用毛囊隆突细胞构建复合皮的实验观察

目的 以人毛囊隆突细胞为种子细胞体外构建组织工程复合皮，在体观察其功能性修复全层皮肤缺损的可行性。方法 胶原酶消化法体外分离培养人毛囊隆突细胞和毛乳头细胞，实验分为A、B两组。A组将毛囊隆突细胞与毛乳头细胞按1：2混合，接种于胶原包被的聚羟基乙酸纤维支架中；B组单纯接种相同数量的毛乳头细胞。而后覆盖角质形成细胞膜片，构成组织工程复合皮，移植于裸鼠全层皮肤缺损创面。观察创面愈合情况，分别于术后2、4、6周在光学显微镜下观察移植物组织学变化。结果 组织工程复合皮能够有效修复A、B两组裸鼠全层皮肤缺损。术后2周，A、B组创面均可见完整的表皮及真皮结构。术后4-6周，A组复合皮表皮层明显增厚并形成基膜的钉突，可见毛囊样结构；B组仅表皮层有所增厚但基膜平整，未见钉突和毛囊结构形成。结论 以聚羟基乙酸真皮基质为支架，用角质形成细胞、毛囊隆突细胞和毛乳头细胞共同构建的组织工程复合皮，可以有效修复裸鼠全层皮肤缺损。其中毛囊隆突细胞参与了创面解剖修复，同时可能引导组织结构和功能的修复。     

毛乳头细胞促进组织工程皮肤血管化的实验研究

目的探讨毛乳头细胞对表皮干细胞与同种异体脱细胞真皮基质构建的组织工程皮肤血管化的影响。方法取门诊包皮环切术患者皮肤,用于表皮细胞培养;取孕19、20周引产胎儿头部皮肤,用于毛乳头细胞培养;患者及家属均知情同意。以中性蛋白酶联合胰蛋白酶消化、分离表皮细胞,IV型胶原黏附法分选表皮于细胞;以Ⅰ型胶原酶消化法分离毛乳头,常规成纤维细胞培养液培养。以同种异体脱细胞真皮基质为支架,在真皮基质的真皮乳头侧种植毛乳头细胞,基底膜侧种植表皮干细胞构建实验组组织工程皮肤替代物;对照组组织工程皮肤替代物不种植毛乳头细胞。取6～8周龄BALB/C-nu裸鼠60只,随机分成实验组和对照组（n=30）;实验动物背部制造1 cm×1 cm大小全层皮肤缺损模型,将两组组织工程皮肤替代物移植修复创面,2周后观察移植皮肤成活率。术后2、4周,取移植皮肤替代物标本,行HE及免疫组织化学染色,观察移植皮肤替代物CD31表达水平,并计算两组标本血管密度。结果Ⅳ型胶原分选后表皮细胞同时表达角蛋白19、β₁整合素,提示为表皮干细胞。由毛乳头培养出的细胞表达α平滑肌肌动蛋白,提示为毛乳头细胞。动物移植术后2周,实验组移植皮肤成活率为93.3%（28/30）,对照组为80.0%（24/30）,比较差异有统计学意义（χ²=2.31,P=-0.00）。HE染色观察示实验组表皮层达12层,表皮细胞体积较大;对照组表皮层达4～6层,表皮细胞体积较小。实验组术后2、4周,血管密度分别为（38.56±2.49）个/mm2和（49.12±2.39）个/mm²,对照组分别为（25.16±3.73）个/mm²和（36.26±3.24）个/mm²,两组比较差异均有统计学意义（P<0.01）。结论毛乳头细胞可促进移植皮肤替代物血管形成,利于表皮层重建,提高组织工程皮肤移植成活率。     

微囊化人头皮毛乳头细胞移植诱导毛发形成

目的探索微囊化人头皮毛乳头细胞（以下简称毛乳头细胞）对裸鼠背部皮肤毛囊形成的诱导作用。方法胶原酶消化法体外分离、培养毛乳头细胞，再将毛乳头细胞以海藻酸钠-多聚赖氨酸-海藻酸钠（alginate-polylysine—alginate，APA）微囊包裹，以胶原凝胶作为载体，植入裸鼠背部皮下；移植空囊、游离毛乳头细胞各作为对照。观察移植部位毛发生长情况，利用组织学方法观测所形成的毛囊结构。结果微囊化毛乳头细胞皮下移植4周后，裸鼠背部移植区有白色、浓密、分布均匀的毛发长出。局部组织切片见大量发育完整的毛囊结构；而空囊及游离毛乳头细胞移植均未能诱导出上述现象。结论聚集性生长的微囊化毛乳头细胞能够保持诱导皮肤毛囊形成的作用，且这种作用无种属特异性。     

