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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2022, Vol. 42 Issue (8): 13-20    DOI: 10.13523/j.cb.2203065
研究报告     
牙源性干细胞复合微渠多孔羟基磷灰石支架成骨性能研究*
赖爽1,2,刘畅1,刘春晖2,刘聪2,任小华1,2,**(),牟雁东1,2,**()
1.电子科技大学附属医院 四川省人民医院 成都 610072
2.电子科技大学医学院 成都 610072
Osteogenic Properties of Dental-derived Stem Cell Composite with Grooved Porous Hydroxyapatite Scaffolds
LAI Shuang1,2,LIU Chang1,LIU Chun-hui2,LIU Cong2,REN Xiao-hua1,2,**(),MU Yan-dong1,2,**()
1. Affiliated Hospital of University of Electronic Science and Technology of China,Sichuan Provincial People’s Hospital, Chengdu 610072, China
2. University of Electronic Science and Technology of China, Medical School,Chengdu 610072, China
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摘要：

目的: 探讨牙源性干细胞复合微渠多孔羟基磷灰石支架(grooved porous hydroxyapatite scaffolds, HAG支架)的成骨性能,为骨缺损修复治疗提供新手段。方法: 从健康成人第三磨牙中提取牙周膜干细胞(periodontal ligament stem cells, PDLSCs)及牙髓干细胞(dental pulp stem cells, DPSCs)分别接种于HAG支架上,进行多向分化鉴定及碱性磷酸酶(alkaline phosphatase,ALP)活性测定;并通过CCK-8检测细胞增殖能力;逆转录聚合酶链反应(qRT-PCR)检测骨形态发生蛋白2(bone morphogenetic protein 2, BMP-2)、骨钙素(osteocalcin, OCN)和骨桥蛋白(osteopontin, OPN)等成骨相关基因的表达。体内研究中将搭载PDLSCs和DPSCs的HAG支架移植到裸鼠的背部皮下,8周后取材,组织切片后采用苏木精-伊红(HE)染色观察新骨形成,提取组织蛋白采用Western blot检测ALP、OCN等成骨相关蛋白的表达。结果: 体外研究中DPSCs复合HAG支架组的细胞增殖能力、ALP活性,以及成骨相关基因ALP、BMP2、OCN等的表达均高于PDLSCs复合HAG支架组。体内研究中HE染色显示,PDLSCs复合HAG支架组及DPSCs复合HAG支架组均较空白HAG支架组有更多细胞生长区、纤维细胞增生及骨基质形成,且DPSCs复合HAG支架组的骨基质面积更大,成纤维细胞数量更多;PDLSCs复合HAG支架组及DPSCs复合HAG支架组成骨相关蛋白的表达量均高于空白HAG组,且DPSCs复合HAG支架组中ALP蛋白表达量显著高于PDLSCs复合HAG支架组。结论: PDLSCs、DPSCs复合HAG支架在体内外均表现出良好的成骨性能,其中DPSCs复合HAG支架的成骨性能更为优异。
关键词： 羟基磷灰石;  牙髓干细胞;  牙周膜干细胞;  骨再生    
Abstract:

