改良脱细胞异种真皮的制备与动物埋藏实验

目的:研制一种新型异种人工真皮。方法:应用鼠尾胶原和异种(猪)脱细胞真皮复合制备成新型复合真皮,将复合真皮与交联型脱细胞真皮分别埋藏于Balb/C小白鼠皮下,观察新型材料的组织相容性及促胶原代谢能力。结果:所有试验小鼠在早期均有毛发脱失现象,交联型真皮组持续时间长并伴有肉芽组织形成。4周后,新型复合真皮显示组织结构致密有序,降解时间更长;组织相容性及促自体胶原代谢能力均较交联型真皮更为优越。结论:新型材料具有更好的组织相容性和更强的促胶原代谢能力,是良好的皮肤组织工程支架材料。     

以活性复合真皮基质为载体构建组织工程皮肤的研究

目的 构建含活性真皮基质的组织工程皮肤. 方法将人成纤维细胞(Fb)与Ⅰ型牛胶原混合接种于猪脱细胞真皮基质(PADM)的表面,构建活性真皮替代物.其上接种人表皮细胞进行气-液面培养,获得组织工程皮肤,进行组织学观察. 结果 Fb在胶原内结构完整,与PADM形成复合真皮基质.所构建的组织工程皮肤表皮层结构与人正常皮肤相似,具备基底层、棘层、颗粒层和角质层,细胞之间有桥粒连接,细胞分化良好. 结论 Fb-胶原-PADM真皮替代物可作为较好的构建组织工程皮肤的真皮支架.     

含内皮祖细胞的组织工程皮肤体外构建及裸鼠移植研究

目的构建含内皮祖细胞（EPC）和成纤维细胞的组织工程复合皮，初步研究EPC在组织工程皮肤移植中促血管发生的作用。方法采用免疫磁珠法从人脐血中分离培养CD133＋血管内皮祖细胞，以聚羟基乙酸（PGA）为真皮基质，接种EPC和成纤维细胞，其上覆盖表皮细胞膜片，构建组织工程复合皮，覆盖裸鼠背部全层皮肤缺损创面。结果EPC和成纤维细胞能均匀贴附于PGA支架纤维材料上，并逐渐伸展为梭形或多极状。裸鼠移植实验表职，实验组创面愈合早于对照组，可见EPC参与大量新生小血管形成，真皮支架5周完全降解。结论EPC参与血管重建，具有促进血管新生，加速缺血组织血管化的作用。应用PGA＋纤维蛋白凝胶，制备含EPC和成纤维细胞的活性真皮替代物，具有良好的生物相容性、降解特性。     

Fibroblasts improve performance of cultured composite skin substitutes on athymic mice

BACKGROUND: This study investigated the impact of adding human fibroblasts to a cultured composite skin substitute model of cultured human keratinocytes and acellular human dermis. METHODS: Skin substitutes were prepared by seeding human keratinocytes on the papillary side of acellular dermis with or without seeding fibroblasts on the reticular side. Performance of the grafts was compared both in vitro by histology and in vivo on surgically created full-thickness wounds on athymic mice. Graft size and contraction were measured and immunohistochemical stains were done to reveal vascularization. RESULTS: Skin substitutes with fibroblasts formed thicker epidermis than skin substitutes without fibroblasts. When transplanted onto athymic mice, skin substitutes with fibroblasts maintained their original size with only 2% contraction. In contrast, skin substitutes without fibroblasts showed 29% contraction. Vascular basement membrane specific mouse CD31staining and endothelial cell specific mouse collagen type IV staining revealed vascularization as early as 1 week posttransplant in grafts with fibroblasts, and was significantly higher than grafts without fibroblasts at 2 weeks. CONCLUSIONS: Addition of fibroblasts to keratinocyte based composite skin substitutes improves epidermis formation, enhances vascularization and reduces contraction.     

