The regenerative repair of deep‑degree (second degree) burned skin remains a notable challenge in the treatment of burn injury, despite improvements being made with regards to treatment modality and the emergence of novel therapies. Fetal skin constitutes an attractive target for investigating scarless healing of burned skin. To investigate the inflammatory response during scarless healing of burned fetal skin, the present study developed a nude mouse model, which was implanted with normal human fetal skin and burned fetal skin. Subsequently, human peripheral blood mononuclear cells (PBMCs) were used to treat the nude mouse model carrying the burned fetal skin. The expression levels of matrix metalloproteinase (MMP)‑9 and tissue inhibitor of metalloproteinases (TIMP)‑1 were investigated during this process. In the present study, fetal skin was subcutaneously implanted into the nude mice to establish the murine model. Hematoxylin and eosin staining was used to detect alterations in the skin during the development of fetal skin and during the healing process of deep‑degree burned fetal skin. The expression levels of MMP‑9 and TIMP‑1 were determined using immunochemical staining, and their staining intensity was evaluated by mean optical density. The results demonstrated that fetal skin subcutaneously implanted into the dorsal skin flap of nude mice developed similarly to the normal growth process in the womb. In addition, the scarless healing process was clearly observed in the mice carrying the burned fetal skin. A total of 2 weeks was required to complete scarless healing. Following treatment with PBMCs, the burned fetal skin generated inflammatory factors and enhanced the inflammatory response, which consequently resulted in a reduction in the speed of healing and in the formation of scars. Therefore, exogenous PBMCs may alter the lowered immune response environment, which is required for scarless healing, resulting in scar formation. In conclusion, the present study indicated that the involvement of inflammatory cells is important during the healing process of deep‑degree burned skin, and MMP‑9 and TIMP‑1 may serve important roles in the process of scar formation.
To optimize the best concentration of neuraminidase (Neu) that enhances the migration of neuraminidase (Neu)-treated donor bone marrow cells (dBMCs) to the liver, and observe the influence of short-term cyclosporin A(CsA) application combined with intravenous injection (i.v.) of Ne treated dBMCs on the survival of skin allografts.The experiment consisted of two parts. For selection of an appropriate concentration of Neu, 26 female Wistar rats were randomly divided into four groups. The dBMCs were prepared by routine method and treated with four concentrations (0, 0.5, 1.0, 2.0 U/ml) of Neu at 37 degrees C for 30 min. The untreated and Neu-treated dBMCs were labeled by 99mTc, and injected via the tail veins to female Wistar rats in each group, respectively. After five hours, the radioactivity of various organs collected from sacrificed rats was measured by a gamma counter, and the values were expressed as percentage of total radioactivity of all organs from the same rat. To observe the survival of skin allograft, 23 male Wistar rats were randomly divided into control group, untreated dBMCs group and Neu-treated dBMCs group. All rats in each group were grafted with skin allografts from male Sprague-Dawley (SD) rats. The dBMCs from the same donor without and with Neu treatment by the concentration selected from the above experiment were injected via the tail veins of female Wistar rats in untreated dBMCs group and Neu-treated dBMCs group, respectively. Rats in untreated dBMCs group and Neu-treated dBMCs group received CsA (10 mg/kg) through intraperitoneal injection (i.p.) at 2 and 5 days post-grafting. Neither dBMCs or CsA were given in the control group. The survival of allograft skin in each group was checked and photographed daily after 5 days post operation.When the concentration of Neu was 1.0 U/ml, the percentage of dBMCs in liver was (75.3 +/- 9.8) %, which was obviously higher than that in 0 U/ml group [(58.9 +/- 4.2%)], (P < 0.01), indicating that the optimal concentration of Neu was 1.0 U/ml. The survival time of skin allografts in rats of Neu-treated dBMCs group was prolonged significantly in comparison with that of the rats in dBMCs group without Neu treatment (P < 0.01). The survival time in both dBMCs group and Neu-treated dBMCs group was longer that of control group (P < 0.01), and it was prolonged in Neu-treated dBMCs group compared with that in dBMC group.Administration of proper concentration of Neu can increase the affinity of dBMCs to the liver, and promote the Neu-treated dBMCs to migrate to liver. The intravenous injection of Neu-treated dBMCs combined with short-term CsA administration can delay the rejection of skin allografts in rats.
To observe the influence of mixed grafting of autologous and allogeneic microskin on burn wound healing.Autologous microskin grafting (expansion rate 5:1) was employed as control. Autologous microskin mixed with the allogeneic microskin with the thickness of 0.3 mm and 0.6 mm, respectively, were designated as experimental groups 1 and 2 (EP1 and EP2). The wound healing rate, wound contraction rate, and histological changes were observed on the 2nd, 3rd and 4th weeks after the grafting.The wound healing rate in two experimental groups (94.58 +/- 3.99)% in EP1, and (95.28 +/- 1.93)% in EP2 was significantly higher than that in the control group (88.28 +/- 6.85)% at the end of the 2nd week after the grafting (P < 0.05) The wound healing rate in experimental group 2 (94.55 +/- 3.47)% was obviously higher than that in control (88.51 +/- 5.59)% and experimental group 1 (89.51 +/- 4.70)% at the end of the 3rd week after grafting (P < 0.05). There was no obvious difference in wound healing rate among the three groups at the end of the 4th week after grafting. Obvious lymphocytic infiltration was observed by histological examination between epidermis and dermis in the two experimental groups at the end of the 2nd week after grafting. But there was no obvious difference among the three groups 4 weeks after grafting.The wound healing could be improved by mixed skin grafting with appropriate quantity of allogeneic and autologous microskin. Furthermore, the wound contraction could be ameliorated if the thickness of allogeneic dermis was increased in the mixed grafting even with the same proportion of allogeneic to autologous microskin.
