兔脱细胞软骨基质颗粒复合SD大鼠脂肪干细胞对软骨内成骨的促进作用

doi:10.13481/j.1671-587X.20220406

摘要/Abstract

摘要： 目的

探讨兔脱细胞软骨基质颗粒（ACM）的生物相容性，阐明其对SD大鼠脂肪干细胞（ADSCs）软骨内成骨的促进作用。

方法

提取原代SD大鼠ADSCs进行三向分化实验，流式细胞术检测细胞干性。采集3月龄新西兰白兔耳软骨，采用研磨浸泡法制备ACM备用。实验分为ACM组和ADSCs+ACM共培养组，在扫描电镜下观察ACM和ACM上ADSCs的超微形态表现，采用钙黄绿素/碘化丙啶（Calcein-AM/PI）荧光染色法观察ADSCs+ACM共培养组中ADSCs的生长情况。将ADSCs分为对照组和实验组（ADSCs与ACM共培养），采用CCK-8法检测2组细胞增殖活性，实时荧光定量PCR （RT-qPCR）法检测成软骨诱导7和14 d时2组细胞中Ⅱ型胶原（COL-Ⅱ）、Ⅹ型胶原（COL-Ⅹ）、SOX转录因子9（SOX-9）、血管内皮生长因子（VEGF）、碱性磷酸酶（ALP）和Runt相关转录因子2（RUNX-2）mRNA表达水平。

结果

成功制备兔ACM，提取的SD大鼠ADSCs可以进行三向分化且表达干细胞特异性表面标志物。ADSCs可以在ACM上黏附生长。与对照组比较，实验组ADSCs增殖活性明显升高（P<0.05）。软骨诱导7 d时，与对照组比较，实验组ADSCs中VEGF、ALP和RUNX-2 mRNA表达水平明显升高（P<0.05），COL-Ⅱ、SOX-9和COL-Ⅹ mRNA表达水平差异无统计学意义（P>0.05）；软骨诱导14 d时，与对照组比较，实验组ADSCs中VEGF、ALP和RUNX-2 mRNA表达水平明显升高（P<0.05），COL-Ⅹ mRNA表达水平明显降低（P<0.05），COL-Ⅱ和SOX-9 mRNA表达水平差异无统计学意义（P>0.05）；与软骨诱导7 d时比较，软骨诱导14 d时对照组ADSCs中VEGF、ALP和RUNX-2 mRNA表达水平明显降低（P<0.05），COL-Ⅱ和COL-Ⅹ mRNA表达水平明显升高（P<0.05）；与软骨诱导7 d时比较，软骨诱导14 d时实验组ADSCs中COL-Ⅱ和COL-Ⅹ mRNA表达水平明显升高（P<0.05），VEGF、ALP和RUNX-2 mRNA表达水平明显降低（P<0.05）。

结论

兔ACM缺乏免疫原性，具有良好的生物相容性和成软骨诱导能力，有利于软骨内成骨过程的发生。

关键词: 脂肪干细胞, 脱细胞软骨基质颗粒, 骨再生, 生物材料

Abstract: Objective

To investigate the biocompatibility of rabbit acellular cartilage matrix particles （ACM）， and to clarify its promotion effect on the endochondral osteogenesis of adipose tissue-derived stem cells （ADSCs） in the SD rats.

Methods

The primary ADSCs of SD rabbits were extracted for three-dimensional differentiation and flow cytometry was used to detect the cell dryness.The ear cartilage of 3-month-old New Zealand white rabbits was collected and treated with grinding and soaking method to prepare ACM. The experiment was divided into ACM and ACM+ADSCs co-culture group.The ultrastructures of ACM and the ADSCs in ACM were observed under scanning electron microscope，and the growth status of ADSCs in ADSCs+ACM co-culture group was by Calcein-AM/PI fluorescence staining method. The ADSCs were divided into control group and experimental group（ADSCs and ACM co-culture）.The CCK-8 method was used to detect the proliferation activities of cells in two groups. Real-time fluorescence quantitative PCR （RT-qPCR） was used to detect expression levels of type Ⅱ collagen （COL-Ⅱ），type Ⅹ collagen （COL-Ⅹ）， SRY-related high mobility group-box 9 （SOX-9），vascular endothelial growth factor （VEGF）， alkaline phosphatase （ALP） and Runt-related transcription factor 2 （RUNX-2） mRNA in the ADSCs in two groups at 7 and 14 d of chondrogenic induction.

