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.     

角质形成细胞在脱细胞异种真皮上培养的实验研究

目的 在脱细胞异种(猪)真皮上培养角质形成细胞探讨体外复合皮的构建。方法 取出生24h内的SD大鼠全厚皮肤，采用低温酶消化法和密度梯度离心法分离获得纯角质形成细胞；以不用任何滋养层的角质形成细胞培养作为空白对照组，脱细胞异种真皮作为支架的角质形成细胞培养为实验组，原代培养后行气——液面培养。形态学，常规组织学HE染色观察，免疫组化染色(SABC法)检测复合皮肤中的Pancyrtokeratin和层粘连蛋白(Laminin)。结果 HE染色显示有4层以上的角质形成细胞和基底膜层形成，并有轻度角质化；免疫组化染色显示：Pancytokeratin( )，提示在脱细胞猪真皮上生长的为角质形成细胞；Laminin( )，提示培养的角质形成细胞产生了新的基底膜。结论 体外培养的角质形成细胞能在脱细胞异种真皮上良好生长、存活，并有基底膜形成，在体外成功构建了具有表皮和真皮的复合皮，可以作为一种新的组织工程化皮肤。     

利用毛囊干细胞和成纤维细胞重建全层皮肤的研究

目的建立利用毛囊干细胞和成纤维细胞进行全层皮肤重建的实验方法.方法取美容手术切取的头皮组织,K19免疫荧光染色定位毛囊干细胞,消化、分离、体外培养,以胶原-成纤维细胞聚合物为基质,采用气-液界面对第2代毛囊干细胞立体培养14 d,建立全层皮肤培养模型,并行组织学及K1免疫荧光染色观察.结果K19免疫荧光染色定位毛囊干细胞存在于毛囊外根鞘处,与成纤维细胞联合体外气-液界面立体培养14 d,获得的皮肤类似物,可见真皮基底膜形成,表皮多层上皮有序、多角形排列,上层细胞出现角化,真皮部成纤维细胞均匀分布于胶原基质中,角质细胞形成分化特异标志物K1染色阳性.结论毛囊外根鞘处的毛囊干细胞具有高增殖能力并向表皮细胞分化,可成功利用毛囊干细胞和成纤维细胞进行皮肤重建,为临床应用奠定基础.     

人工真皮结合角质形成细胞修复猪皮肤缺损

目的探索胰蛋白酶分离香猪角质形成细胞膜片的最佳方案,在胶原-壳聚糖人工真皮支架创面上进行角质形成细胞体内培养,明确该方法修复猪创伤全层皮肤缺损的实用性。方法用不同皮肤厚度、胰蛋白酶浓度和分离温度分离出角质形成细胞,把有活力的角质形成细胞接种于人工真皮支架植入后1周的全层皮肤缺损创面,2周后对创面愈合进行观察并作病理检测。结果在37℃条件下用0.20%的胰蛋白酶消化1mm厚度皮片100分钟为最佳分离方案。2周后肉眼和病理切片均显示接种的角质形成细胞膜片成活并完全覆盖修复创面。结论应用角质形成细胞体内培养加人工真皮支架制成＂人工皮肤＂能较好的修复猪创伤全层皮肤缺损,抑制创面收缩和瘢痕形成。     

组织工程化人工复合皮肤的构建

目的 为皮肤缺失的移植提供具有生物活性的表皮、真皮的组织工程化人工复合皮肤。方法 将体外传代培养的表皮角朊细胞和真皮成纤维细胞分别接种在冷冻干燥及戊二醛交联的I型胶原基质网架的两侧,在液面下培养1周后,改为气－液界面培养,动态观测人工皮肤光镜下组织学形态及电镜下超微结构。结果 人工复合皮肤具有表皮、真皮双层结构。培养过程中表皮逐步增殖、分化、发育,形成基底层、棘细胞层、颗粒层和角质层;真皮基质网架渐渐降解,并逐步被增殖的成纤维细胞及其所分泌的细胞外基质所取代,完成真皮构建。结论 利用组织工程技术体外构建人工复合皮肤,可用于皮肤缺失者的自体移植治疗,体外构建的复合人工皮肤可从根本上解决自体供皮不足,且细胞源于自体皮肤,排除了发生免疫排斥反应和疾病传播的风险,具有安全、有效、实用等优点。     