Biocompatibility of acellular human pericardium

BACKGROUND: Previous studies have shown successful decellularization of human pericardium without affecting the major structural components and strength of the matrix. The aim of this study was to assess the biocompatibility and reseeding potential of the acellular human pericardial scaffold. MATERIALS AND METHODS: Pericardia were treated sequentially with hypotonic buffer, sodium dodecyl sulfate, and a nuclease solution. The presence of cellular attachment factors after decellularization was evaluated using immunohistochemistry. The scaffold was seeded with dermal fibroblasts and cellular attachment to and numbers of cells penetrating were assessed over time. Biocompatibility was also evaluated following subcutaneous implantation into a mouse model for three months. RESULTS: After decellularization, the scaffold stained positively for fibronectin, but collagen IV and laminin staining was reduced. Seeded fibroblasts attached to the mesothelial surface and were visualized in the tissue within a week of seeding. The majority of fibroblasts in the tissue were viable and there was evidence of remodeling of the matrix. Analysis of the explanted tissues from mice showed that fresh/frozen and glutaraldehyde-fixed pericardia were encapsulated with a thick layer of inflammatory cells and fibrous tissue. In contrast, the decellularized scaffold was infiltrated with myofibroblasts, CD34+ cells and macrophages, indicating a healthy repair process. Compared with the glutaraldehyde-fixed tissue, the calcium content of the fresh/frozen and decellularized pericardia was negligible. CONCLUSIONS: The pericardial scaffold was biocompatible in vitro and in the mouse model in vivo.     

Comparison studies of the in vivo treatment of full‐thickness excisional wounds and burns by an artificial bilayer dermal equivalent and J‐1 acellular dermal matrix

The effects upon skin repair were compared between a homemade bilayer dermal equivalent (BDE), composed of a collagen/chitosan porous scaffold and a silicone membrane, and J‐1 acellular dermal matrix (ADM), a commercial ADM that is used widely in China to treat various skin defects. Full‐thickness excisional and burn wounds were prepared on the backs of pigs and then treated with the BDE and J‐1 ADM. Biopsy specimens were harvested on days 7, 14, and 21 after surgery for gross, biochemical, and molecular examinations. In comparison with the burn wounds, the excisional wounds showed accelerated granular tissue formation and superior integration with the equivalents, regardless of their type. Immunohistochemical, immunofluorescence, real time quantitative polymerase chain reaction and Western blotting analyses showed that the vascularization rates in the excisional wounds group were also significantly faster than those of the burn group for both dermal equivalents. There was no significant difference between J‐1 ADM and BDE treatment on the formation of newly formed blood vessels for the excisional wounds at days 7, 14, and 21. However, there was a significant difference in the number of nascent blood vessels formed in the burn wounds after treatment with J‐1 ADM compared with BDE. The highest numbers of newly formed and mature blood vessels were present in the J‐1 ADM‐treated excisional wounds after 21 days. Ultrathin skin grafts were further transplanted on to the regenerated dermis for 28 days, resulting in the repair of the full‐thickness wounds and production of a structure similar to normal skin.     

PDGF-B基因表达在组织工程化皮肤移植后血管重建中的作用

目的构建含有血小板衍化生长因子B(PDGFB)基因的组织工程化皮肤,进行动物移植实验,研究PDGFB基因表达在真皮血管重建中的作用。方法构建PDGFB真核表达质粒,用脂质体LipofectAMINE介导转染成纤维细胞。分别构建3种不同类型的组织工程化皮肤角质形成细胞 脱细胞猪真皮基质(A组),角质形成细胞 脱细胞猪真皮基质 成纤维细胞(B组),角质形成细胞 脱细胞猪真皮基质 PDGFB基因转染的成纤维细胞(C组),分别移植于大鼠背部创面,观察术后2、4、6周真皮血管重建情况。结果术后2周C组真皮浅层内可见较多新生毛细血管长入,B组次之,A组毛细血管长入较少(P<005);术后4周各组真皮浅层内毛细血管数逐渐增加,但C组毛细血管数仍明显高于B、A两组(P<005);术后6周各组织工程化皮肤内毛细血管数差异无显著性意义。结论PDGFB基因在组织工程化皮肤移植后早期真皮血管重建中发挥了重要的作用,为移植后皮片成活提供了保障。     