Results

The rabbit ACM was successfully prepared. The extracted ADSCs could differentiate in three ways and the specific surface markers of stem cells were expressed in the ADSCs. The ADSCs could adhere to ACM. Compared with control group， the proliferation activity of the cells in experimental group was increased （P<0.05）.The RT-qPCR results showed that compared with control group， the expression levels of VEGF，ALP and RUNX-2 mRNA in the ADSCs in experimental group were increased at 7 d of chondrogentic induction （P<0.05）， and the expression levels of COL-2， SOX-9， and COL-Ⅱ mRNA had no significant differences（P>0.05）. At 14 d of chondrogentic induction， compared with control group， the expression levels of VEGF， ALP， and RUNX-2 mRNA in the ADSCs in experimental group were increased （P<0.05），and the expression level of COL-Ⅹ mRNA was decreased （P<0.05）. Compared with 7 d of chodrogenic induction， the expression levels of VEGF， ALP， and RUNX-2 mRNA in the ADSCs in control group at 14 d of chodrogenic induction were decreased （P<0.05）， and the expression levels of COL-Ⅱ and COL-Ⅹ mRNA were increased （P<0.05）. Compared with 7 d of chondrogenic induction， the expression levels of COL-Ⅱ and COL-Ⅹ mRNA in the ADSCs in experimental group were increased at 14 d of chondrogenic induction （P<0.05）， while the expression levels of VEGF， ALP， and RUNX-2 mRNA were decreased（P<0.05）.

Conclusion

The rabbit ACM lack immunogenicity and have good biocompatibility and chondrogenic induction ability， which is beneficial to the occurrence of endochondral osteogenesis.

Key words: Adipose tissue-derived stem cells, Acellular cartilage matrix particles, Bone regeneration, Biomaterial

中图分类号:

Q813.1

边东潇,包幸福,胡敏. 兔脱细胞软骨基质颗粒复合SD大鼠脂肪干细胞对软骨内成骨的促进作用[J]. 吉林大学学报(医学版), 2022, 48(4): 883-891.

Dongxiao BIAN,Xingfu BAO,Min HU. Promotion effect of rabbit acellular cartilage matrix particles combined with SD rat adipose tissue-derived stem cells on endochondral osteogenesis[J]. Journal of Jilin University(Medicine Edition), 2022, 48(4): 883-891.

