Human De-epidermized Dermis as a Stem Cell Carrier

Recently several types of skin equivalents have been developed, consisting of differentiated keratinocytes cultured on various dermal substitutes. Different models of reconstructed human skin have been proposed, such as human and animal de-epidermized dermis, inert filters, collagen matrices, lyophilized collagen membranes populated with fibroblasts, and other models populated with melanocytes and/or Langerhans cells. These skin equivalents mimic native skin in vivo. They have provided information about dermal-epidermal interactions, cell-cell, and cell-matrix interactions; responses of dermal and epithelial cells to biological signals and pharmacological agents; as well as effects of drugs and growth factors on wound healing. Human allodermis from tissue banks has been used for clinical purposes, namely, as support for autologous keratinocyte cultures and as a potentially ideal scaffold for dermal replacement. This bioproduct is considered to be the most suitable clinical carrier for autologous fibroblasts and keratinocytes, as well as an useful experimental model to study angiogenesis and to stimulate vascularization in reconstructed human skin. Because it is human-derived, it is in our opinion the safest of all available types of skin equivalent. Having epidermal and dermal structures, it can be used in one-stage grafting procedures for wound closure.     

Efficacy of gaseous nitric oxide in the treatment of skin and soft tissue infections

Bacterial burden significantly interferes with the healing process in chronic ulcers. Nitric oxide (NO) plays a key role in regulating skin's response to infection and wound healing. In previous studies, we demonstrated that exogenous NO gas (gNO) at 200 parts per million (ppm) exhibits potent antimicrobial effects against a representative range of pathogens. The aim of the present study is to explore the antimicrobial properties of gNO in vivo and to determine skin cells' sensitivity to the cytotoxic effects of gNO. To test gNO's antimicrobial effects, full-thickness wounds were infected with Staphylococcus aureus on the dorsal skin surface of New Zealand White rabbit and treated with 200 ppm gNO for 8 hours/day for 3 consecutive days. Significant reduction in wound bacterial content was observed in the presence of gNO. In a separate experiment, primary cultures of human fibroblasts, keratinocytes, and endothelial cells were established to test gNO's cytotoxicity in the skin. Methyl thiazolyl tetrazolium proliferation assays demonstrated that human skin cells, unlike bacterial cells, exhibited significant resistance toward gNO cytotoxicity. In vitro migration studies on keratinocytes and endothelial cells revealed that gNO treatment does not seem to interfere with reepithelialization and angiogenesis during the process of wound healing. Following 24 hours of gNO treatment, fibroblasts expressed significantly higher levels of procollagen and, to a lesser degree, a decrease in matrix metalloproteinase -1 mRNA. In conclusion, the present study provides evidence for the potential application of high doses of gNO as an antimicrobial agent for the treatment of infection in chronic nonhealing ulcers or burn patients, without compromising the viability, and function of skin cells.     

Approaches to improve angiogenesis in tissue-engineered skin

A problem with tissue-engineered skin is clinical failure due to delays in vascularization. The aim of this study was to explore a number of simple strategies to improve angiogenesis/vascularization using a tissue-engineered model of skin to which small vessel human dermal microvascular endothelial cells were added. For the majority of these studies, a modified Guirguis chamber was used, which allowed the investigation of several variables within the same experiment using the same human dermis; cell type, angiogenic growth factors, the influence of keratinocytes and fibroblasts, mechanical penetration of the human dermis, the site of endothelial cell addition, and the influence of hypoxia were all examined. A qualitative scoring system was used to assess the impact of these factors on the penetration of endothelial cells throughout the dermis. Similar results were achieved using freshly isolated small vessel human dermal microvascular endothelial cells or an endothelial cell line and a minimum cell seeding density was identified. Cell penetration was not influenced by the addition of angiogenic growth factors (vascular endothelial growth factor and basic fibroblast growth factor); similarly, including epidermal keratinocytes or dermal fibroblasts did not encourage endothelial cell entry, and neither did mechanical introduction of holes throughout the dermis. Two factors were identified that significantly enhanced endothelial cell penetration into the dermis: hypoxia and the site of endothelial cell addition. Endothelial cells added from the papillary surface entered into the dermis much more effectively than when cells were added to the reticular surface of the dermis. We conclude that this model is valuable in improving our understanding of how to enhance vascularization of tissue-engineered grafts.     

