小型香猪复方壳多糖组织工程皮肤的构建及组织学特点

目的评价制备的小型香猪复方壳多糖组织工程皮肤在组织学方面的特性,为其修复皮肤缺损创面提供实验依据。方法采用传代培养的角质形成细胞和成纤维细胞作为种子细胞,I型胶原作为真皮基质。采用气—液界面方式进行培养,通过苏木精—伊红(HE)染色、比较小型香猪的体外构建组织工程皮肤与正常皮肤的组织学特征,包括表皮、真皮结构。结果制备的复方壳多糖组织工程皮肤表皮细胞增殖活跃,分层分化良好,厚约150μm,真皮层细胞生长正常,排列有序,与机体皮肤结构相似。结论复方壳多糖组织工程皮肤组织结构良好,符合新型皮肤替代物在治疗皮肤缺损时的组织学要求。     

组织工程皮肤的研究进展

组织工程皮肤是由细胞或细胞外基质或由两者共同结合组成的皮肤产品,是应用生命科学和工程学的原理与技术将种子细胞与适当的支架材料相结合构建出的用于修复、维护和改善损伤皮肤组织功能和形态的生物替代物。从结构来分,可以分为三类：一是培养的表皮膜;二是真皮替代物;三是具有表皮、真皮的复合皮替代物。     

异种脱细胞真皮基质治疗大面积深度烧伤的研究进展

自体皮源的严重缺乏一直是大面积深度烧伤临床治疗的主要难点,因此,制备新型皮肤替代物已经成为大面积深度烧伤临床治疗亟待解决的问题。伴随着组织工程学的发展及制备工艺的改善,异种脱细胞真皮基质作为理想的皮肤替代物,在治疗大面积深度烧伤中被大量应用。本文对近几年异种脱细胞真皮基质的临床应用现状、治疗机理研究现状、不同脱细胞真皮基质的优缺点进行综述。     

酮洛芬及其异丙酯对HaCaT细胞构建的组织工程皮肤的渗透作用

目的体外构建适用于经皮给药研究的组织工程皮肤。方法以表皮角质形成细胞系HaCaT细胞及人真皮成纤维细胞为细胞来源，用I型牛胶原蛋白为真皮基质包埋成纤维细胞，其上接种HaCaT细胞，采用气-液界面方式培养，观察不同的培养介质对组织工程皮肤的影响，HE染色切片观察培养皮肤结构形态。以酮洛芬及其异丙酯为模型药物研究经皮渗透和代谢特性。结果HaCaT细胞经气-液界面培养可形成类表皮层，但不能形成完整的角质层。维生素C可明显促进细胞增殖，维生素D₃可促进细胞分化，雌二醇对此组织工程皮肤没有明显的影响。同皮肤细胞匀浆代谢相似，酮洛芬异丙酯被代谢成原药酮洛芬。结论HaCaT细胞在气-液界面培养条件下可形成多层分化不完全的表皮层，保留了一定的酶活性，可用于药物的经皮渗透和代谢等研究。     

组织工程皮肤种子细胞的研究进展

组织工程皮肤是应用生命科学和工程学的原理与方法,构建出的接近于人体组织结构的生物活性替代物。种子细胞的研究是组织工程皮肤产品研制中的关键部分。现就种子细胞相关研究的进展进行综述。     

新型人工复合皮的构建及临床初步应用

目的 探讨以小肠黏膜下层(SIS)为真皮替代物构建复合皮的可行性。方法 制取家猪小肠黏膜下层为真皮替代物。致密层表面种植呈指数分裂状态的自体表皮细胞。继续体外培养2周，获得人工复合皮。移植复合皮修复26例皮肤全层缺损创面；观察复合皮早期存活质量。并分别于培养第1周、第2周和移植后第1周、第2周取复合皮标本作组织学观察。结果 表皮细胞在SIS表面定位、生长。21例移植的复合皮早期成活良好。SIS内新生血管形成。炎性细胞浸润。无移植排斥反应。结论 以SIS为真皮替代物构建的复合皮，移植修复体表创面切实可行。     

