皮肤组织工程研究进展

组织工程学是近年来发展起来的一门新兴学科 ,现就皮肤组织工程的研究作一简要概述 :早在 195 1年 ,就有学者建议用实验室生长的皮肤来治疗烧伤病人[1] ,但由于表皮细胞培养困难 ,此方法未能实施。直到 1975年Ｒｈｅｉｎｗａｌｄ和Ｇｒｅｅｎ[2 ] 攻克了体外大规模培养表皮细胞的难题 ,皮肤替代物的研究和制作才日渐广泛。皮肤替代物可分为 :表皮替代物、真皮替代物和复合皮肤替代物。一、表皮替代物1.表皮细胞悬液 :Ｂｉｌｌｉｎｇｈａｍ等[3 ] 首先用培养的表皮细胞悬液治疗大面积全厚皮肤缺损 ,但该方法存在两大难题 :一是无法确定转移到…     

皮肤组织工程研究的新进展

组织工程学是以细胞生物学、工程学和材料学相结合，在体外或体内构建组织或器官替代物，用于修复、维持或改善机体形态和功能的一门新兴的交叉学科^[1]。组织工程皮肤为急性或慢性皮肤损伤提供了全新的治疗理念。然而，目前的皮肤替代物仅仅是结构的模仿，且存在构建时间长、修复有限、无皮肤附件及瘢痕形成等缺陷。     

体外培养表皮干细胞构建组织工程皮肤的研究

目的探讨应用表皮干细胞构建组织工程皮肤修复皮肤缺损的可行性。方法运用黏附实验将人表皮干细胞分选出来后大量培养，将成纤维细胞和表皮干细胞转移到真皮底物上构建组织工程皮肤，并将之移植于裸鼠皮肤缺损模型，于术后第1、2、4周处死动物，收集标本，进行组织学、透射电镜及免疫组织化学检查。结果组织工程皮肤移植后4周具备完整的表皮层和真皮层，表皮层中具备基底层、棘层、颗粒层和角质层。表皮细胞间还可以看到有桥粒、半桥粒、角质透明颗粒和粗张力微丝柬。表、真皮层间有连续的、完整的基底膜。真皮网架破坏明显，大量的成纤维细胞增殖活跃，胶原纤维排列整齐，有丰富的毛细血管。结论本研究所构建的组织工程皮肤组织学形态和超微结构上已接近于正常的皮肤，从而为临床修复皮肤缺损的研究打下了坚实的理论和实践基础。     

组织工程皮肤种子细胞的研究进展

组织工程即应用细胞生物学与工程学的原理和技术,在正确认识哺乳动物正常及病理条件下组织结构和功能的基础上,研究和开发用于修复、维护和改善人体各种组织或器官损伤后功能和形态的生物替代物的学科。组织工程包括3个基本的要素：种子细胞（seed cell）、可降解的支架材料及讯息因子。组织工程皮肤作为组织工程研究中较为活跃的领域,是在无细胞的生物材料中引入特定的种子细胞,通过一定的组织构建,形成更加接近于人体组织结构的生物活性替代物。种子细胞作为组织工程的基本要素,其黏附、增殖能力、分化程度及免疫特性是生物活性皮肤构建过程中需重点考虑的问题。目前用于皮肤组织工程构建的种子细胞主要包括：表皮细胞、成纤维细胞、干细胞等。     

组织工程皮肤构建的研究进展

组织工程学是生物工程领域的一门新兴学科,是目前学术界的研究热点。利用组织工程的原理构建人工皮肤替代物,用于皮肤移植,可以有效解决临床上皮肤缺损修复,供皮不足的难题。文中从表皮、真皮、复合皮三个方面就近年来组织工程皮肤构建的研究进展及干细胞在这一领域的应用作一综述,并分析组织工程皮肤目前存在的问题,展望未来发展方向。     

