含人脐血来源富血小板血浆的三维生物打印墨水在裸鼠全层皮肤缺损治疗中的应用  

Application of three-dimensional bioprinting ink containing platelet-rich plasma derived from human umbilical cord blood in the treatment of full-thickness skin defects in nude mice

作　　者：宋薇 李曌[1] 朱世钧 张超[1] 姚斌[1] 孔毅 梁莉婷 恩和吉日嘎拉 付小兵[1] 黄沙[1] Song Wei;Li Zhao;Zhu Shijun;Zhang Chao;Yao Bin;Kong Yi;Liang Liting;Enhejirigala;Fu Xiaobing;Huang Sha(Research Center for Wound Repair and Tissue Regeneration,Medical Innovation Research Department,the PLA General Hospital,Beijing 100048,China)

机构地区：[1]解放军总医院医学创新研究部创伤修复与组织再生研究中心,北京100048

出　　处：《中华烧伤与创面修复杂志》2022年第10期905-913,共9页Chinese Journal of Burns And Wounds

基　　金：国家重点研发计划(2017YFC1103303);国家自然科学基金青年科学基金项目(32000969);上海王正国创伤医学发展基金会生长因子复兴计划(SZYZ-TR-03)。

摘　　要：目的探讨含人脐血来源富血小板血浆(HUCB-PRP)的海藻酸钠-明胶(AG)生物墨水(称为HUCB-PRP-AG生物墨水)的可打印性能和细胞相容性,及该生物墨水的三维打印组织治疗裸鼠全层皮肤缺损创面的效果。方法采用实验研究方法。制备含体积分数2.5%、5.0%、10.0%HUCB-PRP的HUCB-PRP-AG生物墨水,分别命名为1P-AG、2P-AG、4P-AG。取AG、1P-AG、2P-AG、4P-AG,观察室温下外观,采用旋转流变仪检测黏度、储能模量和损耗模量。利用以上4种生物墨水分别进行三维生物打印并观察打印组织外观(后续使用交联后打印组织),将4种打印组织分别与人脐静脉内皮细胞(HUVEC)在Transwell小室内用HUVEC专用培养基共培养24 h,采用细胞计数试剂盒8检测细胞增殖水平(样本数为3);将4种打印组织分别用DMEM培养基培养12、24、48 h,进行干燥称重并计算降解率(样本数为3);将1P-AG、2P-AG、4P-AG打印组织分别用磷酸盐缓冲液培养0.5、24.0、48.0 h,采用酶联免疫吸附测定法检测培养上清液中血管内皮生长因子(VEGF)的表达(样本数为5)。取16只6~8周龄雌性BALB/c-NU裸鼠建立背部全层皮肤缺损创面模型,分为创面仅覆盖医用水胶体敷料的常规对照组和另予HUCB-PRP滴加的HUCB-PRP组、AG打印组织覆盖的AG组、4P-AG打印组织覆盖的4P-AG组(每组4只),观察每组3只裸鼠伤后4、8、14 d创面愈合情况并计算创面愈合率;取每组剩余裸鼠伤后8 d创面组织,行苏木精-伊红染色后观察组织病理学改变,行免疫组织化学染色后观察CD31阳性新生血管情况。对数据行重复测量方差分析、LSD检验及Bonferroni校正。结果在室温下,AG、1P-AG、2P-AG、4P-AG均呈半透明液态;AG为淡黄色,1P-AG、2P-AG、4P-AG均为淡红色但颜色依次逐渐加深。在温度10℃和剪切率0.1~10.0 s-1范围内,AG、1P-AG、2P-AG、4P-AG的黏度均随着剪切率的增加而减小;在温度10℃和角频率1~100 rad/s范围内,4种生物墨水Objective To investigate the printability and cytocompatibility of sodium alginate-gelatin(AG)bioink containing platelet-rich plasma derived from human umbilical cord blood(HUCB-PRP),named HUCB-PRP-AG bioink,and the effect of the three-dimensionally printed tissue with the bioink on full-thickness skin defect wounds in nude mice.Methods The method of experimental research was used.HUCB-PRP-AG bioinks with 2.5%,5.0%,and 10.0%of HUCB-PRP by volume were prepared and named 1P-AG,2P-AG,and 4P-AG,respectively.The appearances of AG,1P-AG,2P-AG,and 4P-AG at room temperature were observed,and their viscosity and storage/loss modulus were measured by a rotational rheometer.The above four bioinks were used for three-dimensional bioprinting respectively,and the appearances of the printed tissue were observed(the printed tissue was subsequently cross-linked and used).The four kinds of bioprinted tissue were respectively co-cultured with human umbilical vein endothelial cells(HUVECs)in Transwell chambers with HUVEC special medium for 24 h,and the cell proliferation level was detected by cell counting kit 8(n=3).The four kinds of bioprinted tissue were respectively cultured in Dulbecco's modified eagle medium for 12,24,and 48 h,which were dried and weighed,and the degradation rate was calculated(n=3).The expression of vascular endothelial growth factor(VEGF)in the culture supernatant of 1P-AG,2P-AG,or 4P-AG cultured in phosphate buffer solution at 0.5,24.0,and 48.0 h was detected by enzyme-linked immunosorbent assay(n=5).Sixteen female BALB/c-NU nude mice aged 6-8 weeks were selected to establish a full-thickness skin defect wound model on the back and were divided into conventional control group with wounds being covered with medical hydrocolloid dressing alone,HUCB-PRP group with additional HUCB-PRP dripping to the wounds,AG group additionally covered with AG printed tissue,and 4P-AG group additionally covered with 4P-AG printed tissue,respectively(with 4 nude mice in each group).The wound healing of 3 nude mice in each group

关 键 词：生物相容性材料 打印 三维 新生血管化 生理性 生物墨水 人脐血来源富血小板血浆 创面修复 

分 类 号：R622[医药卫生—整形外科]