聚乳酸-聚羟基乙酸/Ⅰ型胶原复合支架的生物相容性研究

目的观察聚乳酸-聚羟基乙酸(poly-lactide-co-glycolide,PLGA)/Ⅰ型胶原复合支架的生物相容性,探讨其作为组织工程阴道支架的可行性。方法取经多聚赖氨酸包被的PLGA置于含0.25%Ⅰ型胶原的醋酸水溶液,制备PLGA/Ⅰ型胶原复合支架。取10~12周龄雌性SD大鼠阴道组织,采用酶消化法分离培养阴道上皮细胞,取第2代细胞进行实验。取复合支架浸提液培养阴道上皮细胞,观察材料细胞毒性。将阴道上皮细胞与复合支架共培养48 h(实验组),检测细胞黏附率;以单纯PLGA支架接种细胞作为对照组。将细胞-支架复合物埋植至SD大鼠皮下,于2、4、8周取材行HE染色、免疫组织化学染色,观察细胞在支架上生长情况。将细胞-支架复合物移植至6只切除阴道组织的SD大鼠阴道部位,于术后3、6个月观察阴道生长情况,6个月后取阴道组织进行组织学观察。结果大鼠阴道上皮细胞在PLGA/Ⅰ型胶原复合支架材料浸提液中生长、增殖良好,细胞毒性为1级。实验组细胞黏附率为71.8%±9.2%,显著高于对照组的63.4%±5.7%(t=2.195,P=0.005)。阴道上皮细胞能在PLGA/Ⅰ胶原复合支架材料上黏附、生长;大鼠皮下埋植2周后,细胞在支架孔隙内生长增殖,成纤维细胞生长;4周支架材料表面形成1~3层上皮;8周后支架材料部分降解,上皮层次增加,呈极性排列,角蛋白免疫组织化学染色呈阳性。细胞-支架复合物原位移植3个月后,大鼠阴道黏膜呈粉红色,有光泽,支架材料大部分降解;6个月时阴道深约1.2 cm,无明显狭窄,阴道黏膜外观类似正常阴道黏膜,皱襞较少,组织学观察示上皮层与正常阴道无明显区别,基底层可见钉状突起,数量少于正常阴道,角蛋白免疫组织化学染色呈阳性。结论 PLGA/Ⅰ型胶原复合支架具有良好的生物相容性,可作为构建组织工程阴道的支架材料。     

应用聚乳酸聚乙醇酸膜构建组织工程心脏瓣膜的实验研究

目的;探讨应用聚乳酸聚乙醇酸（PLGA）构建组织工程心脏瓣膜的可行性。方法：扫描电子显微镜观察PLGA结构特点,将PLGA在兔皮下包埋,分别于2周、4周、6周、8周和12周观察材料的生物相容性和降解率,培养犬主动脉瓣间质细胞、主动脉壁间质细胞和皮肤成纤维细胞,对照其生长曲线、平滑肌α肌动蛋白表达和扫描电子显微镜特点。将犬主动脉壁间质细胞和内皮细胞种植于PLGA上,观察其形态并测定细胞合成胶原和前列环素的功能。结果：PLGA呈网孔状结构,孔径179μm。皮下包埋显示PLGA生物相容性好,体内降解时间为12周。犬主动脉瓣间质细胞和主动脉壁间质细胞平滑肌α肌动蛋白均为部分阳性表达,细胞内有大量粗面内质网,生长曲线相似。细胞种植显示细胞在材料表面生长良好,并具有合成胶原和前列环素的功能（P＜0．05）。结论：以PLGA为支架体外构建组织工程心脏瓣膜细胞不仅能在PLGA表面生长,还能合成细胞间质和血管活性物质,初步提示应用本组材料和方法构建组织工程心脏瓣膜是可行的。     

聚二元共聚物多孔支架活性真皮替代物的体外构建

活性皮肤替代物是指含有活细胞成分的皮肤组织工程产品 ,是皮肤组织工程研究的热点。由于人工合成的生物可降解材料具有生物相容性、加工性能良好以及降解速度可控等优点 ,已被广泛用作细胞支架。然而 ,在多孔的人工材料中种植细胞还存在很多问题。本文应用成纤维细胞胶原凝胶 (fibroblastspopulatedcollagenlattice ,FPCL)的制作方法 ,成功地将人成纤维细胞种植于聚二元共聚物 (乙交酯 /丙交酯polylacticacid/ polyglycolicacidcopolymers ,PLGA)多孔真皮支架中 ,体外培养出一种新型的活性真皮替代物。材　料　与　方　法1.胶原和细胞 …     