无细胞异种真皮与自体薄皮片移植的术后护理

无细胞真皮基质是以提供移植皮肤足够量的真皮成分的一种永久性皮肤替代物，他对治疗深度烧伤创面及皮肤缺损具有很大的临床意义，采用无细胞真皮基质与自体薄皮片移植术，对当前解决植皮外观和功能起到相当重要的作用。1999年2月-2004年3月，我院进行无细胞异体真皮基质与自体薄皮片移植，治疗深度烧伤创面及疤痕畸形患者15例均获得成功．收到了明显的效果。该术的成功与术后护理有非常重要的关系。现将术后护理报道如下：     

无细胞真皮基质的研制

目的研制人同种脱细胞真皮基质,用于大面积深度烧伤后真皮成分的替代、美容医学中的填充及皮肤组织工程中的支架材料。方法获得的正常人新鲜尸体皮肤,用高渗盐溶液去除表皮,十二烷基硫酸钠、胰蛋白酶等处理以去除真皮中的细胞成分,得到人同种脱细胞真皮基质,经光镜、电镜和免疫组织化学分析。结果经处理后得到的人无细胞真皮基质,完全去除了表皮和真皮中的细胞成分,保留了完整的基底膜复合物,胶原束结构和排列正常,弹力纤维结构正常。结论用此方法制备无细胞真皮基质,能完全去除表皮和真皮中的细胞成分,不改变真皮中胶原纤维和弹力纤维结构,其制备工艺操作性强,是一种简单有效的制备方法。     

脱细胞异种真皮基质与自体薄皮片复合移植治疗创面全层软组织缺损

人体真皮组织的主要成分是胶原,其富于弹性,质地致密坚韧。因此使皮肤具有柔韧性和耐久性,Ⅲ°烧伤切痂创面,应用薄皮片移植愈合后因缺少真皮,皮肤弹性差,挛缩严重。在真皮替代物中,天然异体真皮、复合皮、人工皮等因排异及其它原因尚未取得满意而可信的临床结果。我科自2000年4月至2005年2月应用异体脱细胞真皮与自体薄皮片复合移植修复16例全层软组织缺损,现报道如下。资料与方法1·一般资料本组16例,男12例,女4例。年龄5~60岁。深度烧伤切痂病人10例14个创面,皮肤撕脱伤及其它外伤后皮肤软组织缺损6例6个创面。均采用异种脱细胞真皮基质…     

人工真皮支架研究进展

人工真皮支架材料是运用生物化学方法将天然或人工合成的聚合物运用于临床,以达到维持皮肤干细胞繁殖,且对人体无毒性作用的生物材料。良好的人工真皮支架材料,可以让皮肤组织工程尽可能地接近正常皮肤组织,以达到皮肤再生的目的。目前的研究重点是将人工合成类聚合物与天然材料通过一定的生物化学方法及比例进行组合,以形成具有良好的可塑性及适宜的力学特性、高度孔隙度的三维立体结构及生物相容性的复合型支架。支架材料作为再生模板和基材,其在创面     

The living skin equivalent: Its manufacture, its organotypic properties and its responses to irritants

A living skin equivalent that resembles human skin has been manufactured as a quality controlled product and commercialized. The skin equivalent consists of a dermal equivalent, reconstituted with collagen and dermal fibroblasts that are biosynthetically active, and a differentiated epidermis that arises from cultured keratinocytes plated onto the surface of the dermal equivalent. The organotypic system is configured so that the dermis is in contact with a nutrient pool while the stratum corneum of the epidermis is exposed to the atmosphere and is thereby accessible for the application of test substances in liquid, gel, emulsion, salve, powder or other forms. The stratum corneum of the living skin equivalent possesses a partial barrier function compared with that of skin. For example, the permeability constant (K_p) for tritiated water is approximately 20-fold higher than that of skin. The living skin equivalent also lends itself to the study of radiation effects. The dermal equivalent has been used to assess the efficacy of sunscreens applied directly upon it, and a good correlation between sun protection factor values and the degree of protection afforded to the living dermal equivalent has been demonstrated. The present report provides evidence that threshold doses for damage caused by various substances to living skin equivalent are similar to those for damage to skin.     

