【Abstract】 Objective To investigate the impact of dermal papillary cells on vascularization of tissue engineered skinsubstitutes consisting of epidermal stem cells and allogeneic acellular dermal matrix. Methods Human foreskins from routinecircumcisions were collected to separate epidermal cells by using dispase with trypsogen. Collagen type IV was used to isolateepidermal stem cells from the 2nd and 3rd passage keratinocytes. Dermal papilla was isolated by the digestion method of collagenaseI from fetus scalp and cultured in routine fibroblast medium. Tissue engineered skin substitutes were reconstructed by seedingepidermal stem cells on the papillary side of allogeneic acellular dermis with (the experimental group) or without (the controlgroup) seeding dermal papillary cells on the reticular side. The two kinds of composite skin substitutes were employed to cover skindefects (1 cm × 1 cm in size) on the back of the BALB/C-nu nude mice (n=30). The grafting survival rate was recorded 2 weeks aftergrafting. HE staining and immunohistochemistry method were employed to determine the expression of CD31 and calculate themicrovessel density at 2 and 4 weeks after grafting. Results Those adhesion cells by collagen type IV coexpressed Keratin 19 andβ1 integrin, indicating that the cells were epidermal stem cells. The cultivated dermal papillary cells were identified by expressinghigh levels of α-smooth muscle actin. The grafting survival rate was significantly higher in experimental group (28/30, 93.3%), thanthat in control group (24/30, 80.0%). HE staining showed that the epithelial layer in experimental group was 12-layered with largeepithelial cells in the grafted composite skin, and that the epithelial layer in control group was 4-6-layered with small epithelial cells.At 2 and 4 weeks after grafting, the microvessel density was (38.56 ± 2.49)/mm2 and (49.12 ± 2.39)/mm2 in experimental group andwas (25.16 ± 3.73)/mm2 and (36.26 ± 3.24)/mm2 in control group respectively, showing significant differences between 2 groups(P lt; 0.01). Conclusion Addition of dermal papillary cells to the tissue engineered skin substitutes can enhance vascularization,which promotes epidermis formation and improves the grafting survival rate.
Objective To evaluate the effect of tissue engineered skin with isogeneic cells on repairing skin defects in inbred rat model so as to provide relevant evidences for the clinical application. Methods The skins of newborn inbred F344 rats were harvested and treated with Dispase trypsin to isolate the epidermal cells. The skins of adult Sprague Dawley rats were obtained and treated with hypertonic sodium-SDS-trypsin to prepare the acellular dermal matrix. The tissue engineered skin was reconstructed by submerging culturing and air-liquid interface culturing in vitro. The full-thickness skin defects of 1.5 cm × 1.5 cm in size were prepared along the dorsal both sides of 36 adult inbred F344 rats, and 72 defects were repaired with tissue engineered skin in experimental group (n=24), with allogeneic acellular dermal matrix in negative control group (n=24), and with autologous full-thickness skin in positive control group (n=24). Finally the gross observation, the survival rate, wound contraction rate, and histological observation were used to evaluate the effect. Results The wound healed by first intension at 4 weeks postoperatively in the experimental group; the grafts connected with the adjacent tissue tightly and had normal appearance. At 4 weeks after operation, the survival rate of the graft was 0 in the negative control group; the survival rates were 62.5% (15/24) in the experimental group and 91.7% (22/24) in the positive control group, showing significant difference between 2 groups (χ2=5.779, P=0.016). The wound contraction rates of the experimental group and positive control group were significantly lower than that of the negative control group (P lt; 0.05), but no significant difference was found between the experimental group and positive control group (P gt; 0.05). Histological observation showed that slight inflammation reaction appeared at 1 week postoperatively in the experimental group; the regeneration of the blood vessel and the proliferation of the fibroblasts in dermis and the gradual maturation of epidermis were observed at 2 weeks, and new collagen deposition and collagen remodeling in the dermis of the graft were found at 4 weeks postoperatively. Conclusion The tissue engineered skin is able to repair full-thickness skin defect of rats effectively, it has similar effect to the autologous full-thickness skin in preventing the wound contraction and promoting the wound healing, which provides experimental evidences for the clinical application.