Locomotion of human skin keratinocytes on polystyrene, fibrin, and collagen substrata and its modification by cell-to-cell contacts

Epithelial wound repair assures the recovery of the epithelial barrier after wounding. During wound healing epithelial cells migrate to cover the wound surface. The presented experiments were carried out to compare the migration of human keratinocytes from primary and secondary culture on polystyrene, collagen, and fibrin glue used in clinical techniques. The images of migrating keratinocytes were recorded and analyzed using computer-aided methods. The results show that the character of the substrate strongly affects the speed and turning behavior of keratinocytes locomoting over it. The highest motile activity of human skin keratinocytes was found on fibrin glue substratum. It was found that locomotion of freely moving isolated cells was much faster than that of cell sheets. The autologous keratinocytes cultured in vitro were applied with fibrin glue to cover trophic wounds. The transplantation of human autologous keratinocyte suspension in fibrin glue upon long-lasting trophic wounds appeared to induce rapid and permanent wound healing.     

Reconstructed human skin produced in vitro and grafted on athymic mice

BACKGROUND: The best alternative to a split-thickness graft for the wound coverage of patients with extensive burns should be in vitro reconstructed autologous skin made of both dermis and epidermis and devoid of exogenous extracellular matrix proteins and synthetic material. We have designed such a reconstructed human skin (rHS) and present here its first in vivo grafting on athymic mice. METHODS: The rHS was made by culturing newborn or adult keratinocytes on superimposed fibrous sheets obtained after culturing human fibroblasts with ascorbic acid. Ten days after keratinocyte seeding, reconstructed skins were either cultured at the air-liquid interface or grafted on athymic mice. We present the macroscopic, histologic, and phenotypic properties of such tissues in vitro and in vivo after grafting on nude mice. RESULTS: After maturation in vitro, the reconstructed skin exhibited a well-developed human epidermis that expressed differentiated markers and basement membrane proteins. Four days after grafting, a complete take of all grafts was obtained. Histological analysis revealed that the newly generated epidermis of newborn rHS was thicker than that of adult rHS after 4 days but similar 21 days after grafting. The basement membrane components (bullous pemphigoid antigens, laminin, and type IV and VII collagens) were detected at the dermo-epidermal junction, showing a continuous line 4 days after grafting. Ultrastructural studies revealed that the basement membrane was continuous and well organized 21 days after transplantation. The macroscopic aspect of the reconstructed skin revealed a resistant, supple, and elastic tissue. Elastin staining and elastic fibers were detected as a complex network in the rHS that contributes to the good elasticity of this new reconstructed tissue. CONCLUSIONS: This new rHS model gives supple and easy to handle skins while demonstrating an adequate wound healing on mice. These results are promising for the development of this skin substitute for permanent coverage of burn wounds.     

Effects of keratinocyte conditioned medium,amniotic fluid and EGF in reepithelialization of human skin wounds in vitro

Growth factors and combinations of growth promoting substances in amniotic fluid, platelet extracts or factors secreted by cultured human keratinocytes have been shown to stimulate cell proliferation and wound healing. In this report, the possibility of studying reepithelialization of wounds in human skin, using small biopsies maintained in vitro, was examined. The effects of fetal calf serum (FCS), keratinocyte conditioned medium (KCM), amniotic fluid and epidermal growth factor (EGF) on the reepithelialization process was investigated after seven days. KCM alone did not affect healing, but when added to a suboptimal concentration of FCS (2%), KCM induced reepithelialization of the wounds. Amniotic fluid (25%) alone stimulated the formation of a new epidermis, whereas EGF (10 ng/ml) alone or added to 2% FCS did not induce reepithelialization. The model used in this study includes an autologous matrix comprising living fibroblasts and endothelial cells and may thus be used to study aspects of wound healing in human skin.     

