基于自组装纳米多肽及脂肪间充质干细胞3D打印组织模型的研究

Biomechamical models constructed by functionalized self-assembing peptide hydrogel and human adipose-derived stem cells using a 3D bio-printer

目的 观察自组装纳米多肽与人脂肪间充质干细胞（Ad-MSCs）3D打印出的组织工程学模型内细胞生长状态及多向分化能力.方法 分离、培养原代Ad-MSCs,流式细胞仪鉴定,以自组装纳米多肽、Ad-MSCs混合溶液为＂生物墨汁＂,利用3D生物打印技术,打印组织工程学模型.诱导其内的Ad-MSCs成骨和成脂分化并分别与对照组进行染色比较.结果 原子力显微镜下观察到纳米多肽纤维结构直径为10-30nm,长200-500nm,具有纳米级材料特点.流式细胞仪鉴定可见分离、培养的细胞CD29（98.51%）、CD90（97.87%）、CD166（98.32%）表达阳性,不表达CD31（1.58%）、CD34（2.42%）、CD45（2.95%）和人类白细胞抗原DR基因（HLA-DR）（0.53%）,符合Ad-MSCs免疫表型.随后利用＂生物墨汁＂打印出组织工程模型,对并其内的Ad-MSCs进行诱导分化,分别对实验组和对照组进行相关染色,茜素红S染色可见成骨诱导实验组中钙结节形成,油红O染色可见成脂诱导实验组Ad-MSCs内脂滴着色,两对照组不着色.结论 以自组装纳米多肽、脂肪间充质干细胞混合溶液为＂生物墨汁＂3D打印出的组织工程学模型内Ad-MSCs生长状态良好,仍具有较强的分化能力.

Objective To observe the growth behavior and multi-directional differentiation capacity of human adipose-derived stem cells （Ad-MSCs） in the tissue engineering models which were constructed by 3D bio-printer with the functionalized serf-assembling peptide hydrogel and AMSC.Methods The Ad-MSCs were isolated and cultured, analyzed and identified by flow cytometry.Thereafter, the tissue engineering models were constructed by 3D bio-printer with the bio-ink which consisted of functionalized serf-assembling peptide hydroge and Ad-MSCs.The Ad-MSCs were induced in tissue engineering models to differentiate into osteoblastic cells and adipocytes.Results The self-assembing peptide hydrogel was observed by an atomic force microscope, and the nanofibers structure with 10-30 nm in diameter and 200-200 nm in length was seen and had the characteristics of nanoscale materials.The cells expressed a cell-surface protein profile positive for CD29 （98.51%）, CD90 （97.87%） and CD166 （98.32%） and negative for CD31 （1.58%）, CD34 （2.42%）, CD45（2.95%） and human leukocyte antigen-DR （HLA-DR, 0.53%）.The tissue engineering models were successfully constructed by the mixture solution.Ad-MSCs in the models had a good cellular growth behavior.After osteoinductive differentiation, Alizarin red S staining verified the formation of mineralized nodules.After adipogenic differentiation, Oil red O staining verified the formation of lipid droplets.Neither of the control groups colored.Conclusion Ad-MSCs in the tissue engineering models which were constructed by 3D bio-printer with the mixture solution had a good cellular growth behavior, and also had a strong ability of differentiation.