Objective The immunogenicity of tissue engineered skins is still vague, though it has been appl ied cl inically for several years. To observe the evidence of immunologic rejection of tissue engineered skins transplanted to severe combined immunodeficiency (SCID) mice, which are implanted human splenic lymphocytes to construct human immunesystem. Methods Tissue engineered skins and acellular dermic matrix were constructed in vitro. Twenty SCID mice, aging4-6 weeks and weighing 16-17 g, were randomly divided into four groups equally (n=5). The tissue engineered skins, human foreskins from circumcision and acellular dermic matrix were transplanted to groups A, B, and C, respectively; group D was used as a control. After 2 weeks of transplanting, 3 × 107 human splenic lymphocytes were injected into every SCID mouse intraperitoneally. After 4 weeks, the morphology, histology, immunohistochemistry and human IgG immunofluorescence were used to observe immunologic rejection. Results Group A showed that transplanted tissue engineered skins had the bilayer structure of dermis and epidermis, which was similar to the normal human skin structure. Group B showed that the transplanted human foreskins still retained normal structure of human skin. Group C showed that acellular dermic matrix were located in situ and had no sign of degradation. After injecting human splenic lymphocytes into the SCID mice, no inflammatory cells infil itration were observed basically in groups A, C, and D; the inflammatory cells infil itration of group B were significantly higher than that of other 3 groups (P lt; 0.05). The results of anti human keratin 14 monoclonal antibody (mAb) staining and anti human type IV collagen mAb staining were positive in group A; no positive cells for CD3, CD4, and CD8 were observed in groups A, C, and D; and many positive cells for CD3, CD4, and CD8 were observed in group B. The results of IgG immunofluorescence staining was negative in group A, C, and D, and positive in the great vessel wells of group B. Conclusion The immunogenicity of tissue engineered skins is very weak, and tissue engineered skins would not be rejected by host immune system after transplantation.
Objective Human acellular amniotic membrane (HAAM) contains collagens, glucoproteins, proteinpolysaccharide,integrin, and lamellar, which can supply rich nutrition to cell prol iferation and differentiation. To explore the possibil ity of HAAM with adi pose-derived stem cells (ADSCs) as a good engineered skin substitute for repairing skin defect. Methods Primary ADSCs were obtained from inguinal fat of 30 healthy 4-month-old SD rats, male or female, weighing 250-300 g, and cultured in vitro and purified. The 3rd passage ADSCs were used to detect CD44, CD49d and CD34 by immunocytochemistry staining. After physical and trypsin preparation, the HAAM was observed by HE staining and scanning electron microscope(SEM) respectively. ADSCs were seeded on epithel ial side of HAAM at the density of 2 × 105/cm2, cocultured, and observed by SEM at different time. MTT test was used to detect viabil ity of cells that seeded on HAAM, the group without HAAM was used as control. Thirty SD rats were made models of full-thickness skin wound and randomly divided into three groups (A, B, and C). Wound was repaired with HAAM/ADSCs composites in group A, with HAAM in group B, and with gauze as control in group C. The rats underwent postoperative assessment of wound heal ing rate and histological observation at the 1st, 2nd, and 4th weeks. Results HE staining showed that the 3rd passage ADSCs was spindle-shaped with an ovoid nucleus which located in the middle of cell; the immunocytochemistry staining showed positive result for CD44 and CD49d and negative result for CD34. There were no residues of cells in the HAAM by HE staining. SEM showed that there were different structures at the two sides of HAAM；one side had compact reticular structure and the other side had fibrous structure. After 3 days of co-culture, ADSCs showed good growth on HAAM; the cells were closely packed onto the HAAM, attached firmly and prol iferated to confluence on the stromal surface of HAAM. MTT test showed that the cells on the HAAM grew well and had b prol iferation vital ity. There was no significant difference between ADSCs cultured in the HAAM and control group (P gt; 0.05). One, 2, 4 weeks after graft, there were significant differences in wound heal ing rate between group A and groups B, C (P lt; 0.05), between group B and group C (P lt; 0.05). HE staining showed that wound healed faster in group A than in groups B, C. Cytokeratin 19 (CK19) immunohistochemical statining showed that there were more CK19 positive cells in group A than in groups B, C. Conclusion The graft of HAAM with ADSCs plays an effective role in promoting the repair of full-thickness skin wound