应用胶原支架构建组织工程心脏瓣膜的初步研究

目的 探讨应用胶原支架构建组织工程心脏瓣膜的可行性。方法 行兔皮下包埋实验。分别于4、6、8、12周观察材料的生物相容性和降解率。将犬主动脉壁间质细胞和主动脉内皮细胞种植于胶原膜上，进而将此瓣叶植入犬腹主动脉内3个月，观察材料吸收和瓣叶结构的变化。结果 胶原膜呈网孔状结构，孔径107μm。皮下包埋实验显示，材料生物相容性好，完全降解时间为12周。体内实验显示，材料于12周末被间质细胞合成的细胞外基质所取代，瓣叶表面有内皮细胞覆盖。结论 以胶原膜为支架构建组织工程心脏瓣膜是可行的，但材料在强度和组织相容性方面尚需改进。     

电纺聚己内酯-明胶纳米纤维膜复合兔骨髓间充质干细胞构建软骨组织工程支架

目的探索构建电纺聚己内酯(PCL)-明胶纳米纤维膜复合体作为软骨组织工程支架的可行性。方法采用静电纺丝技术制作PCL-明胶(50︰50)纳米纤维膜作为支架。取第三代兔骨髓间充质干细胞(BMSC),接种于上述支架,构建细胞-支架复合体并进行成软骨诱导培养。通过电镜分析、力学测试、体内植入试验和细胞实验等检测材料纤维直径、孔径和孔隙率、力学性能,细胞在支架上生长、分化情况以及生物相容性。结果 PCL-明胶纳米纤维膜直径均匀,有较大的孔隙率和比表面积,较好的力学性能。细胞-支架共培养24 h、48 h、72 h后BMSC生长增殖良好,共培养能促进BMSC成软骨诱导分化。体内试验表明材料无毒性,对组织刺激性小,具有良好的生物相容性。结论电纺PCL-明胶纳米纤维膜有较好的力学性能、细胞亲和性和生物相容性,基本满足软骨组织工程支架条件,能够用作种子细胞承载体。     

利用胶原构建皮肤组织工程支架的研究

目的利用胶原构建皮肤组织工程支架。方法用Na2S、弹性蛋白酶预处理胎牛皮得到胶原纤维；经蛋白酶M降解胶原纤维后，0．5mol／L醋酸溶液溶解得到酸溶性胶原；再用蛋白酶N处理上述酸溶性胶原，最终得到生物相容性良好的胶原溶液；将胶原溶液构建皮肤组织工程支架。通过SDS-PAGE试验，分析酸溶性胶原分子量及基本结构；用皮肤组织工程支架材料包覆大鼠创面，观察创面愈合情况，研究支架材料对大鼠创伤愈合的影响；在支架材料上种植成纤维细胞，观察细胞扩增情况，以及支架材料对细胞移植影响。结果通过特异性蛋白酶M处理得到的酸溶性胶原，显示典型的1型胶原SDS-PAGE图谱；构建的皮肤组织工程支架呈多孔海绵状，孔径为50～200μm；在支架材料上种植成纤维细胞扩增情况良好；用于修复大鼠创面，具有促进创面愈合作用。结论酸溶性胶原经蛋白酶N处理后，生物相容性良好，适合于皮肤组织工程支架的构建，并具有良好生物活性。     

