低能量激光照射对人脂肪来源干细胞/海藻酸钠/明胶三维生物打印体成骨能力的影响

doi:10.19723/j.issn.1671-167X.2018.05.018

北京大学学报(医学版) ›› 2018, Vol. 50 ›› Issue (5): 868-875. doi: 10.19723/j.issn.1671-167X.2018.05.018

低能量激光照射对人脂肪来源干细胞/海藻酸钠/明胶三维生物打印体成骨能力的影响

隋华欣1，吕培军2△，王勇2，冯驭驰1

（1. 北京大学第一医院口腔科，北京100034； 2. 北京大学口腔医学院·口腔医院，口腔医学数字化研究中心/口腔修复教研室卫生部口腔医学计算机应用工程技术研究中心国家口腔疾病临床医学研究中心口腔数字化医疗技术和材料国家工程实验室口腔数字医学北京市重点实验室，北京100081）

出版日期:2018-10-18 发布日期:2018-10-18
通讯作者: 吕培军 E-mail: kqlpj@bjmu.edu.cn
基金资助:
教育部科学技术研究项目（113002A）

Effects of low level laser irradiation on the osteogenic capacity of sodium alginate/gelatin/human adipose-derived stem cells 3D bio-printing construct

SUI Hua-xin1, LV Pei-jun2△, WANG Yong2, FENG Yu-chi1

(1.Department of Stomatology, Peking University First Hospital, Beijing 100034, China; 2. Center of Digital Dentistry, Department of Prosthodontics, Peking University School and Hospital of Stomatology & Research Center of Engineering and Technology for Digital Dentistry, Ministry of Health & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China)

Online:2018-10-18 Published:2018-10-18
Contact: LV Pei-jun E-mail: kqlpj@bjmu.edu.cn
Supported by:
Supported by Project of Chinese Ministry of Education，China (113002A)

摘要/Abstract

摘要： 目的：以人脂肪来源干细胞（human adipose-derived stem cells，hASCs）作为种子细胞，通过三维生物打印构建hASCs共混物打印体，以低能量激光为刺激手段，探索低能量激光照射（low level laser irradiation，LLLI）对三维结构成骨能力的影响。方法：制备hASCs/海藻酸钠/明胶三维生物打印体，并随机分为4组：增殖培养基（proliferative medium，PM)组、PM+LLLI组、成骨培养基（osteogenic medium，OM）组、OM+LLLI组，激光照射总能量密度为4 J/cm2。通过肉眼及倒置相差显微镜对打印体进行观察并拍照；用Live/Dead染色评价打印体内细胞存活率；利用免疫荧光染色比较各组成骨向分化因子骨钙素（osteocalcin，OCN）及 Runt相关转录因子2（Runtrelated transcription factor 2，Runx2）的表达。结果：获得规格10 mm×10 mm×1.5 mm、半透明、网格状的三维结构体，微丝宽度约为1 mm，孔隙呈圆形，直径在700 μm左右。打印体内细胞存活率较高，且各组间无明显差异。第7天时，OCN的表达量从高到低依次为OM+LLLI组、PM+LLLI组、OM组、PM组，除PM+LLLI组与OM组间差异无统计学意义外，其他各组间差异均有统计学意义（P<0.01）。到了第14天时，各组OCN表达量较第7天均有不同程度增高，OM+LLLI与OM组间差异不再有统计学意义。Runx2表达量方面，OM+LLLI组在第7天时即达到90%以上，显著高于OM组（P<0.01），PM+LLLI 组也显著高于PM组（P<0.05）；而到第14天，PM+LLLI组及OM+LLLI组的Runx2表达量又明显降低至低于相应未照射组。故OCN及Runx2表达量上，成骨诱导培养组明显高于未成骨诱导组，经低能量激光照射组明显高于未照射组，随着培养时间的延长，这种差异逐渐减小。结论：LLLI不会对hASCs/海藻酸钠/明胶三维生物打印体内hASCs存活率造成影响，且可促进hASCs成骨向分化。

关键词: 低能量激光照射, 生物打印, 细胞分化, 骨生成, 干细胞

Abstract: Objective： To explore the effects of low level laser irradiation (LLLI) on the osteogenic capacity of three-dimensional （3D） structure by 3D bio-printing construct used human adipose-derived stem cells (hASCs) as seed cells. Methods: Using hASCs as seed cells, we prepared sodium alginate/gelatin/hASCs 3D bio-printing construct, and divided them into four groups: PM (proliferative medium), PM+LLLI, OM (osteogenic medium) and OM+LLLI, and the total doses of LLLI was 4 J/cm2. Immunofluorescence microscopy was used to observe the viability of the cells, and analyze the expression of the osteogenesis-related protein Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN). Results: The 3D constructs obtained by printing were examined by microscope. The sizes of these 3D constructs were 10 mm×10 mm×1.5 mm. The wall thickness of the printed gelatin mold was approximately 1 mm, and the pores were round and had a diameter of about 700 μm. The cell viability of sodium alginate/gelatin/hASCs 3D bio-printing construct was high, and the difference among the four groups was not significant. On day 7, the expression of OCN from high to low was group OM+LLLI, PM+LLLI, OM and PM. There were significant differences among these groups (P<0.01), but there was no significant difference between group PM+LLLI and OM. On day 14, the expression of OCN in each group was hi-gher than that on day 7, and there was no significant difference between group OM+LLLI and OM. The expression of Runx2 in group OM+LLLI was more than 90%, significantly higher than that in group OM (P<0.01). But the expression of Runx2 in group PM+LLLI and OM+LLLI were significantly lower than that in the non-irradiated groups. The expression of osteogenesis-related protein Runx2 and OCN were higher in OM groups than in PM groups. Furthermore, the irradiated groups were significantly higher than the non-irradiated groups. Conclusion: LLLI does not affect the cell viability of sodium alginate/gelatin/hASCs 3D bio-printing construct, and may promote the osteogenic differentiation of hASCs.

Key words: Low-level laser irradiation, Bioprinting, Cell differentiation, Osteogenesis, Stem cells

中图分类号:

隋华欣，吕培军，王勇，冯驭驰. 低能量激光照射对人脂肪来源干细胞/海藻酸钠/明胶三维生物打印体成骨能力的影响[J]. 北京大学学报(医学版), 2018, 50(5): 868-875.

SUI Hua-xin, LV Pei-jun, WANG Yong, FENG Yu-chi. Effects of low level laser irradiation on the osteogenic capacity of sodium alginate/gelatin/human adipose-derived stem cells 3D bio-printing construct[J]. Journal of Peking University(Health Sciences), 2018, 50(5): 868-875.

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低能量激光照射对人脂肪来源干细胞/海藻酸钠/明胶三维生物打印体成骨能力的影响

Effects of low level laser irradiation on the osteogenic capacity of sodium alginate/gelatin/human adipose-derived stem cells 3D bio-printing construct

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