Water permeation barrier in isolated cutaneous newborn rat epidermis.

Abnormal water barrier function occurs in irritated skin and certain cutaneous diseases. Methods have been compared for separating the epidermis (site of the barrier) from the whole skin without disturbing the barrier function. The epidermis was separated from newborn rat skin by (1) exposure to 10% trypsin at 4 degrees C for 16 h, (2) exposure to 0.2% dispase at 4 degrees C for 16 h, (3) heating for 50 s at 55 degrees C, (4) or heating for 40 s at 50 degrees C after the whole skin was kept in medium at the air-liquid interface for 1 day at 35 degrees C. Water permeation of the isolated epidermis was then measured immediately or after 3, 5, 8, and 10 days of maintenance at the air-liquid interface. The water permeation barrier constant (kp) was 1.9+/-0.9 cm/h in intact rat skin. At 0 day of maintenance, the kp of the epidermis was 2.1+/-0.9 after treatment with trypsin, 3.8+/-1.2 after dispase, and 4.3+/-1.4 after immediate heating, or 2.2+/-0.7 cm/h after culture and heating. The dispase and heating methods disrupted the barrier to a greater extent than did the trypsin and culture-heating methods. The latter two methods allowed the kp to be maintained at low levels for 8 days (kp for trypsin 2.8+/-0.9 and 2.5+/-0.8 for culture-heating). Epidermis isolated by the trypsin or culture-heating techniques and maintained at the air-liquid interface can be used to study the mechanism by which barrier function is disrupted by chemicals.     

Elastin expression in a newly developed full-thickness skin equivalent

The resilience of the human skin is mediated by elastic fibres mainly consisting of fibrillins and elastin. In order to establish a model system to study the impact of cosmetic and pharmaceutical compounds on the elastic system in vitro, we analyzed the expression of elastin in a newly developed full-thickness skin model. After a 5-week cultivation period the skin model developed a fully differentiated epidermis including a stratum corneum. The dermis contains fibroblasts embedded in extracellular matrix proteins. The models were viable until at least 51 days at the air-liquid interface (ALI) culture. Using immunohistochemistry we detected elastin first on day 7 of ALI. With proceeding culture time, elastin-positive fibres of different lengths and distribution patterns accumulated in the dermal compartment. Elastin mRNA expression started on day 7 of ALI, increased until day 10 and then dropped to a level comparable to that of day 7. Our results demonstrate that in our full-thickness skin model an in vivo-like elastic system, which clearly mimics at least two subsets of dermal elastic fibres, is generated. This physiological property favours the model as a promising animal-free approach to study those processes leading to an environment- and age-dependent decrease in skin elasticity.     

Epidermis: the major site of cutaneous benzo(a)pyrene and benzo(a)pyrene 7,8-diol metabolism in neonatal BALB/c mice

The metabolism of benzo(a)pyrene (BP) and benzo(a)pyrene-7,8-diol (BP-7,8-diol) by microsomes prepared from whole skin, dermis, and epidermis of neonatal BALB/c mice pretreated with topically applied 3-methylcholanthrene (MCA) was compared. In control animals, microsomes prepared from epidermis showed higher rates of metabolism of BP and BP-7,8-diol (1.4-2.6-fold) than did microsomes prepared from whole skin or dermis. A single topical application of MCA increased the rate of metabolism of BP and BP-7,8-diol in microsomes prepared from whole skin, dermis, and epidermis. The greatest increase occurred in the epidermis. The in vivo covalent binding of [3H]BP, [3H]BP-7,8-diol, and 7,12-[3H]dimethylbenz(a)anthracene ([3H]DMBA) to DNA was found to be greater in epidermis (8.7-15.4-fold) than in whole skin or in dermis. A single topical application of MCA to BALB/c mice enhanced the in vivo binding of [3H]BP, [3H]BP-7,8-diol and [3H]DMBA to DNA of whole skin, dermis, and epidermis more than 2-fold. Exposure of Salmonella tester strains TA98 and TA100 to 2-aminoanthracene, a skin carcinogen, in the presence of an epidermal metabolic activation mixture resulted in a greater mutagenic response when compared to activation mixtures derived from whole skin or dermis. These results indicate that epidermis is the major site of polycyclic aromatic hydrocarbon metabolism and of enzyme-mediated covalent binding of polycyclic aromatic hydrocarbon carcinogens to DNA in skin of BALB/c mice and that topically applied MCA has maximum enzyme induction effects in this skin compartment.     