人脂肪来源干细胞与胶原支架共培养的实验研究

目的观察评价人脂肪来源干细胞(Human adipose derived stem cells,hADSCs)在胶原支架中的生长情况,为进一步体内组织修复研究提供依据。方法取人抽脂术后脂肪,经胶原酶解、过滤、离心获得hADSCs,代传代扩增后,接种到胶原支架上。细胞-材料复合物分别体外培养1周、2周,裸鼠体内培养2周、4周后,HE染色观察细胞在支架上的生长情况,免疫组化HLA-Ⅰ检测经裸鼠体内培养后的复合物上的细胞的种属来源。结果原代培养的hADSCs呈"梭形"或"成纤维细胞"样,并以克隆团形式生长。免疫荧光Vimentin染色阳性。第3代hADSCs经流式细胞鉴定,CD29、CD44、CD105表达阳性,CD45、CD34表达阴性。胶原支架复合hADSCs经过体外、体内培养,hADSCs均能长入胶原支架的空隙内,且体内培养比体外培养有更多的细胞长入支架。体外培养1周,已有细胞粘附生长在胶原支架的边缘,体外培养2周后,更多的细胞渗透到材料内部。体内培养2周后,大量细胞占据材料的边缘,有部分细胞能渗透到材料内部甚至材料全层。体内培养4周后,大量细胞渗透支架全层。HLA-Ⅰ抗体检测发现,支架材料内部细胞阳性表达,说明胶原支架内部的细胞来源于hADSCs。结论胶原支架与hADSCs具有较好的相容性,可作为hADSCs的载体材料,用于组织工程缺损修复的研究。     

交联温度对京尼平交联胶原／壳聚糖组织工程支架的影响

[目的]通过研究材料的孔径、交联度、溶胀率、降解率、细胞毒性及组织相容性的变化,探讨交联温度对京尼平交联胶原/壳聚糖支架的影响.[方法]采用冷冻干燥法制备胶原/壳聚糖复合多孔支架,分别于4℃、20℃、36℃条件下,在0.5%京尼平水溶液中交联24 h.以未交联的胶原/壳聚糖复合多孔支架作为对照,评价所得支架的孔径、交联度、溶胀率、降解率、细胞毒性及组织相容性特点.[结果]随交联温度升高,支架的交联度明显增大,溶胀率和降解率逐渐减小.4℃组支架交联度47.88%土6.4%,溶胀率为721%±46%,4周后降解3.95%±6.4%;20℃组支架交联度67.69%±3.6%,溶胀率为662%±72%,4周降解0.91%±5.9%;36℃组支架交联度70.32%±5.7%,溶胀率为635%±27%,4周降解0.66%±7.3%,三组上述观察指标均优于未交联组(P/0.01).[结论]京尼平交联可以显著降低胶原/壳聚糖支架的降解率和溶胀率,且对支架结构和生物相容性无明显影响.交联温度增加可以提高支架的交联度和抗降解能力,较快获得具有良好生物相容性和降解率的组织工程支架.     

O,O-双十二酰基壳聚糖/聚乳酸复合膜的特性和生物相容性的研究

目的评价O,O-双十二酰基壳聚糖/聚乳酸复合膜的特性和生物相容性,从而寻找新型的组织工程支架材料。方法采用FTIR、WAXD及SEM研究复合膜的理化特性;采用小鼠胚胎成纤维细胞(3T3)作为实验细胞,通过细胞粘附实验、细胞活性MTT实验、小鼠全身过敏实验和皮下埋植急性毒性实验对复合膜的生物相容性进行评价。结果OCS与PLLA之间存在强烈的氢键作用,材料结构均一,无相分离;OCS/PLLA的粘附率较PLLA的高;复合膜的细胞毒性为零级;而且用复合膜100%浸提液注射小鼠,对小鼠生长无不良反应,在小鼠皮下埋植复合膜,该膜与皮下组织粘附良好,膜上粘附有肌肉,同时膜有部分降解。结论O,O-双十二酰基壳聚糖可提高聚乳酸的细胞亲和性,OCS/PLLA复合膜对组织的生物相容性好,该复合膜可能成为一种机械性能好、细胞亲和能力强的新型组织工程支架材料。     