自体组织工程化皮肤修复全层皮肤缺损的实验研究

目的为制成含表皮细胞与成纤维细胞的双层皮肤替代物,直接用于修复全层皮肤缺损.方法选用长枫杂交仔猪10只,酶消化法获取皮肤表皮细胞与成纤维细胞,将原代培养处于对数生长期的表皮细胞、成纤维细胞分别与30%氧化异丙烯F-127 混匀成细胞悬液后,种植聚羟基乙酸(polyglycolic acid,PGA)形成细胞-生物材料复合物,用于修复自体动物背部直径4 cm全层皮肤缺损,以单纯生物材料(PGA+氧化异丙烯)充填的创面作为对照组.修复术后1,2,4,8周取材,通过组织学和基底膜特殊染色等方法对新生组织进行评价.结果第1周新生组织即出现表皮与真皮2层结构,特殊染色观察到连续的基底膜.第2周表皮与真皮均较前增厚.修复后第8周组织工程化皮肤的形态结构均与正常皮肤相似.对照组则无皮肤形成,仅见大量肉芽组织.结论应用组织工程技术,以PGA+氧化异丙烯为表皮细胞、成纤维细胞载体构建的组织工程化皮肤可修复全层皮肤缺损.     

Enhancement of keratinocyte performance in the production of tissue-engineered skin using a low-calcium medium

The success of laboratory-expanded autologous keratinocytes for the treatment of severe burn injuries is often compromised by their lack of dermal remnants and failure to establish a secure dermo-epidermal junction on the wound bed. We have developed a tissue-engineered skin substitute for in vivo use, based on a sterilized donor human dermis seeded with autologous keratinocytes and fibroblasts. However, culture rates are currently too slow for clinical use in acute burns. Our aim in this study was to increase the rate of production of tissue-engineered skin. Two approaches were explored: one using a commercial low-calcium media and the other supplementing well-established media for keratinocyte culture with the calcium-chelating agent ethylene glutamine tetra-acetic acid (EGTA). Using commercial low-calcium media for both the initial cell culture and subsequent culture of tissue-engineered skin did not produce tissue suitable for clinical use. However, it was possible to enhance the initial proliferation of keratinocytes and to increase their horizontal migration in tissue-engineered skin by supplementing established culture medium with 0.04 mM EGTA without sacrificing epidermal attachment and differentiation. Enhancement of keratinocyte migration with EGTA was also maximal in the absence of fibroblasts or basement membrane.     

Autologous and Allogeneic Skin Cell Grafts in the Treatment of Severely Burned Patients: Retrospective Clinical Study

Background

Transplantation of skin cells (keratinocytes and fibroblasts) cultured in vitro is a method of choice for the treatment of severe and extensive burns in patients with a deficit of donor sites for free split-thickness skin grafts, and when the grave medical condition of the patient excludes the possibility of an operation under general anesthetic. Appropriate amounts of keratinocytes and/or fibroblasts cultured in vitro are grafted as a suspension in platelet-leukocyte-rich gel directly on the prepared acceptor site. Approximately 3 weeks are needed for autologous cell culture to grow. Allogeneic cells are obtained from patients who died before their own autologous cell transplantation. Therefore allogeneic cells are considered as ready to use product. The aim of the study was to evaluate the efficiency of in vitro cultured autologous/allogeneic skin cell grafts in the treatment of burns.

