【作者】 陈伟；

【导师】 张艳；

【作者基本信息】 上海交通大学 ， 外科学（整形外科学）， 2019， 硕士

【摘要】 目的:本研究尝试运用生物打印技术在体外构建一种新的细胞-材料复合物修复裸鼠颅骨缺损,并进一步采用原位打印形式修复裸鼠背部皮肤缺损,此外基于连续组织切片重建皮肤三维数字模型,为将来多细胞多层次的在体生物打印奠定基础。方法:1.构建裸鼠双侧颅骨缺损(4×4mm),一侧利用生物打印技术构建人骨髓间充质干细胞(BMSCs)-海藻酸钙/氧化纳米细菌纤维素水凝胶(CA/BC)复合物后植入颅骨缺损内;另一侧仅植入CA/BC作为自身对照。8周后通过大体观察、组织学检查、Anti-HLA Class I抗体免疫组化染色和micro-CT等评估颅骨修复的效果。2.构建裸鼠背部全层皮肤缺损(直径7mm)并覆盖CA/BC,以新生C57小鼠表皮/真皮来源原代细胞为种子细胞,每个创面移植5×10~5个细胞:实验组在CA/BC内原位打印10个高浓度细胞墨点;对照组以1ml注射器手工注射。术后4周观察创面恢复及毛发生长情况,通过HE染色、免疫荧光等检查新生毛囊结构。3.对人正常头皮(面部除皱手术患者)行连续石蜡切片(层厚4μm)及HE染色、20倍显微镜下拍照,获得100张连续图像,在Photoshop CS6中进行配准后导入ImageJ软件行三维重建。结果:1.颅骨缺损实验组、对照组骨愈合率分别为0.880±0.023,0.025±0.010,(P<0.05),实验组骨缺损愈合良好,对照组仅边缘略有骨质生长;组织学检查证实新生组织为骨样结构;Anti-HLA Class I抗体免疫组化染色证实新生骨来源为打印的BMSCs。2.皮肤缺损实验组创面恢复良好,毛发生长旺盛、均匀,而对照组毛发稀疏、不均匀甚至没有毛发生长。组织学证实实验组新生皮肤含完整毛囊结构,而对照组几乎没有毛囊形成。3.完成皮肤结构的三维重建,可在ImageJ软件内动态显示并可重建各个角度截面的二维结构。结论:本研究证明了生物打印体外构建BMSCs-CA/BC复合物修复裸鼠颅骨缺损以及原位打印修复皮肤缺损的可行性;此外,以连续切片进行三维重建的方式也为将来多细胞、多层次的生物打印提供了数字化三维数字模型。更多还原

【Abstract】 Purpose:A new cells-scaffold complex was designed to repair skull defects and dorsal full-thickness skin defects of nude mice,and lay the foundation of multi-cells and multi-levels in-situ bioprinting in future with reconstruction of skin three-dimensional digital model based on series section.Methods:1.Bilateral skull defects(4×4mm)was created in nude mice,one side was treated with bioprinted human bone marrow mesenchymal stem cells(BMSCs)-calcium alginate/bacterial nanocellulose hydrogel(CA/BC),and the other was treated with only CA/BC as self-control group.After 8 weeks,gross observation,histological examination,anti-HLA-Class I and micro-CT were used to evaluate the repair effect.2.Dorsal full-thickness skin defects(7mm in diameter)was created and covered with CA/BC in nude mice.5×10~5 primary cells from neonatal C57 mice skin were used as seed cells and transplanted to the wound.10 points of high-concentration cell plotting was printed in the CA/BC via in-situ bioprinting in experimental group while 1ml injector was used manually in control group.After 4 weeks,wound repairing and hair growth were observed by H&E staining and immunofluorescent staining.3.Continuous paraffin section(slice thickness 4μm)of normal human scalp was created.After H&E staining,100 images were taken under a 20×microscope and aligned in Photoshop CS6.The three-dimensional digital model was created in ImageJ.Results:1.The healing percentage of skull defects of experimental group and control group was 0.880±0.023,0.025±0.010,(P<0.05).Much well-formed new bone was created in experimental group but none in control group.Anti-HLA-Class I staining verified the new formed bone was derived from printed BMSCs.2.Integrated skin with hair grew on wounds of experimental group while scarcely any hair seen in control group and Histological examination confirmed the normal intact hair follicle structure.3.Three-dimensional digital model of skin was created and any cross profile could be shown in ImageJ.Conclusion:This study demonstrated that bioprinted BMSCs-CA/BC complex could be used in skull defect and in-situ bioprinting had good potential in skin wound repairing.And three-dimensional digital skin model can be created by series section and it can be used for future multi-cells and multi-levels bioprinting.更多还原