RESUMEN
OBJECTIVE: To investigate the effects of three kinds of artificial dermal scaffolds on vascularization and scar formation of wounds in pigs with full-thickness burn. METHODS: Eighteen Bama miniature pigs were divided into chitosan scaffold (CS) group, sulfonated carboxymethyl chitosan scaffold (SCCS) group, and acellular dermal matrix (ADM) scaffold group according to the random number table, with 6 pigs in each group. Every pig in all groups was inflicted with 4 or 8 full-thickness scald wounds on the back (totally 96 wounds). Forty-eight hours after injury, eschars of all wounds were excised. Twenty-four wounds in CS group were transplanted with double-layer artificial dermis of collagen-chitosan and silicone rubber, those in SCCS group with double-layer artificial dermis of collagen-sulfonated carboxymethyl chitosan and silicone rubber, and those in ADM scaffold group with ADM. The rest 24 wounds in the three groups were dressed with vaseline gauze as control group. After 2 weeks of treatment, all wounds of every group were covered with skin. In post treatment (scaffold transplantation or gauze covering) week (PTW) 1, 2, 3, and 4, gross condition of wound was observed, and specimens from central parts of wounds were harvested for observation and assessment of vessels or cells with positive expression of CD31, α smooth muscle actin (α-SMA), TGF-ß(1) and TGF-ß(3) with SP staining. Data were processed with one-way analysis of variance and LSD test. RESULTS: (1) Degree of vascularization in SCCS group was better than that in the other three groups. (2) The number of vessels with positive expression of CD31 in CS, SCCS, ADM scaffold, and control groups increased gradually from PTW 1 to PTW 3, and decreased in PTW 4. There were statistical differences among 4 groups from PTW 1 to PTW 4 (with F value respectively 24.005, 38.822, 25.274, 3.856, P < 0.05 or P < 0.01). The numbers of vessels that expressed CD31 in SCCS group from PTW 1 to PTW 3 were more than those in the other three groups (with P values all below 0.05). (3) The numbers of vessels that expressed α-SMA in CS, SCCS, and ADM scaffold groups from PTW 1 to PTW 3 showed the similar trend of change to those of vessels that expressed CD31, which increased gradually in control group from PTW 1 to PTW 4. There were obvious differences among 4 groups from PTW 1 to PTW 4 (with F value respectively 22.637, 28.087, 62.651, 18.055, P values all below 0.01). The number of vessels that expressed α-SMA in SCCS group from PTW 1 to PTW 4 was more than that in the other three groups (with P values all below 0.05). (4) From PTW 1 to PTW 4, the number of cells with expression of TGF-ß(1) in CS group was respectively (127 ± 8), (167 ± 19), (170 ± 18), (144 ± 10) per 400 times visual field, that in SCCS group was respectively (171 ± 17), (207 ± 25), (130 ± 30), (69 ± 16) per 400 times visual field, that in ADM scaffold group was respectively (106 ± 8), (159 ± 17), (171 ± 11), (145 ± 11) per 400 times visual field, and that in control group was respectively (100 ± 20), (150 ± 18), (200 ± 14), (172 ± 20) per 400 times visual field. There were statistical differences among 4 groups from PTW 1 to PTW 4 (with F value respectively 29.675, 9.503, 13.107, 54.515, P values all below 0.01). Compared with those in SCCS group, the number of cells that expressed TGF-ß(1) in the other three groups was decreased in PTW 1, 2 but increased in PTW 3, 4 (with P values all below 0.05). (5) The number of cells that expressed TGF-ß(3) in 4 groups increased gradually from PTW 1 to PTW 3, and decreased or increased continually in PTW 4. There were statistical differences among 4 groups from PTW 1 to PTW 4 (with F value respectively 140.612, 945.850, 714.037, 119.147, P values all below 0.01). The number of cells with positive expression of TGF-ß(3) in SCCS group from PTW 1 to PTW 4 was more than that in the other three groups (with P values all below 0.05). CONCLUSIONS: The collagen-sulfonated carboxymethyl chitosan dermal scaffold can rapidly induce growth and maturation of blood vessels during wound healing after burn. It is beneficial for wound repair at early stage with inhibition of scar proliferation.