毛囊干细胞表皮膜片修复裸鼠皮肤缺损的实验研究

目的开发新的组织工程表皮替代物,利用毛囊干细胞-壳聚糖明胶膜片(Chitosan-gelatin membrane,CGM)进行修复裸鼠皮肤缺损的实验研究。方法将体外扩增培养的毛囊干细胞接种于CGM,构建组织工程表皮膜片,裸鼠背部作直径1cm的全层皮肤缺损,将毛囊干细胞-CGM复合物覆盖创面。回植后1周、4周和12周分别进行组织学和免疫组织化学检测。结果毛囊干细胞在CGM上生长良好。毛囊干细胞-CGM修复裸鼠后1周切片示创面有新生上皮覆盖,表皮分化良好,而对照组残留较大未愈创面。修复后4周和12周可见对照组收缩较实验组显著。结论体外构建的毛囊干细胞-CGM可以修复裸鼠皮肤缺损。     

胎鼠表皮干细胞的分离培养及毛囊再生研究

目的：分离培养胎鼠的表皮干细胞及毛囊乳头层细胞，通过动物模型观察两者复合移植后，上皮形成及毛囊的再生情况。方法：采用IV型胶原分离培养胎鼠表皮干细胞，检测β1整合素及角蛋白15的表达水平，测定其细胞周期及克隆形成率（以角质细胞为对照）；分离培养毛囊乳头层细胞，并接种在纤维蛋白胶中，与表皮干细胞膜片复合形成全层皮肤等同物，移植在裸鼠全层皮肤缺损创面作为实验组，同时将胎鼠成纤维细胞替代乳头层细胞作为对照组，8－10周后观察创面及囊的组织学变化。结果：胎鼠表皮干细胞表达高水平的β1整合素及角蛋白15；细胞周期分析显示，G1期细胞百分率为94．9％，S期为3．5％，提示为慢循环细胞周期；角质细胞的G1期细胞有百分率为74．1％，S期为17．5％；表皮干细胞的克隆形成率为15．3％，明显高于对照组的6．7％；裸鼠创面愈合后8－10周，实验组可见新生毛囊形成，对照组未见到此现象。结论：能初步实现对胎鼠表皮干细胞的体外分离培养；在毛囊乳头层细胞的诱导下，胎鼠表皮干细胞可参与形成新的毛囊结构。     

真皮源干细胞复合人血浆促进皮肤创面神经修复的实验研究

目的观察真皮源干细胞复合人血浆移植对皮肤创面神经修复的影响。方法培养人真皮源干细胞（DMSC），观察其体外分化情况；以人血浆为载体构建DMSC移植物；60只裸鼠背部制作皮肤缺损创面，随机分为干细胞移植组（A组）、单纯血浆支架组（B组）和空白组（C组）；创面愈合后第1、2、4、8周取材，免疫荧光检测表皮下及真皮层PGP9、5的表达．以此反映皮肤神经再生情况。结果DMSC体外向神经元样、胶质样、许旺细胞样分化。A组创面愈合后第1、2周表皮下及真皮层PGP9．5荧光密度高于B组、C组（P〈0．01）；B组第2周真皮层和第4周表皮下PGP9．5荧光密度高于C组（P〈0.05）。结论真皮源干细胞复合血浆移植有利于皮肤创面的神经修复。     

人工毛乳头异种移植诱导大鼠足垫毛囊形成

目的观察人头皮毛乳头细胞微囊(人工毛乳头)异种移植诱导大鼠足垫毛囊形成的能力。方法以海藻酸钠-多聚赖氨酸-海藻酸钠(alginate-polylysine-alginate,APA)微囊包裹分离培养的毛乳头细胞;对体外培养1、4周的毛乳头细胞微囊及无APA的微囊对照组行组织学观察;取培养4周的毛乳头细胞微囊移植至大鼠足垫皮下,6周后取材行组织学检查。结果毛乳头细胞微囊体外培养1周后,毛乳头细胞周围出现细胞外基质;4周后,囊中形成“类毛乳头样结构”;人头皮毛乳头细胞微囊移植至大鼠足垫6周后,移植部位及其周围皮下有大量毛囊及皮脂腺结构形成。结论人工毛乳头诱导并参与了无毛区域新生毛囊及皮脂腺的组织构成。     