羟基磷灰石/聚乳酸-聚羟乙酸/骨形态发生蛋白复合生物材料在兔体内生物组织相容性的研究

目的 评价快速成型工艺制作的羟基磷灰石/聚乳酸-聚羟乙酸/骨形态发生蛋白（HA/PLGA/BMP-2）生物材料在新西兰兔体内的生物组织相容性。方法 健康新西兰大白兔20只,随机抽取10只为实验组,10只为对照组。实验组于兔颅顶部植入HA/PLGA/BMP-2生物材料,对照组植入金属钛网。分别于4周、8周、12周通过HE染色、血常规、血生化等评价方法,评价HA/PLGA/BMP-2生物材料在兔体内的组织相容性。结果 实验组植入物周围组织（约1cm）HE染色结果显示:4周见部分中性粒细胞,少量炎性细胞浸润;8周未见中性粒细胞,无炎性细胞浸润;12周未见炎性细胞,周围可见成纤维细胞增生。对照组植入物周围组织（约1cm）HE染色结果显示4周、8周、12周均未见有炎性细胞生成。实验组、对照组肝、肾组织HE染色结果显示均未见有炎性细胞,无新生组织形成。实验组与对照组12周血液学结果比较,差异无显著性意义。结论 HA/PLGA/BMP-2生物材料具有良好的生物相容性,是一种安全、无肝肾毒性的生物材料,在新型骨组织工程材料上有一定的研发潜力。     

023 Development of Tissue Engineering Skin Based on Acellular Dermal Matrix

Aim: In tissue engineering of the skin, the selection of a scaffold or a matrix in which a cultured cells grow is quite important. We have developed a tissue engineering skin composed of human keratinocytes and fibroblasts on an acellular allogenic dermal matrix (ADM), derived from cryopreserved human skin. Methods: ADM was prepared from cryopreserved split‐thickness human skin by treating with Dispase and Triton X‐100. The tissue engineering skin were produced by seeding human keratinocytes and fibroblasts on ADM. Several days after seeding, the tissue engineering skin was exposed to an air‐liquid interface for another 7 days. Then, the histological structure of the skin and the production of growth factors by the skin were investigated. Results: The produced ADM was found to be completely acellular with remaining structure of the basement membrane components, such as type IV collagen and laminin. The developed tissue engineering skin had stratified keratinocytes on the surface of the ADM migrating fibroblasts in the dermal collagen structure, resembling to the normal skin appearance. It was found that several important growth factors in wound healing process, such as TGF‐α, TGF‐β and VEGF, were produced by the tissue engineering skin. Conclusions: It was suggested that ADM is suitable for a scaffold in tissue engineering of the skin.     

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.     

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.     

Culture of subconfluent human fibroblasts and keratinocytes using biodegradable transfer membranes

This study aims to assess the suitability of biodegradable membranes as transfer matrix materials for the culture of subconfluent fibroblasts and keratinocytes. The materials investigated were based on collagen, chitosan and enzyme-digestible cellulose. The proliferation and growth behaviour of human keratinocytes and dermal fibroblasts were analysed and morphology and distribution determined. Cultured fibroblasts exhibited no significant differences in proliferation for the different membrane types, whereas keratinocytes revealed significantly higher proliferation on collagen membranes compared with membranes based on cellulose and chitosan. Co-cultured fibroblasts and keratinocytes from the same donor on collagen membranes showed more homogenous cell distribution, but they segregated in heterologous co-cultures; this effect must be further investigated. Thus, collagen and collagen-coated chitosan membranes are suitable for the subconfluent transfer of human fibroblasts and keratinocytes.     

Promotion of acellular dermal matrix resolution in vitro by matrix metalloproteinase-2

OBJECTIVE: To determine whether acellular human dermis is degraded by matrix metalloproteinases (MMPs), a large class of matrix-degrading enzymes. METHODS: The degradation of acellular human dermis specimens was evaluated in vitro. Wild-type murine fibroblasts with a broad-spectrum MMP inhibitor, GM6001, and MMP-2-deficient fibroblasts were placed on the basement membrane and dermal surfaces of acellular human dermis. Matrix degradation and fibroblast infiltration into the matrix were assessed after a 20-day incubation period. RESULTS: The basement membrane thickness of the specimens cultured with wild-type fibroblasts was significantly less than that of specimens cultured with GM6001 (P<.001), and the infiltration of fibroblasts into the dermal surface was limited by the addition of GM6001 (P=.002). To determine whether MMP-2 was involved in this in vitro phenotype, MMP-2-deficient fibroblasts were assessed in comparison with wild-type fibroblasts. Wild-type fibroblasts degraded the basement membrane surface (P<.001) and infiltrated the dermal surface (P = .003) more efficiently than did MMP-2-deficient fibroblasts. CONCLUSIONS: The results from our in vitro experiments suggest that MMPs and specifically MMP-2 may play an important role in the resorption of acellular human dermis. Addition of MMP inhibitors to implanted dermal matrices may slow fibroblast infiltration and improve their longevity in vivo.     