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参考文献 24

1	PARK C， KIM K H， LEE Y M， et al. Advanced engineering strategies for periodontal complex regeneration［J］. Materials， 2016， 9（1）： 57.
2	FARRELL E， BOTH S K， ODÖRFER K I， et al. In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells［J］. BMC Musculoskelet Disord， 2011， 12： 31.
3	PAPADIMITROPOULOS A， SCOTTI C， BOURGINE P，et al. Engineered decellularized matrices to instruct bone regeneration processes［J］. Bone， 2015， 70： 66-72.
4	MAES C. Role and regulation of vascularization processes in endochondral bones［J］. Calcif Tissue Int， 2013， 92（4）： 307-323.
5	STAMNITZ S， KLIMCZAK A. Mesenchymal stem cells， bioactive factors， and scaffolds in bone repair： from research perspectives to clinical practice［J］. Cells， 2021， 10（8）： 1925.
6	GAWLITTA D， BENDERS K E， VISSER J， et al. Decellularized cartilage-derived matrix as substrate for endochondral bone regeneration［J］. Tissue Eng Part A， 2015， 21（3/4）： 694-703.
7	KANG H J， LU S B， PENG J， et al. In vivo construction of tissue-engineered cartilage using adipose-derived stem cells and bioreactor technology［J］. Cell Tissue Bank， 2015， 16（1）： 123-133.
8	HUANG Y X， YU X F， HE L J， et al. 3D porous acellular cartilage matrix scaffold with surface mediated sustainable release of TGF-β3 for cartilage engineering［J］.Chin Chem Lett，2020，31（7）：1797-1800.
9	BAHNEY C S， HU D P， TAYLOR A J， et al. Stem cell-derived endochondral cartilage stimulates bone healing by tissue transformation［J］. J Bone Miner Res， 2014， 29（5）： 1269-1282.
10	SUGAWARA A， SATO S. Application of dedifferentiated fat cells for periodontal tissue regeneration［J］. Hum Cell， 2014， 27（1）： 12-21.
11	YANG Q， PENG J， GUO Q Y， et al. A cartilage ECM-derived 3-D porous acellular matrix scaffold for in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells［J］. Biomaterials， 2008， 29（15）： 2378-2387.
12	FENG X K， LI Z Y， WEI J H， et al. Injectable cartilaginous template transformed BMSCs into vascularized bone［J］. Sci Rep， 2018， 8（1）： 8244.
13	BENDERS K E M， RVAN WEEREN P， BADYLAK S F，et al. Extracellular matrix scaffolds for cartilage and bone regeneration［J］. Trends Biotechnol， 2013， 31（3）： 169-176.
14	BENDERS K E， BOOT W， COKELAERE S M，et al. Multipotent stromal cells outperform chondrocytes on cartilage-derived matrix scaffolds［J］.Cartilage， 2014，5（4）： 221-230.
15	HORIKOSHI S， KAJIYA M， MOTOIKE S， et al. Clumps of mesenchymal stem cells/extracellular matrix complexes generated with xeno-free chondro-inductive medium induce bone regeneration via endochondral ossification［J］. Biomedicines， 2021， 9（10）： 1408.
16	FARRELL E， O PVAN DER JAGT， KOEVOET W， et al. Chondrogenic priming of human bone marrow stromal cells： a better route to bone repair？［J］. Tissue Eng Part C Methods， 2009， 15（2）： 285-295.
17	杨艳兰徐普.干细胞修复牙槽骨缺损：微环境认知新进展［J］. 解放军医学杂志， 2021，46（4）： 404-409.
18	ZHAI C J， ZHANG X， CHEN J， et al. The effect of cartilage extracellular matrix particle size on the chondrogenic differentiation of bone marrow mesenchymal stem cells［J］. Regen Med， 2019， 14（7）： 663-680.
19	HERRMANN M， VERRIER S， ALINI M. Strategies to stimulate mobilization and homing of endogenous stem and progenitor cells for bone tissue repair［J］. Front Bioeng Biotechnol， 2015， 3： 79.
20	JEYAKUMAR V， AMRAISH N， NICULESCU-MORSZA E， et al. Decellularized cartilage extracellular matrix incorporated silk fibroin hybrid scaffolds for endochondral ossification mediated bone regeneration［J］. Int J Mol Sci， 2021， 22（8）： 4055.
21	AANEI C M， FLANDRIN P， ELOAE F Z， et al. Intrinsic growth deficiencies of mesenchymal stromal cells in myelodysplastic syndromes［J］. Stem Cells Dev， 2012， 21（10）： 1604-1615.
22	DONG J， ZHU G， WANG T C， et al. Ginsenoside Rg1 promotes neural differentiation of mouse adipose-derived stem cells via the miRNA-124 signaling pathway［J］.J Zhejiang Univ Sci B，2017，18（5）：445-448.
23	WANG Z J， AN R Z， ZHAO J Y， et al. Repair of articular cartilage defects by tissue-engineered cartilage constructed with adipose-derived stem cells and acellular cartilaginous matrix in rabbits［J］. Genet Mol Res， 2014， 13（2）： 4599-4606.
24	BORDBAR S， LOTFI BAKHSHAIESH N， KHANMOHAMMADI M， et al. Production and evaluation of decellularized extracellular matrix hydrogel for cartilage regeneration derived from knee cartilage［J］. J Biomed Mater Res A， 2020， 108（4）： 938-946.

Primer	Sequence(5'－3')
GAPDH	F：GCTCTCTGCTCCTCCCTGTTGTA R：GGCCAAATCCGTTCACACCG
COL-Ⅱ	F：AGAACTGGTGGAGCAGCACGA R：ATCTGGACGTTAGCGGTGTTG
SOX-9	F：ACAAGAAAGACCACCCCGATTA R：GTTAGGAGAGATGTGAGTCTGTTCG
COL-Ⅹ	F：CCATGGTTCACACAACCCCTT R：TGGCTGTGGTAAAGCACCTTG
VEGF	F：AAACCTCACCAAAGCCAGCACATAG R：AACATTTACACGTCTGCGGATCTTG
ALP	F：GGAGACGTCATCCTGGCTATC R：CGAGTTTCTGCCCTGTCAATC
RUNX-2	F：GGTACTTCGTCAGCGTCCTATC R：CATCAGCGTCAACACCATCA