Culture of keratinocytes for transplantation without the need of feeder layer cells

Patients with large burn wounds have a limited amount of healthy donor skin. An alternative for the autologous skin graft is transplantation with autologous keratinocytes. Conventionally, the keratinocytes are cultured with mouse feeder layer cells in medium containing fetal calf serum (FCS) to obtain sufficient numbers of cells. These xenobiotic materials can be a potential risk for the patient. The aim of the present study was to investigate if keratinocytes could be expanded in culture without the need of a feeder layer and FCS. Keratinocytes were cultured on tissue culture plastic with or without collagen type IV coating in medium containing Ultroser G (serum substitute) and keratinocyte growth factor (KGF). An in vitro skin equivalent model was used to examine the capacity of these cells to form an epidermis. Keratinocytes in different passages (P2, P4, and P6) and freshly isolated cells were studied. Keratinocytes grown on collagen type IV were able to form an epidermis at higher passage numbers than cells grown in the absence of collagen type IV (P4 and P2, respectively). In both cases the reconstructed epidermis showed an increased expression of Ki-67, SKALP, involucrin, and keratin 17 compared to normal skin. Only 50,000 keratinocytes grown on collagen type IV in P4 were needed to form 1 cm2 epidermis, whereas 150,000 of freshly isolated keratinocytes were necessary. Using this culture technique sufficient numbers of keratinocytes, isolated from 1 cm2 skin, were obtained to cover 400 cm2 of wound surface in 2 weeks. The results show that keratinocytes can be cultured without the need of a fibroblast feeder layer and FCS and that these cells are still able to create a fully differentiated epidermis. This culture technique can be a valuable tool for the treatment of burn wounds and further development of tissue engineered skin.     

Activated protein C prevents inflammation yet stimulates angiogenesis to promote cutaneous wound healing

Activated protein C (APC) is a serine protease that plays a central role in physiological anticoagulation, and has more recently been shown to be a potent anti-inflammatory mediator. Using cultured human cells, we show here that APC up-regulates the angiogenic promoters matrix metalloproteinase-2 in skin fibroblasts and umbilical vein endothelial cells, vascular endothelial growth factor in keratinocytes and fibroblasts, and monocyte chemoattractant protein-1 in fibroblasts. In the chick embryo chorioallantoic membrane assay, APC promoted the granulation/remodeling phases of wound healing by markedly stimulating angiogenesis as well as promoting reepithelialization. In a full-thickness rat skin-healing model, a single topical application of APC enhanced wound healing compared to saline control. APC-treated wounds had markedly more blood vessels on day 7 and a significantly lower infiltration of neutrophils at days 4 and 7. The broad spectrum matrix metallo-proteinase, GM6001, prevented the ability of APC to promote wound healing. In summary, our results show that APC promotes cutaneous wound healing via a complex mechanism involving stimulation of angiogenesis and inhibition of inflammation. These unique properties of APC make it an attractive therapeutic agent to promote the healing of chronic wounds.     

In vitro cytotoxity of silver: implication for clinical wound care

In this study, we look at the cytotoxic effects of silver on keratinocytes and fibroblasts. We have assessed the viability of monolayer cultures using the MTT and BrdU assays. The composition of the culture medium and also the culture technique were modified to assess the effects of culture 'environment' on the susceptibility of the cells to the toxic action of silver. Further in vitro, experiments were performed using tissue culture models to allow cellular behavior in three dimensional planes which more closely simulated in vivo behavior. The silver source was both silver released from silver nitrate solution but also nanocrystalline silver released from a commercially available dressing. The results show that silver is highly toxic to both keratinocytes and fibroblasts in monolayer culture. When using optimized and individualized culture the fibroblasts appear to be more sensitive to silver than keratinocytes. However, when both cell types were grown in the same medium their viability was the same. Using tissue culture models again indicated an 'environmental effect' with decreased sensitivity of the cells to the cytotoxic effects of the silver. Nevertheless in these studies the toxic dose of skin cells ranging from 7 x 10(-4) to 55 x 10(-4)% was similar to that of bacteria. These results suggest that consideration of the cytotoxic effects of silver and silver-based products should be taken when deciding on dressings for specific wound care strategies. This is important when using keratinocyte culture, in situ, which is playing an increasing role in contemporary wound and burn care.     