角质干细胞及其在皮肤组织工程中的应用前景

表皮是一种持续更新的组织，其更新过程始于基底层的角质干细胞的增殖与定向分化。在向上迁移的过程中，经历程序化的终末分化，最后成为死亡的角化扁平细胞从皮肤表面脱落。在正常稳定状态下，干细胞的增殖与表层细胞的丢失保持着精确的平衡^[1]。角质干细胞有两个重要特征：一是在体内属于静止的或慢细胞周期的细胞；二是具有无限的增     

混合接种法构建组织工程皮肤

目的 探讨混合接种法体外构建复合皮的可行性.方法 在异种猪脱细胞真皮的真皮面接种人成纤维细胞7 d后,将其分两组进行实验.实验组:将表皮细胞按5×105/cm2与成纤维细胞0.2 ×105/cm2混合后接种于表皮面,培养液用K-SFM与成纤维细胞上清的1:1混合液.对照组:仅接种表皮细胞5×105/cm2,培养液用K-SFM.培养1、3周后取材观察其形态变化,并行免疫组化鉴定.结果 培养3周后,实验组可见表皮层连续,细胞层数3～4层,与真皮连接紧密,有表皮突形成;对照组表皮细胞层仅1～2层,且与真皮分离.实验组Laminin强阳性提示基底膜形成充分,并经透射电镜也可观察到完整的基底膜.结论 将表皮细胞与少量成纤维细胞混合接种,可促进表皮细胞在脱细胞真皮上黏附增殖,并有助于基底膜充分形成.     

含表皮干细胞组织工程皮肤的构建研究

目的用表皮干细胞和成纤维细胞作为种子细胞研制一种增殖能力强的具有表皮、真皮的组织工程化人工复合皮肤。方法从幼儿包皮中分离表皮干细胞,用胶原Ⅳ纯化、富集表皮干细胞,接种在3T3细胞滋养层上,将体外传代培养的表皮干细胞和真皮成纤维细胞分别接种在经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3周后,改为气-液界面培养,构建含表皮干细胞的复合皮肤,并进行组织学、免疫组织化学及电镜观察。结果表皮干细胞呈克隆状生长,G0/G1期细胞和α6briCD71dim细胞百分率明显高于对照组,差异有统计学意义(P<0.05),实验组K19免疫细胞化学染色呈阳性,对照组呈阴性。光镜下观察可见体外培养的含表皮干细胞的人工复合皮肤,具有表皮和真皮,表皮层由基底至浅层可见3~5层细胞,角蛋白免疫组化染色阳性。结论表皮干细胞种植于胶原海绵膜上,可生成全层组织工程皮肤,具备了同正常皮肤类似的结构,表明其具有在体外分化成表皮的能力。     

皮肤组织工程

目的 综述近阶段皮肤组织工程研究领域中的进展。方法 广泛查阅国内外近期在皮肤组织工程研究的文献,着重阐述表皮替代物,真皮替代物,培养的表皮真皮复合皮片的研究进展和重要问题。结果 多数学者认为理想的皮肤替代物应及时重建已人的表皮和真皮结构。目前的研究主要集中在如何尽早移植表皮细胞,并保护移植后细胞的活性和功能,以及研制能更有效地促进细胞功能,诱导创床血植入,移植后可降解,无毒性,无病原携带风险的细胞     

Development of a tissue-engineered bypass graft seeded with stem cells

The gold standard conduit for bypass of diseased small-diameter arteries remains autologous vascular tissue. In the absence of such tissue, patients are offered bypass with prosthetic material, with far less durable results. Vascular tissue engineering, the creation of a vascular conduit by seeding a tubular scaffold with various cells, may offer an alternative approach to this difficult situation. Herein we review some of the significant challenges that remain in designing an ideal vascular conduit and outline potential solutions offered by a graft created by seeding natural vascular tissue (decellularized vein allograft) with readily available autologous cells (adipose-derived stem cells).     

