from 2 to 4 weeks after BDE transplantation, and so did in the control wounds from 3 to 4 weeks. However, apoptosis signals in the scaffold wounds on 2nd, 3rd, and 4th week after BDE transplantation were significantly more than those in the corresponding control wounds, and there was no difference between apoptosis signals in the scaffold wounds on 1st week after BDE transplantation and those in the corresponding control wounds. 4) Observation by picrosirius red polarized light method: self collagen began to synthesize in the scaffold wounds on 1st week after BDE transplantation, and scaffolds had been replaced by self collagen from 2 to 3 weeks after BDE transplantation. CONCLUSIONS: Collagen-chitosan porous scaffold plays a very important role in wound healing of full thickness skin defect. The mechanisms of wound repair by dermal scaffold are different from those by granulation and scar healing. It has a good future in repairing skin defect.

[Study on repair of full-thickness skin defect with collagen-chitosan dermal stent in pigs].

OBJECTIVE: To investigate angiogenesis of collagen-chitosan porous scaffold, and to study survive of skin grafts on the scaffold after bilayer dermal equivalent (BDE) was transplanted on wounds with full thickness skin defects. METHODS: The full thickness skin defects were made on 10 Bama miniature pigs and the BDE composed of collagen-chitosan porous scaffold and silicone membrane was transplanted on wound. Angiogenesis in dermal equivalent, wound healing, and healing and survive of skin grafts on dermal equivalent were observed in 1, 2, and 3 weeks after the BDE transplantation. At the same time, CD34 positive signals (neo-forming micro-vessels) were detected by immunohistochemical staining. RESULTS: Inflammatory cells and fibroblasts infiltrated into dermal equivalent and a few new micro-vessels had been formed in 1 week after the BDE transplantation; neo-forming micro-vessels perpendicular to wound bed had increased significantly in 2 weeks after the BDE transplantation; neo-forming micro-vessels could be observed in almost all dermal equivalents in 3 weeks after the BDE transplantation. CD34 positive signals (neo-forming micro-vessels) in 3 weeks after the BDE transplantation was much more than those in 2 weeks after the BDE transplantation, and CD34 positive signals in 2 weeks after the BDE transplantation was much more than those in 1 week after the BDE transplantation. Survival rate of intermediate split thickness skin graft were 10%, 70% and 100% respectively after the skin grafts had been grafted for 2 weeks on surface of the scaffold which had been transplanted for 1, 2 and 3 weeks. Epidermis which had been grafted on surface of the scaffold for 1 or 2 weeks could perfectly survive after BDE had been transplanted for 1 or 2 weeks. CONCLUSIONS: Collagen-chitosan porous scaffold plays a very important role in wound healing of full thickness skin defect and can induce fibroblast infiltration and new micro-vessel formation. Epidermis grafted on surface of collagen-chitosan porous scaffold can perfectly repair wounds, and it has brilliant applied prospects in repairing skin defect.

[Cordyceps sinensis polysaccharide enhances apoptosis of HL-60 cells induced by triptolide].

OBJECTIVE: To investigate the effects of polysaccharide fraction of Cordyceps sinensis (PSCS) on triptolide (TPL)-induced apoptosis in the HL-60 cells and the involved molecular mechanism. METHODS: The cultured leukemia HL-60 cells were divided into three groups: control group, TPL group (cells were treated with 5 ng/ml TPL only), and PSCS+TPL cells group (cells treated with 5 ng/ml TPL and 100 microg/ml or 200 microg/ml PSCS for 18 h). Cell viability was tested by MTT assay and apoptotic cells were quantitatively measured by flow cytometry with Annexin V/PI double stain.The expressions of Caspase-3, 6, 7, 9 and NF-kappa B proteins were tested by Western blot. RESULT: MTT assay showed that different concentrations of PSCS inhibited the cell viability. Flow cytometry indicated that TPL markedly increased the apoptosis rate of the HL-60 cells, and PSCS enhanced the apoptosis in a dose-dependent manner. Western blot showed that TPL did not inhibit the expression of the Caspase-3, 6, 7, 9 and NF-kappa B proteins, and when cells were treated with PSCS, the expression of proteins decreased with the PSCS concentration rising. CONCLUSION: PSCS can enhance TPL-induced apoptosis in HL-60 cells and inhibit the expression of NF-kappa B and Caspase 3,6,7,9,which might be the possible signaling pathway of inducing apoptosis.

[Experimental study of increasing effect of the fat flap tissue after delay operation on its free graft survival volume and duration].