【Abstract】 Objective To search for a feasibil ity of repairing full-thickness cutaneous deficiency with tissueengineered skin substitute composited by human epidermal stem cells and fibroblasts in fibrin frame. Methods Epidermal stem cells and fibroblasts were harvested from human epidermis and dermis by trypsin digestion. Cells were cultured and subcultured in non-serum medium. Epidermal stem cells (5×104/mL) and dermal fibroblasts (1×104/mL) in 0.5 mL medium were coagulated in 0.5 mL fibrin frame to construct tissue engineered skin substitute. The tissue engineered skin substitute was grafted onto full-thickness cutaneous deficiency of nude mice. Forty-five male mice, 4-5 week old, weighted 20 g on average, were randomly divided into 5 groups. Oil yarn (group C), fibrin frame membrane without cell inoculation (group F), composite skin substitute with epidermal stem cells (group S) and composite skin substitute with fibroblasts (group Fb) were used as controls, while tissue engineered skin substitute (group T) was experimental group. The wounds were observed 1, 3, 6, 8 weeks after surgery. Samples were harvested 3, 6, 8 weeks after surgery, and were examined by means of histology、immunohistochemistryand scanning electron microscopy (SEM). Results Four weeks after cell culture, there were some round cells in the culture capsule of epidemic cells, and some fusiform cells in the culture capsule of fibroblast. Six days after cells were cultured in the BrdU culture medium, there were some BrdU positive cells appeared. There were some CK19 positive cells and Nestin positive cells appeared in the chaff of group T before transplanting. The new formed skin of group T grew faster and had less scar than other groups. Six weeks after surgery, the average thickness of new formed skin was (0.460 ± 0.049) mm in group C, (0.480 ± 0.055) mm in group F, (0.540 ± 0.043) mm in group S, (0.510 ± 0.032) mm in group Fb, (0.660 ± 0.047) mm in group T. The thickness of new formed skin in group T was thicker than other groups (P lt; 0.05). By histology and SEM observation, 3, 6, 8weeks after surgery, the new formed cuticular layer, fibroblast and blood vessels in the group T were more than those in theother groups. The al ignment of blood vessels and collagen fibers in group T were much regular than those in the other groups. Three weeks after surgery, the new formed skin of group T had a continuous color zone of positive collagen Ⅳ staining, while no continuous color zone was found in the other groups. Six weeks after surgery, CK14 positive cells appeared in the new formed skin of group T, while no positive cell was found in the other groups. Conclusion Tissue engineered skin substitute which is composited with epidermal stem cells and fibroblasts in fibrin frame has potential prospects in appl ication of repairing fullthickness cutaneous deficiency with advantage of faster wound heal ing.
Objective The amniotic carrier complex membrane, which contains bFGF and vitamin C (VitC) and is loaded with BMSCs, is planted into the deeply-partial wounds of rabbits. To explore its influence on the epidermis renascence and regenerating speed in the process of the dermis restore. Methods BMSCs were isolated from the marrows of 24 healthy3-month-old New Zealand rabbits, male or female, weighing 1.0-1.5 kg. The BMSCs were cultured in vitro and purified, and then amniotic carrier complex membrane was prepared, whose size was 4.52 cm2. Three deep-partial wounds, with the area of about 3.14 cm2, were produced on the back of each rabbit. All the wounds were randomly divided into 3 groups: group A, group B and group C. Group A was the experimental group in which the amniotic carrier complex membrane was planted, including 1 ml BMSCs, 10 mL bFGF (0.2 mg/L) and 10 mL VitC (0.02 g/L). In group B, the amniotic carrier complex membrane was planted, including only 1 mL BMSCs. In group C, the amniotic carrier complex membrane alone was planted. After the operation, general observation was conducted. At postoperative 7, 14 and 21 days, respectively, the observation by HE, Masson, Van Giesonr staining and immunohistochemical staining of collagen type I was performed. The ink perfusion method was performed to evaluate the velocity and the qual ity of the wound heal ing after the transplantation. Results All the wounds obtained good heal ing. At 14 days after the operation, the ratio of wound heal ing was 60%, 41% and 23% in groups A, B and C, respectively. At 21 days after the operation, the the ratio of wound heal ing was 99%, 90% and 81% in groups A, B and C, respectively. There were significant differences between any two groups (P lt; 0.05). The depth of the newborn dermis, the number of the active collagen type I mascul ine cells and the number of the blood vessels in group A were better and more than in group B. And those in group B were better and more than in group C. At the exterior area of the newborn dermis, there was lots of regenerated epidermis from the peripheral normal skin, which in group A was better than in group B, and in group B was better than in group C. onclusion The amniotic carrier complex membrane transplanted to deep-partial wounds, which is appended withBMSCs, bFGF and VitC, can accelerate repair and reconstruction of the dermis. There has an optimal time of the renascence and regeneration of the epidermis in the process of dermis repair.