Group	COL-Ⅱ mRNA	SOX-9 mRNA	COL-Ⅹ mRNA	VEGF mRNA	ALP mRNA	RUNX-2 mRNA
Control
7 d	1.00±0.07	1.00±0.11	1.01±0.12	0.02±0.01	1.00±0.09	1.00±0.04
14 d	2.05±0.19^△	0.77±0.32	1.55±0.09^△	0.01±0.01^△	0.10±0.01^△	0.13±0.01^△
Experimental
7 d	1.16±0.24	1.23±0.25	0.86±0.03	0.13±0.02^*	4.69±0.09^*	7.10±0.27^*△
14 d	2.59±0.07^△	0.93±0.12	1.16±0.08^*△	0.04±0.01^*△	1.75±0.11^*△	2.10±0.21^*△

[1]	谭紫凝,甄昱,李珊山. 干细胞与白癜风发生发展的关系及其对白癜风治疗作用的研究进展Research progress in relationship between stem cells and occurrence and development of vitiligo and its therapeutic effect on vitiligo[J]. 吉林大学学报(医学版), 2022, 48(1): 256-264.
[2]	赵竹兰,张庆宇,夏德庚,仲杨,张天翼,黄玉,张莉,马宁. 重度侵袭性牙周炎正畸种植修复联合治疗的临床修复效果1例报告及文献复习[J]. 吉林大学学报(医学版), 2021, 47(5): 1292-1297.
[3]	黄玉, 赵竹兰, 仲杨, 许立硕, 刘晨光, 马宁, 张莉. 重度慢性牙周炎患者软硬组织增量术联合即刻种植修复1例报告及文献复习[J]. 吉林大学学报(医学版), 2020, 46(05): 1082-1086.
[4]	孔维健, 方红娟, 潘肃, 杨利丽, 郑爽, 齐治平, 付川, 钟磊. 搭载紫杉醇脂质体的聚乳酸-羟基乙酸共聚物静电纺丝膜对神经干细胞增殖分化的影响[J]. 吉林大学学报(医学版), 2020, 46(03): 509-514.
[5]	焦鹏, 陈飞, 金权, 陈楠楠, 张莉, 马宁. 重度牙周炎致牙齿松动脱落患者前牙美学区综合治疗1例报告及文献复习[J]. 吉林大学学报(医学版), 2018, 44(02): 421-424.
[6]	田大川, 李海乐, 肖大伟, 周山健, 苏永蔚, 刘丹平, 綦惠. 联合应用软骨再生支架与突变型HIF-1α修饰BMSCs分泌的外泌体对晚期软骨缺损修复的促进作用[J]. 吉林大学学报(医学版), 2018, 44(02): 216-222.
[7]	陈飞, 史金先, 焦鹏, 金权, 许立硕, 张莉, 马宁. 不良修复体致前牙区残根残冠患者的牙周手术治疗1例报告及文献复习[J]. 吉林大学学报(医学版), 2018, 44(01): 170-174.
[8]	孙宏晨，朱阳，姜力铭，李道伟，王丹丹，唐宇欣. 在骨缺损修复中生物材料引起的免疫反应对骨微环境影响的研究进展[J]. 吉林大学学报(医学版), 2013, 39(5): 1063-1066.
[9]	韩超，廖怀伟，刘丽忠，简雪平. 同种异体富血小板血浆提取液对脂肪干细胞的体外促增殖作用[J]. 吉林大学学报(医学版), 2013, 39(2): 259-263.
[10]	平飞云\|苗卓伟\|王新木\|董研. 壳聚糖在引导骨再生膜中的应用[J]. J4, 2012, 38(4): 813-816.
[11]	高心，何钟勤，王成坤，张志民，王志刚，钟丞，欧阳喈. 骨缺损修复材料煅烧牛骨粉的理化特性[J]. J4, 2009, 35(1): 138-141.
[12]	吴哲，刘畅，郭雷，陈远，孙宏晨. 聚乳酸-聚羟基乙酸植入大鼠拔牙创对骨愈合的影响[J]. J4, 2007, 33(2): 234-236.