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Quemaduras/cirugía , Cicatriz/patología , Dermis/trasplante , Neovascularización Fisiológica , Piel Artificial , Cicatrización de Heridas , Dermis Acelular , Animales , Quitosano/análogos & derivados , Colágeno , Femenino , Trasplante de Piel , Porcinos , Andamios del TejidoRESUMEN
OBJECTIVE: To investigate the effects of vascular endothelial growth factor (VEGF) DNA plasmids, encoded in collagen-sulfonated carboxymethyl chitosan porous scaffold, on the angiogenesis in full-thickness burn wounds. METHODS: Wounds and pathological changes were observed at Week 1, 2, 3 and 4 after pure collagen-sulfonated carboxymethyl chitosan porous scaffold (group A) and collagen-sulfonated carboxymethyl chitosan porous scaffold encoding VEGF DNA plasmids (group B) were transplanted onto eachar-removed wounds of full-thickness burn in 6 Bama miniature pigs respectively. Wound healing was observed by pathologic slides after epidermal grafting for 2 weeks (at Week 4) onto the surface of different dermal scaffolds transplanted on wounds for 2 weeks. At the same time, new-forming vessels expressing CD31 and mature vessels expressing α-SMA (α-smooth muscle actin) and the expression of VEGF in wounds at Week 1, 2, 3 and 4 were detected in situ by immunohistochemical staining. The burn wounds without any transplanted scaffold (group C) were studied as the controls. RESULTS: Wounds with various scaffolds were different from those without any scaffold. Angiogenesis of the group B was better than that of the group A. Neo-forming micro-vessels expressing CD31 in the wounds of group A or B at Week 1, 2, 3 and 4 were as follows: 18.7 ± 3.1, 25.7 ± 2.3, 36.8 ± 2.5 & 26.2 ± 2.9; 24.5 ± 3.8, 32.3 ± 2.8, 39.2 ± 2.2 & 27.3 ± 3.0. Mature vessels expressing α-SMA: 11.7 ± 1.9, 20.5 ± 1.9, 35.0 ± 4.5 & 24.0 ± 2.8; 20.2 ± 3.1, 33.5 ± 3.7, 38.2 ± 4.5 & 26.5 ± 2.3. The expressions of VEGF: 48.7 ± 7.9, 141.7 ± 9.1, 201.5 ± 8.6 & 107.0 ± 8.2; 97.3 ± 7.9, 172.3 ± 8.1, 208.7 ± 8.3 & 114.0 ± 5.8. Expressing intensity of CD31, α-SMA and VEGF in the wounds of group C at Week 1, 2, 3 & 4: 6.0 ± 2.0, 9.8 ± 3.4, 19.3 ± 2.5 & 18.7 ± 2.2; 3.7 ± 2.0, 8.7 ± 1.8, 13.0 ± 2.5 & 14.0 ± 2.8; 3.5 ± 2.3, 10.3 ± 3.5, 23.0 ± 5.6 & 21.5 ± 5.1. There were significant statistical differences among three groups. CONCLUSION: Artificial dermal scaffold encoding exogenous pDNA-VEGF can promote a quicker angiogenesis through a high expression of VEGF. Thus a full-thickness burn wound may be better repaired.
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Quitosano , Factor A de Crecimiento Endotelial Vascular , Animales , Quemaduras , Colágeno/metabolismo , ADN , Plásmidos , Trasplante de Piel , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de HeridasRESUMEN
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.
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Quemaduras/cirugía , Quitosano/análogos & derivados , Piel Artificial , Andamios del Tejido , Animales , Apoptosis , Quemaduras/patología , Quitosano/farmacología , Colágeno , Modelos Animales de Enfermedad , Femenino , Trasplante de Piel , PorcinosRESUMEN
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.
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Quemaduras/cirugía , Piel Artificial , Andamios del Tejido , Animales , Quitosano/análogos & derivados , Colágeno , Modelos Animales de Enfermedad , Femenino , Siliconas , Trasplante de Piel , Porcinos , Porcinos EnanosRESUMEN
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.
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Apoptosis , Quitosano/metabolismo , Colágeno/biosíntesis , Dermis , Animales , Colágeno/metabolismo , Matriz Extracelular , Femenino , Pruebas de Irritación de la Piel , Piel Artificial , Stents , Porcinos , Porcinos Enanos , Ingeniería de Tejidos , Factor de Crecimiento Transformador beta1/metabolismo , Cicatrización de HeridasRESUMEN
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.