组织工程技术修复皮肤缺损的动物实验

目的　探索应用组织工程技术修复包括皮下组织的皮肤缺损的方法。方法　长枫杂交仔猪 2 0只 ,取腹部 2cm× 2cm全厚皮肤 ,酶消化法获取表皮细胞与成纤维细胞。经原代培养 ,将处于对数生长期的表皮细胞、成纤维细胞分别与 30 %氧化异丙烯F 1 2 7(pluronicF 1 2 7)混匀成细胞悬液后 ,种植于聚羟基乙酸 (polyglycolicacid ,PGA)形成细胞 生物材料复合物 ,用于修复自体背部直径 4cm皮肤缺损 ,以单纯生物材料 (PGA 30 %氧化异丙烯F 1 2 7)修复作为对照组。术后 1、2、4、8周取材 ,通过组织学方法评价新生组织。结果　实验组 :第 1周 ,形成含表皮与真皮两层结构的组织工程化皮肤 ;第 2周 ,表皮与真皮均较前增厚 ;第 8周 ,组织工程化皮肤的结构与正常皮肤相似 ,仅缺乏毛发、毛囊和汗腺等附属器。对照组 :则无皮肤形成 ,仅见大量肉芽组织。结论　以原代培养的表皮细胞与成纤维细胞作为种子细胞 ,PGA 30 %氧化异丙烯F 1 2 7为细胞载体的方法可修复皮肤缺损。     

Inosculation of Tissue-Engineered Capillaries with the Host''s Vasculature in a Reconstructed Skin Transplanted on Mice

The major limitation for the application of an autologous in vitro tissue-engineered reconstructed skin (RS) for the treatment of burnt patients is the delayed vascularization of its relatively thick dermal avascular component, which may lead to graft necrosis. We have developed a human endothelialized reconstructed skin (ERS), combining keratinocytes, fibroblasts and endothelial cells (EC) in a collagen sponge. This skin substitute then spontaneously forms a network of capillary-like structures (CLS) in vitro. After transplantation to nude mice, we demonstrated that CLS containing mouse blood were observed underneath the epidermis in the ERS in less than 4 days, a delay comparable to our human skin control. In comparison, a 14-day period was necessary to achieve a similar result with the non-endothelialized RS. Furthermore, no mouse blood vessels were ever observed close to the epidermis before 14 days in the ERS and the RS. We thus concluded that the early vascularization observed in the ERS was most probably the result of inosculation of the CLS network with the host's capillaries, rather than neovascularization, which is a slower process. These results open exciting possibilities for the clinical application of many other tissue-engineered organs requiring a rapid vascularization.     

Persistent Ischemia Impairs Myofibroblast Development in Wound Granulation Tissue a New Model of Delayed Wound Healing

Skin substitutes are slowly finding a position in the treatment of burns, scar reconstructions and chronic wounds. Some of the substitutes consist of extracellular matrix replacement material only, such as Integra and Alloderm; some include allogeneic cells (Dermagraft, Appligraf). The ideal skin substitute has not been developed yet, since none of the presently available products can ultimately prevent scar formation.
Study of the role of autologous fibroblasts in the healing process might give further insight into the scarring process and eventually lead to improved skin substitutes.
We compared wound reepithelialization of experimental wounds treated with proliferating keratinocytes and a dermal substitute with either dermal fibroblasts, adipose tissue derived fibroblasts or no fibroblasts. We also investigated the rate of keratinocyte migration of human skin equivalents cultured in vitro in the presence of dermal or adipose tissue derived fibroblasts.
We reached successful wound closure in 8 days with transfer of proliferating keratinocytes on a dermal substitute seeded with dermal fibroblasts. However, the wounds treated with substitutes which contained adipose tissue derived fibroblasts or no fibroblasts at all were not closed even after 21 days.
Keratinocytes seeded onto collagen lattices populated with either dermal or fat‐derived fibroblasts showed similar findings: a retarded migration and/or proliferation of keratinocytes on the collagen lattices with fat‐derived fibroblasts. The collagen lattices populated with fat‐derived fibroblasts also showed a marked contraction, up till 50% of the original area.
In both models, more alpha‐smooth muscle actin positive cells were found in the fibroblast population from adipose origin.
We conclude that epidermal regeneration is negatively influenced by the presence of fat‐derived fibroblasts in a dermal matrix; possibly, myofibroblasts play a role in this.     