Objective To observe the change of the immunogenity of keratinocytes when cultured in vitro. Methods Health children foreskins were digested bydispase and trypase. The human keratinocytes were cultured in vitro and passaged in succession until the fifth passage. Different passage keratinobytes signed by SP method to show the percentage of langerhans cells and melanocytes. Every passage keratinocytes were respectively mixed with allogenic lymphocytes which isolated from peripheral blood, and then the proliferation degree of allogenic lymphocytes was tested. Results Keratinocytes were cultured well in KSFM medium. When keratinocytes conjugated, every passage cells grew like paving stone. After cryopreservation and then rewarming, the survival exceeded 80%. The percentages of langerhans cells and melanocytes in the primary passage were 5.8% and 8.1% respectively. In the 1st passage they were 2.1% and 2.8% respectively. They were not detected in the second passage. The values of cpm were respectively 482.13±46.61 (primary passage), 362.50±35.12(1st passage), 228.38±51.46(2nd passage), 171.86±34.63(3rd passage), 143.63±15.95(4th passage), and 123.25±14.39(5th passage), showing statistically significant differences when compared withcontrol (53.67±8.61) (Plt;0.05). There were statistically significant differences between the primary passage, the 1st passage respectively and the other passages(Plt;0.05). There were statistically significant differences between the4th passage, the 5th passage respectively and the 2nd passage (Plt;0.05). There was no statistically significant difference between the 2nd passageand the 3rd passages(Pgt;0.05). There was not statistically significant difference among the 3rd, the 4th and the 5th passages (Pgt;0.05). Conclusion Allogenic keratinocytes were cultured in vitro and passaged, and their immunogenity gradually decreased.
Objective To observe the clinical effect of the human tissue engineered activeskin (ActivSkin) with full thickness on the donor site of the split thickness skin graft. Methods Nine patients with 18 wounds of the donor sites, and every p atient had 2 wounds. The wounds of each patient were randomly assigned to the therapy group and the control group. Autocontrol observation was performed. Nine donor sites of the split thickness skin graft were repaired with ActivSkin in the therapy group. Nine donor sites of the split thickness skin graft were repaired with the vash oil gauze in the control group. The wound pain, the time to complete closure, and the ratio of the complete healing in the ActivSkin therapy gro up was measured and compared with those in the control group. The donor sites of the split thickness skin graft were assessed at 180 days of the follow-up visit . Results The wound pain was obviously reduced after the harvest ing of the skin grafts in the therapy group. The time to complete closure on the donor sites of the split thickness skin graft was significantly shorter in the ActivSkin therap y group than in the control group (9.67±2.92 d vs.16.56±2.96 d, Plt;0.05 ). Both the ratios of the complete healing in the ActivSkin therapy group and the control group were 100%（Pgt;0.05）. The subsequent results showed that neit her the blister nor the residual wound occurred with an alleviated scar after the Ac tivSkin treatment. Conclusion ActivSkin can promote wound closure, prevent blister and residual wound, and alleviate scarring on the donor sites of the splitthickness skin graft after the ActivSkin treatment.