组织工程化皮瓣的构建

目的应用脂肪组织来源干细胞(ASCs)在体外构建组织工程脂肪并与已构建的复合皮组装培育,试构建出含脂肪层的组织工程化皮瓣。方法(1)构建组织工程脂肪从脂肪组织中分离培养ASCs并进行三系分化诱导鉴定。ASCs与胶原凝胶混合并进行体外成脂诱导,诱导15 d后分别进行倒置显微镜、油红O染色及HE染色观察。(2)构建复合皮从包皮组织中分离培养表皮细胞(Kc)和成纤维细胞(Fb)。Fb与胶原凝胶混合后培养5 d,将Kc接种到含有Fb的凝胶表面,在液面下培养4 d后移至气-液界面继续培养10 d形成复合皮。(3)构建含皮下脂肪组织的3层结构的皮肤复合组织将培养的复合皮与脂肪组织按正常皮肤结构顺序组装后继续培养3 d并行HE染色观察。结果(1)ASCs具有成脂、成软骨和成骨分化能力。ASCs凝胶复合物体外成脂诱导15 d后,可见细胞内充满圆形脂滴,呈单房或多房形,油红O染色及HE染色均显示为成熟脂滴。(2)经过气-液界面培养后,Kc成层分化,形成基底上层。(3)组装培育后的皮肤复合组织分3层,表层Kc成层分化并形成基底上层;中层和底层连接处细胞聚集;成脂分化的ASCs位于底层。结论分别应用Kc、Fb和ASCs三种细胞,在体外构建出组织工程化脂肪和复合皮,且按序组装培育,可构建出组织工程化皮瓣。

Construction of tissue-engineered skin flap in vitro

Objective To construct a tissue-engineered skin flap using composite skin and adipose tissue constructed by adipose-derived stem cells(ASCs).Methods Human ASCs isolated from adipose tissue were cultured and identified for their adipogenic,osteogenic and chondrogenic differentiation potentials.ASCs were then mixed with collagen gel for adipogenic induction and observed 15 days later with inverted microscope,oil-red O staining and HE staining.To construct the composite skin,keratinocytes and fibroblasts were isolated from human foreskin.The fibroblasts were mixed with collagen gel and cultured for 5 days,and keratinocytes were seeded on the gel for 4 days before transfer of the culture to air-liquid interface for culture for another 10 days.The adipose tissue and composite skin were then assembled according to the structure of normal skin and cultured for 3 days with HE staining observation.Results The cultured ASCs were capable of adipogenic,osteogenic and chondrogenic differentiation,and adipogenic induction of the ASCs-gel complex for 15 days resulted in adipogenic differentiation of the ASCs in gel.The assembled tissue-engineered skin consisted of 3 layers,including a suprabasal layer formed by the stratified and differentiated keratinocytes,the middle layer and sublayer containing numerous cells,and a underlying sublayer formed by the adipogenic ASCs.Conclusion Tissue-engineered skin flap can be constructed by assembling composite skin and adipose derived from cultured keratinocytes,fibroblasts,and ASCs.