A pre‐clinical functional assessment of an acellular scaffold intended for the treatment of hard‐to‐heal wounds

The majority of the population experience successful wound‐healing outcomes; however, 1–3% of those aged over 65 years experience delayed wound healing and wound perpetuation. These hard‐to‐heal wounds contain degraded and dysfunctional extracellular matrix (ECM); yet, the integrity of this structure is critical in the processes of normal wound healing. Here, we evaluated a novel synthetic matrix protein for its ability to act as an acellular scaffold that could replace dysfunctional ECM. In this regard, the synthetic protein was subjected to adsorption and diffusion assays using collagen and human dermal tissues; evaluated for its ability to influence keratinocyte and fibroblast attachment, migration and proliferation and assessed for its ability to influence in vivo wound healing in a porcine model. Critically, these experiments demonstrate that the matrix protein adsorbed to collagen and human dermal tissue but did not diffuse through human dermal tissue within a 24‐hour observation period, and facilitated cell attachment, migration and proliferation. In a porcine wound‐healing model, significantly smaller wound areas were observed in the test group compared with the control group following the third treatment. These data provide evidence that the synthetic matrix protein has the ability to function as an acellular scaffold for wound‐healing purposes.     

Human plasma as a dermal scaffold for the generation of a completely autologous bioengineered skin

BACKGROUND: Keratinocyte cultures have been used for the treatment of severe burn patients. Here, we describe a new cultured bioengineered skin based on (1) keratinocytes and fibroblasts obtained from a single skin biopsy and (2) a dermal matrix based on human plasma. A high expansion capacity achieved by keratinocytes grown on this plasma-based matrix is reported. In addition, the results of successful preclinical and clinical tests are presented. METHODS: Keratinocytes and fibroblasts were obtained by a double enzymatic digestion (trypsin and collagenase, respectively). In this setting, human fibroblasts are embedded in a clotted plasma-based matrix that serves as a three-dimensional scaffold. Human keratinocytes are seeded on the plasma-based scaffold to form the epidermal component of the skin construct. Regeneration performance of the plasma-based bioengineered skin was tested on immunodeficient mice as a preclinical approach. Finally, this skin equivalent was grafted on two severely burned patients. RESULTS: Keratinocytes seeded on the plasma-based scaffold grew to confluence, allowing a 1,000-fold cultured-area expansion after 24 to 26 days of culture. Experimental transplantation of human keratinocytes expanded on the engineered plasma scaffold yielded optimum epidermal architecture and phenotype, including the expression of structural intracellular proteins and basement-membrane components. In addition, we report here the successful engraftment and stable skin regeneration in two severely burned patients at 1 and 2 years follow-up. CONCLUSIONS: Our data demonstrate that this new dermal equivalent allows for (1) generation of large bioengineered skin surfaces, (2) restoration of both the epidermal and dermal skin compartments, and (3) functional epidermal stem-cell preservation.     

成纤维细胞-无细胞真皮替代物的生物学活性及移植实验

目的 研究含成纤维细胞的无细胞真皮替代物的生物学活性及真皮支架作用。方法 将成纤维细胞种植于无细胞真皮表面培养,形成活性真皮替代物。采用ELISA法和RIA法测定培养上清中IL－6、IL－8、TGF－β1及细胞外基质层粘连蛋白、透明质酸的分泌。并将真皮替代物植入BALB／c-nu小鼠（裸鼠）全层皮肤缺损创面,观察血管化速度、创面收缩率。结果 成纤维细胞种植于无细胞真皮表面生长良好,可形成单层细胞膜片,并分泌多种细胞因子和细胞外基质成份。活性真皮替代物植入创面后,与单纯无细胞真皮移植相比,血管化速度加快,收缩率减小。结论 无细胞真皮上种植成纤维细胞后具有较强的生物学活性,可作为较好的真皮替代物。     