无细胞异种真皮与表皮细胞悬液复合移植的研究

目的　研究用无细胞异种真皮与表皮细胞悬液复合移植的效果及组织学变化。方法　将 4 2只裸鼠分为实验组及对照组 ,实验组于背部全层皮肤缺损创面移植无细胞异种真皮及人表皮细胞悬液 ,对照组单纯移植人表皮细胞悬液。于术后 2、3和 5周计算创面愈合率 ,术后 3、6和 12周计算创面收缩率 ,并取活检行组织学检测。结果　实验组创面愈合率高 ,分别为 79.1%± 4 .5 % ,89.6 %± 4 .4 % ,98.1%± 3.4 % ,收缩程度轻为 16 .3%± 5 .2 % ,2 5 .5 %±7.2 % ,32 .5 %± 7.1% ,外观平整 ,质地良好 ,胶原纤维排列整齐 ,基底细胞桥粒 -半桥粒结构及基底膜等结构重建明显 ,未见明显的急性排斥反应 ;对照组创面愈合率分别为 6 9.2 %± 4 .9% ,78.2 %± 7.6 % ,90 .6 %± 5 .0 % ,收缩率为2 0 .5 %± 6 .0 % ,31.3%± 6 .9% ,4 4 .6 %± 7.2 % ,与实验组相比 ,均有统计学意义 (P<0 .0 5 ) ,愈合后的表皮质地薄 ,易破溃 ,胶原纤维紊乱 ,表皮 -真皮连接结构重建不明显。结论　无细胞异种真皮与表皮细胞悬液复合移植修复创面 ,可改善创面愈合质量。     

人工毛乳头异种移植诱导大鼠足垫毛囊形成

目的观察人头皮毛乳头细胞微囊（人工毛乳头）异种移植诱导大鼠足垫毛囊形成的能力。方法以海藻酸钠-多聚赖氨酸-海藻酸钠（alginate- polylysine - alginate, APA）微囊包裹分离培养的毛乳头细胞；对体外培养1、4周的毛乳头细胞微囊及无APA的微囊对照组行组织学观察；取培养4周的毛乳头细胞微囊移植至大鼠足垫皮下，6周后取材行组织学检查。结果毛乳头细胞微囊体外培养1周后，毛乳头细胞周围出现细胞外基质；4周后，囊中形成“类毛乳头样结构”；人头皮毛乳头细胞微囊移植至大鼠足垫6周后，移植部位及其周围皮下有大量毛囊及皮脂腺结构形成。结论人工毛乳头诱导并参与了无毛区域新生毛囊及皮脂腺的组织构成。     

以胶原海绵为载体培养的人表皮细胞移植

目的：将体外培养的人表皮细胞接种到胶原海绵上,构建一种表皮替代物,移植到鼠创面后研究观察皮肤的再生,方法：将手术切下的包皮经中性蛋白酶分离表皮,真皮,再经胰蛋白酶消化制成表皮细胞悬液,移入培养瓶中培养,将处于对数生长期的人表皮细胞直接接种到培养皿中的胶原海绵 ,再加上表皮细胞液培养3天后,移植到裸鼠全层皮肤缺损的创面上,以单纯无接种细胞的胶原海绵作为对照,并进行组织学,免疫组织化学及电子显微镜观察。结果：这种表皮替代物移植到创面后,表皮细胞继续增殖分化,形成一层新生表皮,与对照组相比,创面闭合早且收缩程度小,表皮成熟早且分层较多,基底膜形成较早,表皮下胶原纤维较少。结论：体外培养的表皮细胞能在胶原海绵上生长,移植到创面上后,该细胞可自动移行到创面并形成多层表皮结构,抑制创面收缩,可用于修复皮肤缺损。     

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.     

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.     

壳多糖-胶原-糖胺聚糖凝胶人工皮肤的初步研究

目的 研制一种新型的胶原凝胶类人工皮肤。方法 制备壳多糖—胶原—糖胺聚糖(GAGs)—成纤维细胞真皮替代物(DE)，观察成纤维细胞(FB)在凝胶中的生长情况和不同因素对凝胶收缩的影响，并绘制FB生长曲线和凝胶收缩曲线。了解不同含量壳多糖对FB和角质形成细胞(KC)生长的影响，不同含量壳多糖DE的抗感染能力。再于“成熟。的DE表面接种KC，先浸没培养，后行气液界面培养，构建完整的人工皮肤。对DE和人工皮肤行组织学和电镜分析。结果 DE收缩度与FB数量呈正比，终末收缩度与胶原蛋白浓度成反比；FB在凝胶中2—9天呈指数增生。DE基质配方对FB的生长无明显抑制作用，但可促进KC的生长，对金黄色葡萄球菌的抑制作用随壳多糖的含量增大而增强。扫描电镜示DE有丰富的微孔结构。人工皮肤组织学观察见类似于正常皮肤，有分化良好的表皮和致密的真皮。结论 壳多糖—胶原—GAGs胶原凝胶人工皮肤是一种新型、有一定抗感染能力的活人工皮肤。     

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.     