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。     

人羊膜活性皮肤替代物修复巨痣切除术后创面的可行性及疗效研究

目的探讨临床应用以人羊膜为基质的活性皮肤替代物(amniotic membrane-living skin equivalent,AM-LSE)修复皮肤缺损的可行性。方法 2016年7月收治1例5岁颈肩背部先天性巨痣患儿。首先取其正常皮肤分离扩增表皮角化细胞和真皮成纤维细胞,再以自愿捐赠的人去上皮AM为基质,经气液面培养10 d构建含有真皮基质和重层分化表皮的AM-LSE。对AM-LSE行大体及组织学观察。手术切除巨痣至深筋膜层,遗留创面面积为20 cm×15 cm;覆盖胶原蛋白海绵人工真皮,2周后改用AM-LSE贴敷于肉芽创面,之后常规换药。术后观察AM-LSE成活以及创面修复效果。结果气液面培养10 d后,成功构建具有真皮层以及重层分化良好的表皮层的AM-LSE。临床修复术后见AM-LSE贴敷良好,逐渐扩大融合,至术后6个月其色泽接近正常皮肤,质地柔软,皮片间隙为淡红色瘢痕。组织学观察,可见呈柱状紧凑排列的基底层细胞,以及分化的棘细胞层、颗粒层以及角质层。真皮层内分布大量成纤维细胞以及血管,未见明显炎性细胞浸润。结论采用自体皮肤细胞构建的AM-LSE移植修复创面可行,术后能长期存活,且无排斥反应。     

bFGF与皮肤创面修复过程中瘢痕形成的关系研究

碱性成纤维细胞生长因子（basicFibroblastGrowthFactor简称bFGF）是一种多肽类生长因子，主要作用于中胚层和神经外胚层起源的组织和细胞。也就是说来源于中胚层、神经外胚层的细胞表面分布有bFGF受体。bFGF与之结合进而启动细胞内合成过程，促使细胞生长、分裂和分化。皮肤由来源于外胚层的表皮和中胚层的真皮及皮下组织构成，在皮肤各层的组织和细胞中分布着bFGF受体。在表皮，基底层细胞可进行分裂，产生新生表皮细胞，并最终形成有角质蛋白的角质细胞。真皮则由胶原纤维、弹力纤维和基质等组成。成纤维细胞是分泌这三种成分的最…     

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。     

整合素及其与创面修复关系的研究进展

皮肤损伤后其创面的修复过程一般包括表皮的再生和真皮的重构.表皮的再生主要依靠位于表皮基底层细胞的增殖,分化为成熟的角质细胞,而真皮的构建则靠刨底成纤维细胞不断合成胶原等细胞外基质并重新排列形成.     

Tissue engineered artificial skin composed of dermis and epidermis

Abstract: We made an artificial skin comprised of a stratified layer of keratinocytes and a dermal matrix with a type I collagen containing fibroblasts. In this work, we showed keratinocyte behavior under primary culture, gel contractions varying with concentration of collagen solution, and cell growth plots in the collagen gel. The optimum behavior of dermal equivalent could be obtained using 3.0 mg/ml collagen solution and attached gel culture. The attached gel culture had a jumping effect of growth factor on cell growth at the lag phase. To develop the artificial skin, 1× 10⁵ cells/cm² of keratinocytes were cultured on the dermal equivalent at air-liquid interface. Finally, to overcome the problem that artificial skin of collagen gel was torn easily during suturing of grafting, we prepared histocompatible collagen mesh and attached the mesh to the bottom of the gel. Cultured artificial skins were successfully grafted onto rats.     