Cultured autologous keratinocytes on a cell-free dermis in the treatment of full-thickness wounds.

The purpose of this study was to establish a method for transplantation of autologous keratinocytes on an allogenic cell-free dermis. From four healthy volunteers two full thickness skin grafts, 1 x 1 cm, were taken. The epidermis was separated from the dermis enzymatically and the cells of the dermal part were removed by incubation in Triton X-100. Keratinocytes were seeded on a cell-free dermis and the combined graft transplanted back to one of the wounds of the donor of the keratinocytes. The other wound was covered with cell-free dermis without keratinocytes. After 2, 3, 4 and 6 weeks, respectively, the grafted wounds were removed from the subjects and investigated histologically and immunohistochemically regarding re-epithelialisation, fibroblast ingrowth and angiogenesis. The wounds covered with cell-free dermis and keratinocytes showed a complete epidermal coverage 2 weeks after transplantation, in contrast to the wounds covered with un-seeded dermis which only showed epidermal coverage at the wound edges. There was also a marked difference concerning fibroblast ingrowth and angiogenesis. In this study we have shown that autologous keratinocytes can be seeded on a cell-free dermis and transplanted as a kerato-dermal graft which stimulate re-epithelialisation as well as fibroblast ingrowth and angiogenesis in the wound.     

A novel bioreactor microcarrier cell culture system for high yields of proliferating autologous human keratinocytes

Rapid and efficient resurfacing of various skin defects by autologous keratinocyte transplantation is significant in skin wound healing. We developed a novel bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous human keratinocytes on a large scale. In this Bio-MCCS we used porcine gelatin microbeads as microcarriers for autolgous keratinocytes and spinning bottles as fermentation tanks. First, the microbeads were modified by culturing them with autologous dermal fibroblasts that were subsequently killed when they proliferated to confluence on the microbeads. We then performed the Bio-MCCS by expanding ketatinocytes on the microbeads in spinning bottles at 37 degrees C, 5% CO2. Our results showed that keratinocytes rapidly attached to and actively proliferated on the modified microbeads in the Bio-MCCS, achieving high cell densities on the modified microbeads (MTT assay and PI staining). Keratinocytes cultured on the modified microbeads in the Bio-MCCS remained proliferating potentials as shown by positive PCNA staining and BrdU labeling. In contrast, keratinocytes cultured on nonmodified microbeads in the Bio-MCCS proliferated slowly, rapidly ceased to proliferate, and finally dislodged from the microbeads. When removed from the Bio-MCCS and cultured under static conditions, keratinocytes were able to leave the modified microbeads and formed a multilayered epidermal equivalent on the culture surfaces. While stored at room temperature, keratinocytes remained at higher viabilities on the modified microbeads when compared to those on nonmodified microbeads. The achievement of high yields of proliferating autologous keratinocytes by this Bio-MCCS offers a practical potential of resurfacing various skin defects by direct administration of autologous keratinocyte microbeads on various skin defects.     