Ex vivo calcium percutaneous eggression in normal and tape-stripped human skin

Ions play a crucial role in skin homeostasis. Calcium, a participant in wound healing and keratinization, is localized at an increasing gradient from the stratum basal to the stratum granulosum. In vivo and in vitro studies show disturbances in this gradient in damaged skin. We developed here a model to study ex vivo calcium outward flux from normal and tape-stripped human skin. We measured here, with a calcium specific electrode, ex vivo calcium percutaneous eggression from dermis to epidermis in normal and tape-stripped skin places in franz cells, with a calcium source or not in the dermis compartment. Tape-stripped skin released a greater calcium concentration and had a higher rate over time than normal skin in both cases. The rate went from 8.1.10(-3) ± 8.9.10(-3) nmol.cm(-2).min(-1) to 4.8.10(-2)?±?1.8.10(-2)?nmol.cm(-2).min(-1) with no calcium in the dermis compartment and from 1.7.10(-1) ± 1.2.10(-1) nmol.cm(-2).min(-1) to 5.9?±?3.4 nmol.cm(-2).min(-1) with calcium. Also calcium uptake from the dermis is greater in tape-stripped skin during the first 5?h of the experiment. This ex vivo method reproduced the increased of calcium skin permeability with a disrupted barrier. This approach aims to develop a non-invasive method to measure calcium flux concentration in normal and damaged skin that might be reproduced and validated in vivo.     

Skin substitutes to enhance wound healing

Biologically-based skin substitutes have developed as commercial products over the last 5 years. The first generation includes the collagen-based synthetic device, Integra, and Alloderm, which is based on devitalised and cross-linked human dermis. These are used as dermal replacements for third degree burns. Within the last year, the tissue-engineered product, Dermagraft-TC, has become available. While originally intended as a temporary covering for severe burns, Dermagraft-TC has proved to markedly improve the healing of deep second degree burns. The earliest living skin substitutes used autologous keratinocytes expanded in vitro. Two new products containing living cells, Dermagraft and Apligraf, are expected to be approved shortly for diabetic foot ulcers and venous stasis ulcers, respectively. Dermagraft is produced by growing human fibroblasts on a three-dimensional scaffold. The cells actively proliferate and lay down extracellular matrix to generate a papillary dermis-like device that shows a combination of angiogenic, growth factor and cell adhesion properties that enhance healing in diabetic foot ulcers. The production of Apligraf includes casting human fibroblasts in collagen, in order to generate a dermal equivalent on which is grown an epidermis. The structure is akin to a skin graft and is so applied. Despite Dermagraft and Apligraf being of allogeneic origin, rejection has not been an issue in clinical trials and possible contamination by pathogens has been eliminated as a concern through extensive testing. These developments represent a new concept and are expected to revolutionise wound care. They may also provide a platform for gene therapy applications.     

A method for preparing mouse skin for assessing in vitro dermal penetration of xenobiotics

A method is described for preparing mouse skin for assessment of in vitro penetration of dermally applied compounds. Separation of the epidermis from the dermis was attempted using a dermatome, heat, trypsin and collagenase. When mouse skin was incubated in a collagenase solution and separated using water, the hypodermis and part of the dermis were separated from the epidermis while leaving the hair follicles and hair shafts intact. Morphologically, the skins prepared for in vitro use appeared to offer similar barriers to topically applied compounds as those found in vivo.