Transplantation of acellular dermis and keratinocytes cultured on porous biodegradable microcarriers into full-thickness skin injuries on athymic rats

In search of an optimal transplantation regime for sufficient dermal and epidermal regeneration after a full-thickness skin injury, wounds on athymic rats were grafted with split-thickness skin grafts or acellular human dermis followed by transplantation with human keratinocytes either in single-cell suspension or cultured on porous biodegradable microcarriers. After 2 weeks, all wounds grafted with acellular human dermis showed a well organised and vascularised dermal component and reepithelialisation on the grafted dermal matrix was complete 21 days after transplantation with human keratinocytes. Wounds grafted with human keratinocytes seeded on biodegradable microcarriers or split-thickness skin grafts displayed over time (i.e. 16-21 days post-transplantation) a significantly thicker epithelial cell layer in comparison to wounds grafted with keratinocytes in single-cell suspensions or microcarriers not seeded with cells. Furthermore, measurements of dermal thickness in the closed wounds 21 days after grafting showed a significantly thicker and well organised neodermal component in wounds transplanted with keratinocytes seeded on microcarriers or split-thickness skin grafts compared to all other wounds. Positive immunostaining towards von Willebrand factor revealed the plausible proangiogenic effects of transplantation with keratinocytes seeded on microcarriers. Analysis of representative tissue sections after fluorescence in situ hybridisation visualised that grafted human keratinocytes were present in the epidermal layers covering the wounds 16 and 21 days after transplantation, strongly indicating preservation of cell viability. These results shows that the use of biodegradable microcarriers in the culture of autologous keratinocytes for treatment of full-thickness wounds not only facilitate the cultivation, transportation and transplantation processes but also enhances the dermal regeneration induced by a dermal scaffold which results in a clinical result that is significantly superior to the one obtained when keratinocytes are transplanted in a single-cell suspension.     

三种组织工程鼓膜的构建

目的 构建3种不同材料的组织工程鼓膜,并对其进行生物学特性检测. 方法 体重 30 kg 健康雄性小白猪1只,制备脱细胞硬脑膜及脱细胞真皮,并从牛皮中提取液态胶原,将第3代豚鼠鼓膜成纤维细胞 1×106个/mL,各 0.2 mL 种于3种材料表面进行培养,倒置相差显微镜下观察成纤维细胞的生长状态.培养1周后,将3种材料分别进行扫描电镜及HE染色观察,测定材料厚度及其张力. 结果 成纤维细胞在3种材料表面生长良好,呈束状平行排列,其中以胶原与脱细胞真皮表面成纤维细胞生长情况较佳.HE 染色及扫描电镜下可见成纤维细胞呈单层于3种材料表面连续生长.胶原膜、脱细胞真皮及脱细胞硬脑膜的平均厚度分别为 9.4、10.0 及 10.4 μm.张力强度测定,复合成纤维细胞的胶原组织工程鼓膜张力 (1.417±0.030) N/mm2,复合成纤维细胞的脱细胞真皮张力为 (24.500±2.040)N/mm2,复合成纤维细胞的脱细胞硬脑膜张力为(53.300±2.600)N/mm2;复合成纤维细胞的脱细胞真皮张力与正常豚鼠鼓膜张力26.700 N/mm2最接近. 结论 脱细胞猪真皮为支架复合成纤维细胞构建的组织工程鼓膜,其张力与厚度均与正常豚鼠鼓膜相接近,是构建组织工程鼓膜的理想材料.     