Enhanced secretion of TIMP-1 by human hypertrophic scar keratinocytes could contribute to fibrosis

Hypertrophic scars are a pathological process characterized by an excessive deposition of extracellular matrix components. Using a tissue-engineered reconstructed human skin (RHS) method, we previously reported that pathological keratinocytes induce formation of a fibrotic dermal matrix. We further investigated keratinocyte action using conditioned media. Results showed that conditioned media induce a similar action on dermal thickness similar to when an epidermis is present. Using a two-dimensional electrophoresis technique, we then compared conditioned media from normal or hypertrophic scar keratinocytes and determined that TIMP-1 was increased in conditioned media from hypertrophic scar keratinocytes. This differential profile was confirmed using ELISA, assaying TIMP-1 presence on media from monolayer cultured keratinocytes and from RHS. The dermal matrix of these RHS was recreated using mesenchymal cells from three different origins (skin, wound and hypertrophic scar). The effect of increased TIMP-1 levels on dermal fibrosis was also validated independently from the mesenchymal cell origin. Immunodetection of TIMP-1 showed that this protein was increased in the epidermis of hypertrophic scar biopsies. The findings of this study represent an important advance in understanding the role of keratinocytes as a direct potent modulator for matrix degradation and scar tissue remodeling, possibly through inactivation of MMPs.     

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.     

Role of wound healing myofibroblasts on re-epithelialization of human skin

In human skin, large burned surfaces heal using two concomitant phenomena: re-epithelialization and dermal neoformation. Numerous studies report the role of interactions between keratinocytes and fibroblasts, but the relationship between wound healing myofibroblasts and keratinocytes is not clear, even though these two cell types coexist during healing. We investigated the influence of myofibroblasts on keratinocyte growth and differentiation using an in vitro skin model. A histological study was performed to determine the speed and quality of epithelialization. When the dermis was populated with fibroblasts, a continuous epidermis was formed in 7-10 days. In contrast, with wound healing myofibroblasts or without cell in dermis, the complete reepithelialization never occurred over the 10-day period studied. After 7 further days of epidermal differentiation, histology showed an epidermis more disorganized and expression of basement membrane constituents was reduced when wound healing myofibroblasts or no cells were added in the dermis instead of fibroblasts. These results suggest that wound healing myofibroblasts are not efficient to stimulate keratinocyte growth and differentiation. Treatment of fibroblasts with TGFbeta1 induced an increase of epidermal cell differentiation as seen when myofibroblasts were present. However, this cytokine did not change re-epithelialization rate and induced an increase of basement membrane matrix deposition in opposition to myofibroblasts. Thus, TGFbeta1 action is not sufficient to explain all the different keratinocyte reactions towards fibroblasts and wound healing myofibroblasts. Our conclusion is that myofibroblasts seem to have a limited role in the re-epithelialization process and might be more associated with the increased extracellular matrix secretion.     

Reconstruction of living bilayer human skin equivalent utilizing human fibrin as a scaffold

Our aim of this study was to develop a new methodology for constructing a bilayer human skin equivalent to create a more clinical compliance skin graft composite for the treatment of various skin defects. We utilized human plasma derived fibrin as the scaffold for the development of a living bilayer human skin equivalent: fibrin-fibroblast and fibrin-keratinocyte (B-FF/FK SE). Skin cells from six consented patients were culture-expanded to passage 1. For B-FF/FK SE formation, human fibroblasts were embedded in human fibrin matrix and subsequently another layer of human keratinocytes in human fibrin matrix was stacked on top. The B-FF/FK SE was then transplanted to athymic mice model for 4 weeks to evaluate its regeneration and clinical performance. The in vivo B-FF/FK SE has similar properties as native human skin by histological analysis and expression of basal Keratin 14 gene in the epidermal layer and Collagen type I gene in the dermal layer. Electron microscopy analysis of in vivo B-FF/FK SE showed well-formed and continuous epidermal-dermal junction. We have successfully developed a technique to engineer living bilayer human skin equivalent using human fibrin matrix. The utilization of culture-expanded human skin cells and fibrin matrix from human blood will allow a fully autologous human skin equivalent construction.