Objective: To investigate the osteogenic properties of dental-derived stem cell composite with grooved porous hydroxyapatite (HAG) scaffolds to provide a new approach for bone defect repair. Methods: Periodontal ligament stem cells (PDLSCs) and dental pulp stem cells (DPSCs) were separated from healthy adults’ third molars and seeded on HAG scaffolds for multi-directional differentiation identification and alkaline phosphatase (ALP) activity detection; the cell proliferation ability was detected by CCK-8; reverse transcription polymerase chain reaction (qRT-PCR) was used to detect bone morphogenetic protein 2 (BMP-2), osteocalcin (OCN) and osteopontin (OPN).In vivo study, the HAG scaffolds loaded with two kinds of cells were transplanted into the back subcutaneous tissue of nude mice, and the tissue engineered bones were taken after 8 weeks. After tissue sections, hematoxylin and eosin (HE) staining was used to observe the formation of new bones. Western blot was used to observe expressions of osteogenesis-related proteins such as ALP and OCN. Results: In the in vitro study, the cell proliferation ability, ALP activity, and the expressions of osteogenesis-related genes ALP, BMP2, and OCN in the DPSCs combined with HAG scaffold group were higher than those in the PDLSCs combined with HAG scaffold group. In vivo study, HE staining showed that the PDLSCs combined with HAG scaffold group and the DPSCs combined with HAG scaffold group had more cell growth areas, fibrocyte proliferation and bone matrix formation than the blank HAG scaffold group, and the DPSCs combined with HAG scaffold group had more bone matrix area. The expression of bone-related proteins in the PDLSCs combined with HAG scaffold group and the DPSCs combined with HAG scaffold group was higher than that in the blank HAG group, and the expression of ALP protein in the DPSCs combined with HAG scaffold group was significantly higher than that in the PDLSCs combined with HAG scaffold group. Conclusion: PDLSC and DPSC composites with HAG scaffolds showed good osteogenic properties in vitro and in vivo, and the osteogenic properties of DPSC composite with HAG scaffolds were even better.
Key words: Hydroxyapatite    Periodontal stem cells    Dental pulp stem cells    Osteogenesis
收稿日期: 2022-03-28 出版日期: 2022-09-07
ZTFLH:  X703.5  
基金资助: * 国家自然科学基金(8207033598)
通讯作者: 任小华,牟雁东     E-mail: 1072718910@qq.com;995448060@qq.com
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赖爽
刘畅
刘春晖
刘聪
任小华
牟雁东

引用本文:

赖爽,刘畅,刘春晖,刘聪,任小华,牟雁东. 牙源性干细胞复合微渠多孔羟基磷灰石支架成骨性能研究*[J]. 中国生物工程杂志, 2022, 42(8): 13-20.

LAI Shuang,LIU Chang,LIU Chun-hui,LIU Cong,REN Xiao-hua,MU Yan-dong. Osteogenic Properties of Dental-derived Stem Cell Composite with Grooved Porous Hydroxyapatite Scaffolds. China Biotechnology, 2022, 42(8): 13-20.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2203065    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I8/13

名称 序列
ALP (F)5'-CCTCATCATCTTCCTGGGCG-3'
(R)5'-CATGGCCAGGGGTATCTCAG-3'
BMP2 (F)5'-CGCTGTCTTCTAGCGTTGC-3'
(R)5'-CAACTCGAACTCGCTCAGGA-3'
OCN (F)5'-CTCACACTCCTCGCCCTATTG-3'
(R)5'-CGCCTGGGTCTCTTCACTAC-3'
COL-1 (F)5'-AGGGGTCTCCATGGTGAGTT-3'
(R)5'-GAAGGACCTCGGCTTCCAAT-3'
Runx2 (F)5'-TTCCAGAATGCTTCCGCCAT-3'
(R)5'-AACTGCTGTGGCTTCCATCA-3'
GAPDH (F)5'-TGCAATAACCACCCCTGACC-3'
(R)5'-ATTTGCCGAAGAGCCCTCAG-3'
表1  PCR实验引物序列
名称 来源
ALP 兔抗人多克隆抗体;abcam, ab83259
OCN 鼠抗人单克隆抗体;abcam, ab13420
VEGF 鼠抗人单克隆抗体;Santa Cruz, sc-7269
β-actin 兔抗人单克隆抗体;Abclonal, AC026
表2  Western blot实验抗体
图1  PDLSCs(a)和DPSCs(b)成骨诱导分化结果
图2  PDLSCs(a)和DPSCs(b)成脂诱导分化结果
图3  细胞增殖检测
图4  细胞复合HAG支架进行成骨诱导后ALP活性
图5  成骨相关基因的表达
图6  组织工程骨的组织切片染色
图7  组织工程骨中ALP、OCN和VEGF的Western blot条带
图8  组织工程骨中ALP、OCN和VEGF蛋白表达的定量分析
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