OBJECTIVE: To observe the effects of the fat flap tissues after delay operation on free fat-graft survival rate and duration. METHODS: The delay operation of fat flaps was performed in the inguinal region of a rabbit. Expression of VEGF was assayed using Elisa method after 12 hours of flap delay. The fat flaps were harvested and cut into pieces after 21 days. A subdermal pocket was created in each side of the dorsal midline of a rabbit, the fat pieces were grafted randomly into a pocket and the normal fat pieces into the other pocket as control. After 1, 3, 6, 9 and 12 months of implantation, the grafted fats were harvested, gross observation, weight measurement and histology were carried out. Number of the vessels stained with anti-CD34 antibody was counted out. RESULTS: VEGF concentrations in flaps were significantly higher (P < 0.05). The density of vessels in experimental groups increased significantly compared with that in control groups at 1 and 3 months, respectively (P < 0.01), and no significant differences in the survival rate of fat tissues between experimental and control groups were observed at 1 and 3 months (P > 0.05). The fat cells from the flaps survived after 12 months of fat plantation, while those in control groups disappeared after 6 months. CONCLUSIONS: The survival rate and duration of grafted fat could be increased implanting the fat tissues from delayed fat flap, which may provide researchers with a new method for fat graft.

[Characteristics of genesis and development of peritoneal adhesion by different causes: experiment with rats].

OBJECTIVE: To investigate the characteristics of genesis and development of peritoneal adhesion by different causes. METHODS: 236 rats underwent laparotomy with their vermiform processes lifted up and were randomly divided into 5 groups: Group A (control group), with the vermiform process exposed to air for 5 min, Group B, with the vermiform process smeared with talcum powder; Group C, with the vermiform process scraped by scalpel; Group D, with the tip of vermiform process stabbed by needle so as to squeeze the contents of intestine to cause infection; and Group E, with the artery of vermiform process ligated. Then the abdominal incision was sutured. 1, 2, 4, and 6 weeks after the treatment 11-12 rats from each group were randomly to undergo laparotomy. The degree of adhesion was graded blindly by Bhatia's method. The vermiform process was resected to undergo pathological examination and examination of the level of organ hydroxyproline (OHP) was detected. RESULTS: (1) At different time points the adhesive grades of Groups B-E were all significantly higher than that of Group A (all P < 0.05) and the adhesive grades of Groups B and D were both significantly higher than those of Groups C and E (both P < 0.05). There were no significant differences in the adhesive degree 1, 2, and 4 weeks after the treatment between Groups C and E, however, the adhesive degree of Group E was significantly lower than that of Group C (P < 0.05). (2) There were not significant differences in the OHP levels at any time points in Group A (all P > 0.05). There were not significant differences in the OHP levels 1, 2, and 4 weeks after the treatment (all P > 0.05), and the levels 8 weeks after the treatment were all significantly lower than those 1, 2, and 4 weeks after the treatment (all P < 0.05) in Groups B, D, and E. In Group C the OHP level 2 weeks after the treatment was 0.275 +/- 0.031 microg/mg protein, significantly lower than that 1 week after (0.221 +/- 0.036 microg/mg protein, P < 0.05), and the OHP level 8 weeks after the treatment was 0.254 +/- 0.039 microg/mg protein, significantly lower than those 1, 2, and 4 weeks after (all P < 0.05). The OH levels 1, 2, and 4 weeks after the treatment of the 4 experimental groups were all significantly higher than that of the control group (all P < 0.05). 8 weeks after the treatment the level of OHP of Groups B was significantly higher than that of Group A (P < 0.05), however, the OHP levels of Group C, D, and E had all decreased to almost similar to that of Group A (all P > 0.05). (3) The adhesive degrees of Groups C and E were significantly positively correlated with the OHP level (both P < 0.05), however, the adhesive degrees of Groups B and D were not significantly correlated with the OHP level (both P > 0.05). The adhesive degrees 1, 2, and 4 weeks after the treatment of the 5 groups were all significantly positively correlated with the OHP levels (all P < 0.05, however, the adhesive degrees 8 weeks after the treatment of the 5 groups were all not significantly correlated with the OHP levels (all P > 0.05). (4) The main pathological changes of Group B were foreign body granuloma reaction and fibroplasia in Group B and unspecific inflammatory reaction and fibroplasia in Groups C, D, and E. CONCLUSION: Abdominal adhesions resulting from different causes show different characteristics. The abdominal adhesion caused by foreign bodies and that caused by infection are relatively severe and more difficult to recover than those caused by injury and ischemia. It is more reliable to use OHP level as a marker of abdominal adhesion in the early stage.