CXCR4 antagonist delivery on decellularized skin scaffold facilitates impaired wound healing in diabetic mice by increasing expression of SDF‐1 and enhancing migration of CXCR4‐positive cells

C‐X‐C chemokine receptor type 4 (CXCR4) is an alpha‐chemokine receptor specific for stromal cell‐derived factor 1 (SDF‐1 also called CXCL12). The antagonist of CXCR4 can mobilize CD34+ cells and hematopoietic stem cells from bone marrow within several hours, and it has an efficacy on diabetes ulcer through acting on the SDF‐1/CXCR4 axis. In this study, we investigated for the first time whether the antagonist of CXCR4 (Plerixafor/AMD3100) delivered on acellular dermal matrix (ADM) may accelerate diabetes‐impaired wound healing. ADM scaffolds were fabricated from nondiabetic mouse skin through decellularization processing and incorporated with AMD3100 to construct ADM‐AMD3100 scaffold. Full‐thickness cutaneous wound in streptozotocin (STZ)‐induced diabetic mice were treated with ADM, AMD3100, or ADM‐AMD3100. 21 days after treatment, wound closure in ADM‐AMD3100‐treated mice was more complete than ADM group and AMD3100 group, and it was accompanied by thicker collagen formation. Correspondingly, diabetic mice treated with ADM‐AMD3100 demonstrated prominent neovascularization (higher capillary density and vascular smooth muscle actin), which were accompanied by up‐regulated mRNA levels of SDF‐1 and enhanced migration of CXCR4 in the granulation tissue. Our results demonstrate that ADM scaffold provide perfect niche for loading AMD3100 and ADM‐AMD3100 is a promising method for diabetic wound healing mainly by increasing expression of SDF‐1 and enhancing migration of CXCR4‐positive cells.     

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.     

Acellular dermal matrix reduces capsule formation in two‐stage breast reconstruction

Acellular dermal matrix (ADM) is commonly employed to create an inferior pocket for the tissue expander in two‐stage breast reconstruction. The authors sought to determine whether placement of ADM during the first stage of reconstruction decreases the amount of capsule formation at implant exchange. Patients who underwent mastectomy and tissue expander reconstruction were included in this study. Two biopsies were obtained at the time of implant exchange, one from the pocket adjacent to the ADM and the other from the area adjacent to the pectoralis muscle. Pathology analysis was performed on each sample. Ten patients underwent immediate breast reconstruction with Alloderm during the 3‐month study period. Capsule thickness was significantly greater in the areas where the expander was in direct contact with the pectoralis muscle (782 ± 194 µm) compared to those in contact with human acellular dermal matrix (hADM) (47·91 ± 110·82 µm; P < 0·05). Analysis of the sub‐pectoral capsule demonstrated diffuse deposition of collagen, neutrophils, contractile myofibroblasts and synovia‐like metaplasia, characteristic of a foreign body response. Conversely, within the inferior pocket where the hADM was in direct contact with the expander, we noted migration of host epithelial cells, fibroblasts, mesenchymal cells and angiogenesis, indicating host tissue regeneration. Acellular dermal matrix, when placed at the first stage of breast reconstruction, significantly reduces thickness and inflammatory character of the capsule in comparison to the patient's native tissue.     

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.     

Biomimetic bilayered gelatin-chondroitin 6 sulfate-hyaluronic acid biopolymer as a scaffold for skin equivalent tissue engineering