Cultured keratinocytes in fibrin with decellularised dermis close porcine full-thickness wounds in a single step

The purpose of this study was to assess the feasibility of combined keratinocyte and dermal scaffold transplantation performed in a single step for treatment of full-thickness wounds. Cultured autologous keratinocytes were suspended in fibrin and grafted together with decellularised human dermis (Alloderm) in a porcine animal model, involving 10 animals over 4 weeks. Wound healing was evaluated by planimetry. Histology included morphological analysis as well as immunohistochemistry at regular intervals (1, 2 and 4 weeks). The results showed both successful histo-integration of the in vivo composite grafts and reduced wound contraction, compared with the control group (plain epithelial grafts). Histologically a neo-epithelium originated from the grafted cells on top of the decellularised dermis, as well as a reconstituted basement membrane. After 4 weeks cellular ingrowth into the dermal matrix could be observed. The successful combination of a keratinocyte-fibrin suspension and acellular dermis applied in a single step onto full-thickness wounds resulted in closure.     

Use of an in vitro model of tissue-engineered human skin to study keratinocyte attachment and migration in the process of reepithelialization

To produce a stable epidermis, keratinocytes need to be firmly attached to the basement membrane. However, following wounding, keratinocytes are required to develop a migratory phenotype in order to reepithelialize the wound. To investigate some of the issues underlying reepithelialization, we have developed a three-dimensional in vitro model of tissue-engineered skin, comprising sterilized human dermis seeded with human keratinocytes and dermal fibroblasts. Using this model, we have shown that the inclusion of fibroblasts within the model increases the stability of keratinocyte attachment. We have also demonstrated that keratinocyte migration occurs most effectively in the absence of a basement membrane and following the inclusion of fibroblasts in the model. In addition, subjecting the keratinocyte layer to mechanical trauma induces a migratory phenotype. We conclude that this three-dimensional in vitro wound model can be used to increase our understanding of the factors that enhance keratinocyte migration and hence wound healing in vivo.     

Evaluation of dermal-epidermal skin equivalents ('composite-skin') of human keratinocytes in a collagen-glycosaminoglycan matrix(Integra artificial skin).

Integra artificial skin (Integra LifeSciences Corp., Plainsboro, NJ, USA) is a dermal template consisting of bovine collagen, chondroitin-6-sulphate and a silastic membrane manufactured as Integra. This product has gained widespread use in the clinical treatment of third degree burn wounds and full thickness skin defects of different aetiologies. The product was designed to significantly reduce the time needed to achieve final wound closure in the treatment of major burn wounds, to optimise the sparse autologous donor skin resources and to improve the durable mechanical quality of the skin substitute. The clinical procedure requires two stages. The first step creates a self neodermis, the second creates a self epidermis on the neodermis. However, it is desirable to cover major burn wounds early in a single step by a skin substitute consisting of a dermal equivalent seeded in vitro with autologous keratinocytes ('composite-skin') out of which a full thickness skin develops in vivo.The goal of this experimental study was to develop a method to integrate human keratinocytes in homogeneous distribution and depth into Integra Artificial Skin. The seeded cell-matrix composites were grafted onto athymic mice in order to evaluate their potential to reconstitute a human epidermis in vivo. We were able to demonstrate that the inoculated human keratinocytes reproducibly displayed a homogeneous pattern of distribution, adherence, proliferation and confluence. The cell-matrix composites grafted in this model exhibited good wound adherence, complete healing, minor wound contraction and had the potential to reconstitute an elastic, functional and durable human skin. Histologically we were able to show that the inoculated human keratinocytes in vivo colonised the matrix in a histomorphologically characteristic epidermal pattern (keratomorula, keratinocyte bubbling) and developed a persisting, stratified, keratinising epidermis which immunohistologically proved to be of human origin. These experimental results demonstrate the establishment of an effective cell cultivation process which may be suitable for scale-up production of the epidermal component as large-scale composite-skin grafts. When seeded into Integratrade mark and grafted onto the nude mouse a replacement skin with normal functioning dermal-epidermal components was developed. These results encourage the design of a clinical trial to assess the function of this composite graft in man.     

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.