氧化锆多孔表面显微形貌对成骨细胞增殖及分化的影响

doi:10.19723/j.issn.1671-167X.2022.01.006

北京大学学报（医学版） ›› 2022, Vol. 54 ›› Issue (1): 31-39. doi: 10.19723/j.issn.1671-167X.2022.01.006

氧化锆多孔表面显微形貌对成骨细胞增殖及分化的影响

王铮¹,丁茜^1,^2,^△(),高远¹,马全诠¹,张磊^1,^△(),葛兮源³,孙玉春^1,⁴,谢秋菲¹

1.北京大学口腔医学院·口腔医院修复科,国家口腔医学中心,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,国家卫生健康委员会口腔医学计算机应用工程技术研究中心,国家药品监督管理局口腔生物材料重点实验室,北京 100081
2.佛山(华南)新材料研究院,广东佛山 528000
3.北京大学口腔医学院·口腔医院中心实验室,北京 100081
4.北京大学口腔医学院·口腔医院口腔医学数字化研究中心,北京 100081

收稿日期:2021-10-07 出版日期:2022-02-18 发布日期:2022-02-21
通讯作者: 丁茜,张磊 E-mail:dingqian.623@163.com;drzhanglei@yeah.net
基金资助:
首都卫生发展科研专项(首发2020-2-4104)(2020-2-4104);北京市自然科学基金(7192233);广东省基础与应用基础研究基金(2019A1515110889)

Effect of porous zirconia ceramics on proliferation and differentiation of osteoblasts

WANG Zheng¹,DING Qian^1,^2,^△(),GAO Yuan¹,MA Quan-quan¹,ZHANG Lei^1,^△(),GE Xi-yuan³,SUN Yu-chun^1,⁴,XIE Qiu-fei¹

1. Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology & NHC Research Center of Engineering and Technology for Computerized Dentistry & NMPA Key Laboratory for Dental Materials, Beijing 100081, China
2. Foshan (Southern China) Institute for New Materials, Foshan 528000, Guangdong, China
3. Central Laboratory, Peking University School and Hospital of Stomatology, Beijing 100081, China
4. Center for Digital Dentistry, Peking University School and Hospital of Stomatology, Beijing 100081, China

Received:2021-10-07 Online:2022-02-18 Published:2022-02-21
Contact: Qian DING,Lei ZHANG E-mail:dingqian.623@163.com;drzhanglei@yeah.net
Supported by:
Capital Health Development Research Special Fund(2020-2-4104);Beijing Natural Science Foundation(7192233);Guangdong Basic and Applied Basic Research Foundation(2019A1515110889)

摘要/Abstract

摘要：

目的: 探索两种氧化锆多孔表面显微形貌对小鼠前成骨细胞增殖、分化的影响。方法: 根据加工及造孔方式不同,将氧化锆试件分为4组,分别为切削终烧结组(milled sintering group, M-Ctrl)、切削多孔组(milled porous group, M-Porous)、3D打印终烧结组(3D printed sintering group, 3D-Ctrl)和3D打印多孔组(3D printed porous group, 3D-Porous)。通过扫描电镜(scanning electron microscope, SEM)、激光显微形貌测量显微镜进行表面显微形貌分析,用接触角测量仪测量静态接触角,通过能量色散X射线仪进行表面元素分析。将小鼠胚胎成骨细胞前体细胞MC3T3-E1接种于试件表面,SEM观察细胞培养第1、7天的黏附形态;使用细胞增殖-毒性检测试剂盒(cell counting kit-8, CCK-8)测量细胞培养第1、3、5天的增殖情况;采用实时荧光定量聚合酶链式反应检测分化诱导第14天时碱性磷酸酶(alkaline phosphatase, ALP)、Ⅰ型胶原(type Ⅰ collagen, Colla1)、Runt相关转录因子2(Runt-related transcription factor-2, Runx2)及骨钙素(osteocalcin, OCN ) mRNA的相对表达量。结果: 3D-Porous组孔径[(419.72±6.99) μm]及孔深度[(560.38±8.55) μm]均显著大于M-Porous组的孔径[(300.55±155.65) μm]及孔深度[(69.97±31.38) μm,(P<0.05)], 且3D-Porous组圆孔形状更规则,分布更均匀。各组静态接触角均小于90°,其中3D-Ctrl组的静态接触角(73.83°±5.34°)与M-Porous组(72.7°±2.72°)最大,两组间差异无统计学意义(P>0.05)。M-Porous及3D-Porous组表面均可见细胞在孔内黏附,且培养第3、5天时两组试件表面细胞增殖活性均显著高于M-Ctrl组及3D-Ctrl组(P<0.05)。细胞分化诱导第14天时3D-Porous组ALP、Colla1、Runx2、OCN mRNA的相对表达量均显著低于M-Ctrl组及3D-Ctrl组(P < 0.05)。M-Porous组Colla1、Runx2、OCN mRNA的相对表达量均显著高于3D-Porous组(P<0.05)。结论: 氧化锆表面多孔形貌能促进小鼠前成骨细胞MC3T3-E1的增殖及黏附,但对其成骨向分化有一定抑制作用。

