荷载角质细胞生长因子纳米微囊脱细胞真皮的制备、特性及其对表皮干细胞群生长的作用

Construction and characteristics of acellular dermal matrix loaded with keratinocyte growth factor nanocapsules and its effects on the growth of epidermal stem cells

目的:构建一种荷载有角质细胞生长因子且能控制释放的新型组织工程化皮肤。方法:实验于2006-03/12于中山大学附属第二医院干细胞研究中心及中山大学高分子研究所进行。①材料:角质细胞生长因子(CytolLAB公司),牛血清白蛋白(Amresco公司),聚乳酸-羟基乙酸共聚物(由中山大学高分子化学研究所合成,聚乳酸与聚羟基乙酸摩尔比为75∶25,相对分子质量为50000),脱细胞真皮(北京桀亚莱福生物技术有限公司)。②方法:采用超声乳化-溶剂挥发及低温干燥方法,将角质细胞生长因子以牛血清白蛋白作联接包裹在聚乳酸-羟基乙酸共聚物内制成纳米微囊,根据公式计算微囊成球率、载药量及包封率;将微囊溶解于二氯甲烷中,进行体外释放实验检测不同时间牛血清白蛋白吸光度,作出牛血清白蛋白释放曲线;将角质细胞生长因子纳米微囊交联于脱细胞真皮表面,制成荷载角质细胞生长因子纳米微囊脱细胞真皮(KGF-ADM),在扫描电镜下观察微囊形态、分布及其与脱细胞真皮连接情况;采用Ⅰ型胶原酶复合胰蛋白酶消化的方法,从门诊健康患者无菌手术切除的包皮组织中获取表皮细胞单细胞悬液,经免疫荧光检测,证实其中含表皮干细胞,将表皮干细胞群分别接种于KGF-ADM及聚乳酸-羟基乙酸共聚物上,于恒温箱中培养3d后,在扫描电镜下观察细胞形态、生长情况及其与材料连接情况。结果:①纳米微囊成球率为85%,载药量为17.3%,包封率为73.5%。②纳米微囊中牛血清白蛋白可以控制缓慢释放,其释放曲线显示初期释放速度较快(前5d累计释放约40%),后进入缓慢释放期(30d累计释放约75%)。③扫描电镜检测显示纳米微囊形态规则,在KGF-ADM表面分布均匀,与KGF-ADM联接良好,部分分布于KGF-ADM深面;表皮干细胞群在KGF-ADM上生长活跃,细胞形态良好,部分形成克隆。结论:采用该方法可成功构建荷载角质细胞生长因子纳米微囊的新型组织工程化皮肤。

AIM： To construct a new kind of tissue-engineered skin loaded with keratinocyte growth factor （KGF） nanocapsules, which can be released by control. METHODS： All of the experiments were carded out in the Stem Cell Research Center of the Second Affiliated Hospital of Sun Yat-sen University and the Macromolecular Institute of Sun Yat-sen University From March to December in 2006. Experimental materials included KGF （CytolLAB Company）, bovine serum albumin （BSA, Amresco Company）, polylactic-co-glycolic acid （PLGA, Macromolecular Institute of Zhongshan University, molar ratio was 75：25 and relative molecular mass was 50 000）, acellular dermal matrix （ADM, Beijing Jayya Life Tissue Engineering Co., Ltd）. The experimental procedures were as follows： Ultrasound emulsification-impregnant volatilization and low temperature drying methods were used to connect KGF to BSA and make them encapsuled in PLGA shell. The microsphere rate, the drug loading amount and the encapsulation efficiency were calculated according to the formula; The KGF nanocapsules were dissolved into dichloromethane, the absorbency of BSA of in vitro release test was mensurated to draw the releasing curve of BSA; The KGF nanocapsules were dropped onto the ADM surface to make them linked tightly （KGF-ADM）. The shape of the capsules, their distribution on the KGF-ADM and their connection with ADM were observed with the scanning electron microscope （SEM）; The epidermal cell suspension were captured from foreskin that was excised from healthy out-patients, by using the type Ⅰ collagenase and trypsin digesting method. Then the epidermal cells were confirmed through the immunofluorescence technique to contain stem cells, which were cultivated on the KGF-ADM and the PLGA bracket for three-day culture at constant temperature. SEM was adopted to observe the shape, the growth and the connection with materials of the cells. RESULTS： （1）The nanocapsules were successfully made with the microsphere rate of 85%, the drug loading amount of 17.3%, and the encapsulation efficiency of 73.5%. （2）The encapsuled BSA could be slowly released by control, which released at a rather rapid speed in the first days （40% at former 5 days）, and then showered to a smooth level （75% at 30 days）. （3）SEM results displayed that the nanocapsules were in good spheral shape, distributed evenly on KGF-ADM surface, and linked tightly to the KGF-ADM; The epidermal stem cells grew well on the KGF-ADM and formed clones. CONCLUSION： A new kind of tissue-engineered skin loaded with KGF nanocapsules can be constructed following the method mentioned in this study.