Effect of Atropa belladonna L. on skin wound healing: Biomechanical and histological study in rats and in vitro study in keratinocytes, 3T3 fibroblasts, and human umbilical vein endothelial cells

The effect of Atropa belladonna L. (AB) aqueous extract on skin wound healing was studied in male Sprague–Dawley rats subjected to two parallel full-thickness skin incisions on the back. Specimens for histological evaluation were collected on days 2 and 5 whereas for biomechanical testing, they were collected on day 5. In the in vitro study, a different concentration of AB extract was used to test the differentiation of keratinocytes using a panel of selected antibodies, proliferation, and cell survival of 3T3 fibroblasts and human umbilical vein endothelial cells using the MTT-assay. Results of the in vivo experiments showed in AB-treated wounds a shortened process of inflammation and accelerated collagen formation, as well as significantly increased wound stiffness as compared with control tissues. The in vitro examination showed that control keratinocytes were cytokeratin 19 free, while samples exposed to the highest AB extract concentration expressed CK19. Moreover, all concentrations were stimulatory to human umbilical vein endothelial cell proliferation. In addition, only the AB extract at the lowest tested concentration increased fibroblast growth, but higher concentrations decreased cell survival. In conclusion, our results indicate that the AB water extract positively affects early phases of skin wound healing in rats. However, the in vitro results on the inverse relation between the concentration of the AB extract and its effects on cell proliferation may be important for future research.     

The importance of both fibroblasts and keratinocytes in a bilayered living cellular construct used in wound healing

Cross talk between fibroblasts and keratinocytes, which maintains skin homeostasis, is disrupted in chronic wounds. For venous leg ulcers and diabetic foot ulcers, a bilayered living cellular construct (BLCC), containing both fibroblasts and keratinocytes that participate in cross talk, is a safe and effective product in healing chronic wounds. To show the importance of both cell types in BLCC, constructs were generated containing only fibroblasts or only keratinocytes and compared directly to BLCC via histology, mechanical testing, gene/protein analysis, and angiogenesis assays. BLCC contained a fully differentiated epithelium and showed greater tensile strength compared with one‐cell‐type constructs, most likely due to formation of intact basement membrane and well‐established stratum corneum in BLCC. Furthermore, expression of important wound healing genes, cytokines, and growth factors was modulated by the cells in BLCC compared with constructs containing only one cell type. Finally, conditioned medium from BLCC promoted greater endothelial network formation compared with media from one‐cell‐type constructs. Overall, this study characterized a commercially available wound healing product and showed that the presence of both fibroblasts and keratinocytes in BLCC contributed to epithelial stratification, greater tensile strength, modulation of cytokine and growth factor expression, and increased angiogenic properties compared with constructs containing fibroblasts or keratinocytes alone.     

自异体皮混合移植成活机制中角质形成细胞及细胞因子作用的实验研究

目的　观察细胞因子及角质形成细胞在自异体皮混合移植成活机制中的作用。　方法建立自异体皮混合移植大鼠模型和人体表皮细胞 淋巴细胞混合培养 (MELC)模型 ,检测大鼠移植术后血清及创面局部组织标本培养上清液中白细胞介素 (IL)1 0的水平 ,观察体外表皮细胞在诱导免疫抑制中的作用。　结果　术后 7d,移植大鼠血清IL 1 0含量为 (2 5 .89± 2 .82 )ng/L,明显高于正常大鼠的 (1 4 .2 0± 2 .4 3)ng/L(P <0.0 5 )。术后 4～ 1 4d,大鼠组织标本培养上清液中IL 1 0含量显著不同(P <0.0 5或 0 .0 1 ) ;术后 2 1、2 8d与正常大鼠比较 ,差异无显著性意义 (P >0.0 5 )。自体表皮细胞、角质形成细胞在MELC体系中的抑制作用与其剂量相关。自体角质形成细胞加入到MELC中 ,辅助性T淋巴细胞 (Th)1分泌的细胞因子谱主要通过IL 1 0介导而抑制Th2细胞因子谱的分泌 ;当加入抗IL 1 0单克隆抗体后该抑制作用消失。　结论　自异体皮混合移植后 ,嵌入自体皮片中的角质形成细胞通过激活Th2使局部IL 1 0水平升高 ,这可能是移植成活的重要原因之一。