Objective: To construct a burn model of early and midterm human Foetal Skin (hFS) in vitro, to observe its morphological and histological changes after transplantation, and to examine the expression of Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Metalloproteinases (TIMPs), and further to analyse their roles in foetal wound healing preliminarily.
Methods: hFS harvested from aborted foetuses (gestational age 14-24 weeks) was transplanted into the backs of nude mice. Four weeks later, a burn wound was made in the transplanted foetal skin with a thermostatic electrical scald apparatus. To examine the role of exogenous mature immune cells, human Peripheral Blood Mononuclear Cells (hPBMCs) were injected into the subcutaneous layer of transplanted hFS. The histological characteristics and the expression of MMP-2, MMP-7, MMP-9, and TIMP-1 were examined during the wound-healing process above.
Results: The transplanted foetal skin survived on the backs of mice, the burn wounds healed rapidly and without scarring, and skin appendages regenerated after the epidermis were repaired. Injection of hPBMCs prolonged the burn wound-healing process and caused obvious scarring. MMP-2 expression was observed in the surviving grafts, but MMP-7 expression was not observed throughout the normal growth process after transplantation. MMP-7 expression was observed around the burn wound, but MMP-2 expression was observed only in surviving follicles. MMP-9 expression was observed in the cytoplasm of deep epithelial cells of skin appendages. TIMP-1 expression level was slightly lower than that of MMP-9, although its pattern of expression was similar to that of MMP-9. Injection of exogenous mature immune cells (hPBMCs) into the burn injury changed the patterns of MMP-9 and TIMP-1 expression.
Conclusion: Our method for constructing an early or midterm hFS burn model is simple and reliable. HFS maintains the ability for scarless wound healing and regeneration when transplanted into nude mice. This new animal model may be suitable for investigating the mechanisms responsible for hFS wound healing. Injection of hPBMCs prolonged burn wound healing and caused the formation of scars in the wound. MMPs and TIMPs expression might be important factors for scarless wound healing.
To observe the characteristics of keratin 19 and integrin beta1 expressions in the wound after microskin grafting , and to investigate the healing mechanism.Full layer skin defects were created in twenty Sprague-Dawley rats and they were divided into two groups, i.e., A group (with grafting of autologous microskin accounting 10% in weight of epidermis loss from skin defect), B group (with grafting of autologous microskin and allogeneic microskin, accounting 10% and 40% weight of epidermis loss respectively in skin defect). The wound healing rate and contraction rate were observed at 2,3,4 post-grafting week (PGW), and the expression and distribution of keratin 19 and integrin beta1 were observed at 2 and 4 PGW.The wound healing rate in the B group on 2 and 3 PGW was obviously higher than that in A group [(85 +/- 5)% vs. (53 +/- 10)%, (84 +/- 8)% vs. (65 +/- 9)%, P < 0.01]. No obvious difference in wound contraction rate between the two groups was observed on the 2, 3 and 4 PGW (P > 0.05). Cells with expression of keratin 19 and integrin beta1 were observed in the suprabasal layers of the epidermis in healing wound, but not in the basal membrane. Integrin beta1 positive expression cells were not observed in the suprabasal layers until 4 PGW.Mixed grafting with autogenous and allogenous microskin can improve wound healing. Ectopic expression of keratin 19 and integrin beta1 exists during wound healing process after microskin grafting.
To investigate the influence of different amount of allogeneic microskin in mixed grafting with certain quantity of autologous microskin on wound healing in rats. Methods Male Wistar rats served as alloskin donor rats. Forty female SD rats with full thickness skin defect were enrolled in the study, and they were randomly divided into four groups, i.e. group I (n=10, with allogeneic microskin graft at area expansion rate of 10:3); group II (n=10, with autologous microskin graft at area expansion rate of 10:1); group III (n=10, with mixed grafting of autologous and allogeneic microskin at area expansion rate of 10:1, respectively); group IV (n=10, with mixed grafting of autologous and allogeneic microskin at area expansion rate of 10:1 and 10:3, respectively). The wound healing rate, wound contraction rate and histological changes were observed at the 2, 3 and 4 post graft weeks (PGW).(1) In group I, there was mainly granulation tissue with some de novo epithelial cells appearing at the wound edge along with the rejection of grafted allogenous skin in the rat wound. In group II, there was still some granulation tissue remaining at 2 PGW due to insufficient amount of microskin. However, the wounds in the mixed grafting group appeared almost totally epithelialized. (2) Various amounts of mononuclear inflammatory cell infiltration and different degrees of angiectasis were observed in the dermal layer after the skin grafting in all groups, especially in group II and IV. There was thickening of the epithelial layer in all groups except group I. (3) The wound healing rate decreased obviously along with the development of rejection in group I at 2 to 4 PGWs. The wound healing rate was (55 +/- 26)% in group II, which was obviously lower than that in group III (88 +/- 6)% and in group IV (76 +/- 10)% at 3 PGWs (P < 0.01). (4) The contraction rate of the wound in group IV (69 +/- 7)% was much higher than that in group I (58 +/- 11)% at 3 PGWs (P < 0.05), and there was no difference among all the other groups.Wound healing can be obviously accelerated by mixing some autologous microskin with appropriate amount of alloskin. Moreover, certain amount of autologous microskin (expansion rate 10:1) mixed with the same proportion of allogeneic microskin seems to be more beneficial in promoting wound healing.
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