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Quitosano , Colágeno , Trasplante de Piel , Andamios del Tejido , Animales , Modelos Animales de Enfermedad , Femenino , Supervivencia de Injerto , Neovascularización Fisiológica , Siliconas , Piel/lesiones , Porcinos , Porcinos Enanos , Cicatrización de HeridasRESUMEN
OBJECTIVE: To investigate the effects of intralesional steroid, interferon alpha-2b or verapamil injection on proliferation, apoptosis and TGF-beta1 expression in keloid and hypertrophic scar in vivo. METHODS: 6 patients with keloids and 6 patients with hypertrophic scar were treated with intralesional injection of triamcinolone acetonide (40 mg/ml) or IFN alpha-2b (15 x 10(5) U/ml) or verapamil (2.5 mg/ml). Samples were collected on the 7th day after intralesional injection. Samples of untreated keloid and hypertrophic scar and normal skin were used as control. Expression of PCNA and TGF-beta1 was detected in situ by immunohistochemical staining, and apoptosis was detected in situ by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL). RESULTS: 1) Triamcinolone acetonide could prohibit proliferative scars through inhibiting cell proliferation and TGF-beta1 expression, as well as inducing apoptosis. 2) IFN alpha-2b could prohibit proliferative scars through inhibiting cell proliferation and TGF-beta1 expression, but not inducing apoptosis; 3) Verapamil could also prohibit proliferative scars through inhibiting proliferation and TGF-beta1 expression in fibroblasts, as well as inducing apoptosis. While the effect of inducing apoptosis was stronger than that of triamcinolone acetonide, the effect of inhibiting TGF-beta1 expression was weaker than those of triamcinolone acetonide and IFN alpha-2b. CONCLUSIONS: Although intraleional injection of steroid, interferon alpha-2b or verapamil were all effective in the treatment of keloid and hypertrophic scar, their mechanisms are not similar.
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Cicatriz Hipertrófica/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Esteroides/uso terapéutico , Verapamilo/uso terapéutico , Adolescente , Adulto , Apoptosis , Niño , Femenino , Humanos , Interferón alfa-2 , Masculino , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Recombinantes , Factor de Crecimiento Transformador beta1/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: This paper is to investigate the effects of steroid or IFN alpha-2b on apoptosis and cell pathway of fibroblasts from keloids, hypertrophic scars and normal skins and different responses of different fibroblasts. METHODS: 6 samples from keloid, hypertrophic scar and normal skin were collected respectively and fibroblasts from different sources were cultured in vitro. After different fibroblasts were treated with dexamethasone (0.1 mg/ml) or IFN alpha-2b (1000 U/ml), Bax and Bcl-2 protein expressions were detected in situ by immunohistochemical staining; DNA ladders of different fibroblasts were observed by gel electrophoresis; and relative activated (phospho-) ERK1/2 and JNK pathways were detected by method of FACE ELISA. RESULTS: Dexamethasone could induce apoptosis of fibroblasts from keloids, hypertrophic scars and normal skins through activating (phospho-) ERK1/2 and JNK pathways; IFN alpha-2b could not induce apoptosis of fibroblasts from different sources. IFN alpha-2b could inhibit (phospho-) ERK1/2 pathway and could not affect (phospho-) JNK pathways of fibroblasts from keloid and hypertrophic scar. IFN alpha-2b could affect neither (phospho-) ERK1/2 pathway nor (phospho-) JNK pathways of fibroblasts from normal skin. CONCLUSIONS: The responses of different fibroblasts to steroid or IFN alpha-2b were different.
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Apoptosis/efectos de los fármacos , Cicatriz Hipertrófica/metabolismo , Fibroblastos/metabolismo , Queloide/metabolismo , Adulto , Células Cultivadas , Cicatriz Hipertrófica/inducido químicamente , Cicatriz Hipertrófica/patología , Femenino , Fibroblastos/patología , Hormonas/farmacología , Humanos , Interferón alfa-2 , Interferón-alfa/farmacología , Queloide/inducido químicamente , Queloide/patología , Masculino , Persona de Mediana Edad , Proteínas Recombinantes , Transducción de SeñalRESUMEN
AIM: The aim of the present study was to understand the role of retinoic acid (RA) in the development of isolated patent ductus arteriosus and the features of arterial duct-derived vascular smooth muscle cells (VSMC). METHODS: The VSMC were isolated, and the biological characteristics and the response to RA were investigated in the arterial duct, aorta, and pulmonary artery VSMC from 6 human embryonic samples. Western blotting, immunostaining, and cell-based ELISA were employed to analyze the proliferation regulation of VSMC. RESULTS: The VSMC from the arterial duct expressed proliferating cell nuclear antigen (PCNA) at a significantly lower rate than those from the aorta and pulmonary artery, but expressed a higher level of Bax and Bcl-2. The expression level of PCNA or Bcl-2 was associated with the embryonic age. The effects of RA on the VSMC from the arterial duct were quite different from those from the aorta and pulmonary artery. In arterial duct VSMC, RA stimulated PCNA expression, but such stimulation could be suppressed by CD2366, an antagonist of nuclear retinoid receptor activation. In aorta or pulmonary artery VSMC, the expression response of PCNA to RA was insignificant. The ratio of Bax/Bcl-2 decreased in arterial duct VSMC after RA treatment due to the significant inhibition of Bax expression. CONCLUSION: The VSMC from the arterial duct possessed distinct biological behaviors. RA might be important in the development of ductus arteriosus VSMC.