毛乳头细胞和表皮细胞构建带附属器的组织工程皮肤

目的根据毛囊形成原理,以人毛乳头细胞和表皮细胞为种子细胞,构建带附属器的组织工程皮肤替代物。方法分实验组和对照组,实验组皮肤替代物真皮层接种人毛乳头细胞和表皮细胞;对照组真皮层不接种任何细胞。在裸鼠背部作一圆形全层皮肤缺损,将两组皮肤替代物气-液界面培养5 d后移植到裸鼠背部。观察创面愈合情况,并在移植后第4、6、8周取材,进行组织学观察。结果皮肤替代物移植后2周,实验组创面已完全愈合,移植后4周对照组创面才完全愈合,并且对照组创面收缩比实验组明显。移植后6周,实验组真皮层内见到毛囊样结构和皮脂腺结构,对照组未见毛囊样结构和皮脂腺样结构。结论将人的毛乳头细胞和表皮细胞共同种植在皮肤替代物的真皮层,可以构建出带附属器的组织工程皮肤替代物,这种替代物对创面的修复能力更强。     

In vitro construction of a potential skin substitute through direct human keratinocyte plating onto decellularized glycerol-preserved allodermis

This work demonstrates that glycerol-preserved acellular allodermis can be used as support for the proliferation of human keratinocytes and that the characteristics of this bioengineered tissue suggest its possible use as a permanent skin substitute for therapeutic challenges such as extensive burns as well as its possible use as an in vitro model for pharmacological studies. The removal of all basal membrane components during preparation of the dermal support also provides an original in vitro situation that allows observation of the reorganization of the dermal-epidermal junction. The tissue composite obtained is constituted of dermis covered by a well attached, multistratified epithelium with morphological characteristics that resemble human epidermis as evidenced by light and transmission electron microscopy, including the neoformation, albeit incomplete, of the dermal-epidermal junction. Assessment of involucrin and cytokeratin 14 expression by immunohistochemical assays established differentiation patterns. Both immerse and air-liquid interface culture systems were tested.     

Cellular artificial skin substitute produced by short period simultaneous culture of fibroblasts and keratinocytes.

We developed a novel artificial skin substitute consisting of two collagen sponge layers with different pore sizes and cross-link densities. Fibroblasts suspended in 0.5 ml Dulbecco-modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) were seeded on the lower dermal sponge layer, then epidermal collagen sponge and 0.1 ml suspension of keratinocytes in KGM were layered in this order. After a few hours, the medium was changed to DMEM + 5% FBS. These processes were carried out in one day, and the composite layers were then cultured by the air-liquid interface culture method. Three to five days after seeding, keratinocytes had grown to about ten layers, and fibroblasts had grown three-dimensionally into the lower dermal sponge layer. This novel cellular artificial skin substitute was grafted onto nude mice and took in 4 weeks. This skin substitute has the advantage of a shorter culturing period than previously cultured skins, and may be clinically useful for grafting that is urgently required in patients with severe generalised burns.     

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.     

利用聚羟基乙酸构建组织工程皮肤的实验研究

目的 以聚羟基乙酸(PGA)为培养支架，用组织工程方法在体外构建双层、有活性皮肤。方法应用酶消化法分别获取人成纤维细胞(FB)和表皮角质形成细胞(KC)，体外扩增培养。接种HF与PGA上1周后，在复合物表面接种KC，共培养1周将复合物置于气-液界面继续培养。于不同时间段取材。行组织学、超微结构及免疫组织化学检测。结果接种表皮角质形成细胞在“FB-PGA”复合物表面1周后可见2～3层连续的HK在复合物上生长良好；4周时PGA已部分降解，表皮分层分化较完善，抗involucrin染色阳性，透射电镜观察可见表皮细胞分层分化良好。细胞的相邻面有桥粒连接。结论 采用组织工程技术可以在体外构建双层、有活性的人工皮肤；组织工程皮肤具有和正常皮肤相似的组织学特征及生化成分。