关键词: 氧化锆, 多孔表面, 成骨细胞, 骨结合

Abstract:

Objective: To investigate the effect of porous surface morphology of zirconia on the proliferation and differentiation of osteoblasts. Methods: According to different manufacturing and pore-forming methods, the zirconia specimens were divided into 4 groups, including milled sintering group (M-Ctrl), milled porous group (M-Porous), 3D printed sintering group (3D-Ctrl) and 3D printed porous group (3D-Porous). The surface micromorphology, surface roughness, contact angle and surface elements of specimens in each group were detected by scanning electron microscope (SEM), 3D laser microscope, contact angle measuring device and energy-dispersion X-ray analysis, respectively. MC3T3-E1 cells were cultured on 4 groups of zirconia discs. The cell morphology of MC3T3-E1 cells on zirconia discs was eva-luated on 1 and 7 days by SEM. The cell proliferation was detected on 1, 3 and 5 days by cell counting kit-8 (CCK-8). After osteogenic induction for 14 days, the relative mRNA expression of alkaline phosphatase (ALP), type Ⅰ collagen (Colla1), Runt-related transcription factor-2 (Runx2) and osteocalcin (OCN) in MC3T3-E1 cells were detected by real-time quantitative polymerase chain reaction. Results: The pore size [(419.72±6.99) μm] and pore depth [(560.38±8.55) μm] of 3D-Porous group were significantly larger than the pore size [(300.55±155.65) μm] and pore depth [(69.97±31.38) μm] of M-Porous group (P<0.05). The surface of 3D-Porous group appeared with more regular round pores than that of M-Porous group. The contact angles of all the groups were less than 90°. The contact angles of 3D-Ctrl (73.83°±5.34°) and M-Porous group (72.7°±2.72°) were the largest, with no significant difference between them (P>0.05). Cells adhered inside the pores in M-Porous and 3D-Porous groups, and the proliferation activities of them were significantly higher than those of M-Ctrl and 3D-Ctrl groups after 3 and 5 days’ culture (P<0.05). After 14 days’ incubation, ALP, Colla1, Runx2 and OCN mRNA expression in 3D-Porous groups were significantly lower than those of M-Ctrl and 3D-Ctrl groups (P<0.05). Colla1, Runx2 and OCN mRNA expressions in M-Porous group were higher than those of 3D-Porous group (P<0.05). Conclusion: The porous surface morphology of zirconia can promote the proliferation and adhesion but inhibit the differentiation of MC3T3-E1 cells.

Key words: Zirconia, Porous surface, Osteoblasts, Osseointegration

中图分类号:

R781.3

王铮,丁茜,高远,马全诠,张磊,葛兮源,孙玉春,谢秋菲. 氧化锆多孔表面显微形貌对成骨细胞增殖及分化的影响[J]. 北京大学学报（医学版）, 2022, 54(1): 31-39.

WANG Zheng,DING Qian,GAO Yuan,MA Quan-quan,ZHANG Lei,GE Xi-yuan,SUN Yu-chun,XIE Qiu-fei. Effect of porous zirconia ceramics on proliferation and differentiation of osteoblasts[J]. Journal of Peking University (Health Sciences), 2022, 54(1): 31-39.

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参考文献 37

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Gene	Forward primer (5'-3')	Reverse primer (5'-3')
GAPDH	TGGTGAAGGTCGGTGTGAAC	GCTCCTGGAAGATGGTGATGG
ALP	TGCCTACTTGTGTGGCGTGAA	TCACCCGAGTGGTAGTCACAATG
Colla1	TGAAGAACTGGACTGTCCC	TTTGGTGATACGTATTCTTCCG
Runx2	GACTGTGGTTACCGTCATGGC	ACTTGGTTTTTCATAACAGCGGA
OCN	CCAAGCAGGAGGGCAATAAGGT	CTCGTCACAAGCAGGGTTAAGC

Items	M-Porous	3D-Porous
Diameter of pores/μm, $\bar{x}\pm s$	300.55±155.65	419.72±6.99^*
Depth of pores/μm, $\bar{x}\pm s$	69.97±31.38	560.38±8.55^*
Pitch of pores/μm, $\bar{x}\pm s$		349.87±12.43

氧化锆多孔表面显微形貌对成骨细胞增殖及分化的影响

Effect of porous zirconia ceramics on proliferation and differentiation of osteoblasts

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