In order to develop an adequate scaffold for skin tissue engineering, a bilayered gelatin-chondroitin 6 sulfate-hyaluronic acid membrane with a different pore size on either side was prepared. A rete ridges-like topographic microporous structure, which provided the paracrine crosstalk in the epithelial-mesenchymal interactions, was formed. Chondroitin-6-sulfate and hyaluronic acid were incorporated within the gelatin membrane to mimic skin composition and create an appropriate microenvironment for cell proliferation, differentiation, and migration. In the study, the lower layer of the membrane (pore size: 150 microm) was seeded with dermal fibroblasts and acted as the feeder layer for keratinocyte inoculation. Meanwhile, the upper layer (pore size: 20-50 microm) was seeded with keratinocytes for epidermalization. The dermal fibroblasts were dynamically seeded in a self-designed spinner flask for more even cell distribution. The keratinocytes were cultured in submerged conditions for 5 days and then in an air-liquid interface condition for further differentiation. After being cultured for 21 days, the upper layer, seeded with keratinocytes, developed into an epidermis-like structure while the lower part, which was seeded with dermal fibroblasts developed into a dermis-like structure. A histological examination and immunostain were used to prove that keratinocytes maintain their phenotype and stratified epidermis layers were formed within 21 days. In brief, the bilayered skin substitute with biological dermal analog and epidermal structure was successfully fabricated. From this study, we can suggest that the culture model is suitable for autologous skin equivalent preparation.     

用含活性细胞的胶原支架复合皮修复小鼠全层皮肤缺损的研究

目的构建具有活性细胞成分的复合皮，观察其在修复小鼠全层皮肤缺损中的作用。方法将异种成纤维细胞种植于脱软骨细胞胶原支架上培养3d，形成真皮替代物。再将其放置于培养的上皮细胞膜上共同培养10d，组成含活性细胞成分的复合皮。将复合皮移植于小鼠的全层皮肤缺损处，记录其生长情况并于术后定期活检，进行组织学观察。结果胶原支架中可见生长良好的成纤维细胞和复层表皮细胞。移植术后1周，复合皮与小鼠缺损创面粘连紧密，可见明显的血管化。术后6周，创面愈合良好，移植物与创缘融合，未见明显排斥反应。结论以脱软骨细胞胶原为支架构建的复合皮，可作为皮肤替代物修复全层皮肤组织缺损。     

Culture of rat keratinocytes with acellular pig dermis.

This study evaluated the growth rate and the cell activity of cultured keratinocytes on acellular pig dermis in order to develop a composite skin in vitro for burn injuries or other skin defects. Full thickness skin was cultivated from neonatal SD rats, and separated into epidermal layer and dermal layer with enzyme digestion. The keratinocytes were then seeded on the prepared acellular pig dermis soaked in the culture medium. The cultures were incubated and the growth status of keratinocytes on acellular pig dermis evaluated by phase contrast microscope, histological examination with hematoxylin-eosin staining and acridine orange staining, immunohistochemistry, observation of growth curve plotted by MTT colorimetry and analysis of changes in keratinocytes proliferation cycle with flow cytometer. Almost all keratinocytes anchored in 48-72 h, and most inosculated at days 6 and 7. The growth curve showed that the keratinocytes grew in logarithmic phase at days 3-6 after seeding. More than four layers of keratinocyte structure and the basement membrane between keratinocytes and porcine dermis were observed. Pancytokeratin was strongly positive in the cultured keratinocytes. Laminin and collagen IV were positive in the basement membrane. It is concluded that the cultured keratinocytes on acellular pig dermis grow well and the structure of composite skin which has been established is satisfactory.     

皮肤组织工程学在我国

Great progress has been made in the basic research and clinical application of skin tissue engineering in China over the past 20 years. It includes culture of epithelial cells and their preliminary clinicaluse , research and development of various dermal substitutes such as acellular dermal matrix, spongi form collagen membrane and high molecular weight polymer membrane , and modification of physical properties of dermal substitutes for the sake of raising their bioaffinity and vascular ization , based on which composite skin containing epithelial cell layers has been constructed and used successfully in the repair of full-thickness skin defects. More recently , greater efforts have been made in the study of new epithelial seeding cells such as epithelial stem cell and hair follicle stem cell. With the work going into the center, it is hopeful into constructing an artificial skin that mimics the normal human skin in terms of structure and function with better viability of the transplant, so that it can eventually be used in clinical practice as a skin source for large area deep burn patients to improve the wound healing quality.