力学因素在体外构建组织工程化软骨中的应用

力学因素是软骨组织工程中的重要影响因素之一。近年来的研究表明,力学作用可以刺激细胞外基质的分泌,改变三维支架上培养的软骨细胞的新陈代谢,从而促进软骨组织的生长与重建。本文就力学因素对软骨细胞增殖分泌的促进、力学刺激的传导机制及生物反应器在软骨组织工程中的应用等方面做一综述。     

骨髓间充质干细胞复合组织工程化脱细胞真皮基质构建组织工程皮肤

目的：体外培养扩增SD大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs），复合组织工程化脱细胞真皮基质构建组织工程皮肤，为进一步临床应用奠定基础。方法：将SD大鼠骨髓间充质干细胞进行体外培养扩增后，以生长状态良好的骨髓间充质干细胞接种于制备好的组织工程化脱细胞真皮支架上，进行体外联合培养，构建组织工程皮肤。观察细胞生长情况及组织工程皮肤结构。结果：体外培养的SD大鼠骨髓间充质干细胞生长良好，传代扩增容易，组织工程化脱细胞真皮基质去细胞完全，骨髓间充质干细胞在脱细胞真皮基质中生长良好，可体外构建组织工程皮肤。结论：利用体外扩增培养的骨髓间充质干细胞及制备的组织工程化脱细胞真皮基质可以体外联合构建组织工程皮肤。     

Quantitative study of tissue-engineered cartilage with human bone marrow mesenchymal stem cells

OBJECTIVES: To assess the possibility of cartilage tissue engineering using human mesenchymal stem cells (hMSCs) and to investigate the quantitative relationship between hMSCs and engineered cartilage. DESIGN: Human mesenchymal stem cells were cultured, cryopreserved, and expanded in vitro. Surface antigens were detected by flow cytometry. In vitro chondrogenesis of hMSCs and cryopreserved hMSCs was performed. The chondrogenesis-induced hMSCs were seeded onto polyglycolic acid scaffolds, cultured in vitro for 3 weeks in chondrogenic medium, and then implanted into nude mice. The implants were harvested after 10 weeks and examined with histologic and immunochemical staining. RESULTS: The construction of cartilages was identified grossly and histologically: 1.9 to 2.5 x 10(7) nucleated cells were obtained from 1 mL of bone marrow, and about 1 to 2 x 10(6) hMSCs were obtained from the primary culture. The number of hMSCs tripled at every passage and reached 1.4 to 2.8 x 10(12) at passage 15. The purity of hMSCs was 95% and 98% at the primary and the fourth passages, respectively. Twenty-one days was the optimal (induction rate, 95%) induction time, with no apparent differences in induction rates among different passages. Based on our findings, hMSCs from 0.07 to 0.14 mL of bone marrow, expanded during 4 passages and induced for 21 days, would be sufficient to engineer 1 cm(2) of cartilage, 3-mm thick. CONCLUSION: Quantitative standards of hMSCs as seed cells for cartilage tissue engineering were established and may have value for later clinical work.     

Cosmetic improvement in various acute skin defects treated with tissue-engineered skin

Acute skin defects often cause many adverse events such as abnormal pigmentation and scar formation, the satisfactory healing of which remains a significant clinical challenge. Over the past several decades, a number of skin equivalents have been available for clinical purposes to promote wound closure. However, the true values of skin equivalent - tissue-engineered skin (TE-skin) composed of neonatal fibroblasts and keratinocytes - in improving the quality of wound healing are not yet elucidated. A total of 158 patients were enrolled, 129 of which were used in this study. In these patients, acute skin defects were treated with TE-skin as experimental group, and treated with Vaseline primary dressing as control group. The differences in average healing times between the two groups were determined with statistical analysis according to different depths of skin defects. Wound quality, including pigmentation, cicatrization, and pliability, was assessed by investigators from different clinical centers over a 6-month period. The cosmetic outcome of the wound was further evaluated with histological method. In the study, the average time of wound closure in the experimental group was significantly shortened by 6.5 to 20 days according to different depths of skin defects. The cosmetic quality of reconstructed skin was satisfactory, with the patients enjoying better pliability, less abnormal pigmentation, and cicatrization. Safety analysis demonstrated that the wounds treated with TE-skin did not show clinical or laboratory evidence of rejection during the trial. These results indicate that TE-skin is a suitable and clinically effective treatment for various acute skin defects. Furthermore, the TE-skin appears to produce more satisfactory cosmetic results when compared with the conventional therapy.     

Construction of bilayered tissue-engineered skin with human amniotic mesenchymal cells and human amniotic epithelial cells

Human amniotic mesenchymal cells (hAMCs) and human amniotic epithelial cells (hAECs) have attracted increasing attention in recent years as a possible reserve of stem cells that may be useful for clinical application in regenerative medicine. The object of this study was to establish a new model for reconstruction of bilayered tissue-engineered (TE) skin with hAMCs and hAECs (amniotic cells TE skin, AC-TE skin). We studied these two types of cells and confirmed that they possessed the properties of stem cells. Mesenchymal-epidermal interactions are responsible for organogenesis. On the basis of this mechanism, we modified the constructing methods of traditional TE skin (TE skin with human fibroblasts and keratinocytes) and then established a new bilayered TE skin-AC-TE skin. Histological and immunochemical methods were carried out to assess AC-TE skin. The results showed that AC-TE skin was similar in morphology to human skin which had stratified epidermis and underlying dermis. AC-TE skin expressed proliferative cells marker Ki67 and epithelial stem cells marker K19; moreover, the constructed AC-TE skin could successfully repair full thickness skin defects on athymic mice. Our findings suggest that AC-TE skin is a useful skin equivalent which has good application prospects in regenerative medicine.     

以骨髓基质干细胞构建带内支撑的组织工程化软骨

目的 探讨构建特定形态带内支撑组织工程化软骨的医用假体的可能性.方法 以直径3 mm、长5 mm的圆柱状多孔高密度聚乙烯(Medpor)外裹厚1 mm的聚羟基乙酸为支架,将体外培养的骨髓基质干细胞(bone marrow stromal cells,BMSCs).按10×107/ml的细胞浓度均匀接种于支架,常规培养液培养5 d后,用含诱导因子的培养液立体诱导4周,同时以相同浓度的软骨细胞和BMSCs分别接种,常规体外培养4周作为阳性对照组和阴性对照组,分别种植于裸鼠皮下,4、8周后取材,行大体观察、组织学、组织化学、免疫组化及糖氨聚糖(GAG)定量等检测.结果 各组细胞均与材料粘附良好.实验组和阳性对照组均形成了大体形态良好的Medper-软骨复合体,内部的Medper与外层软骨结合紧密.组织学可见成熟软骨陷窝并渗入Medper孔隙内部、异染基质及Ⅱ型胶原表达,实验组GAG含量4、8周时分别为(5.13 ±0.32)mg/g、(5.37±0.12)mg/g.结论 以BMSCs作为种子细胞可于体内构建特定形态、组织学良好的Medpor-软骨复合体.     

不同支架材料体外构建组织工程软骨的研究

目的:体外构建组织工程软骨,筛选更为适合组织工程软骨构建的支架材料。方法:体外获取SD大鼠肋软骨细胞。采用第一代软骨细胞作为种子细胞,接种于壳聚糖/明胶和BMG/生物蛋白胶支架,体外培养的不同时间对其进行HE、甲苯胺蓝染色、Mas s on染色、免疫学检测、扫描电镜观察。结果:在培养2周时,BMG/生物蛋白胶各种染色结果显示软骨细胞在其表面以及内部分布均匀,蛋白多糖和Ⅱ型胶原染色阳性;壳聚糖/明胶表面细胞稍多于前者,但内部细胞数量极少且分布不均,染色结果不如前者明显。随着培养时间的延长各种检测均显示有大量的软骨细胞特异性的蛋白多糖和Ⅱ型胶原的表达,壳聚糖/明胶凸显出明显的优势。结论:体外成功构建组织工程软骨,软骨细胞在BMG/生物蛋白胶上的生长、增殖和分泌基质情况优于壳聚糖/明胶支架。