A-PRF促进兔膝关节骨软骨损伤愈合的观察

doi:10.3969/j.issn.1674-8115.2024.01.002

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (1): 13-22.doi: 10.3969/j.issn.1674-8115.2024.01.002

A-PRF促进兔膝关节骨软骨损伤愈合的观察

朱泽宇(), 吕成奇, 刘旭凌, 陈昱璐, 邹德荣, 陆家瑜()

上海交通大学医学院附属第六人民医院口腔科，上海 200233

收稿日期:2023-05-30 接受日期:2023-09-12 出版日期:2024-01-28 发布日期:2024-02-28
通讯作者: 陆家瑜 E-mail:2929441470@sjtu.edu.cn;angelinelu@sjtu.edu.cn
作者简介:朱泽宇（1998—），男，硕士生；电子信箱：2929441470@sjtu.edu.cn。
基金资助:
上海交通大学医学院“双百人”项目(20191832);国家自然科学基金(82071160)

Observation on A-PRF promoting regeneration of osteochondral defects in rabbit knee joints

ZHU Zeyu(), LÜ Chengqi, LIU Xuling, CHEN Yulu, ZOU Derong, LU Jiayu()

Department of Stomatology, Shanghai Sixth People′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200233, China

Received:2023-05-30 Accepted:2023-09-12 Online:2024-01-28 Published:2024-02-28
Contact: LU Jiayu E-mail:2929441470@sjtu.edu.cn;angelinelu@sjtu.edu.cn
Supported by:
"Two-hundred Talents" Program of Shanghai Jiao Tong University School of Medicine(20191832);National Natural Science Foundation of China(82071160)

摘要/Abstract

摘要：

目的·探讨改良型富血小板纤维蛋白（advanced platelet-rich fibrin，A-PRF）在骨软骨再生中的作用。方法·获取新西兰兔骨髓间充质干细胞（bone-marrow mesenchymal stem cells，BMSCs）和膝关节软骨细胞；通过低速离心兔心脏血液获得A-PRF。采用光学显微镜观察A-PRF的组织学结构；ELISA法检测A-PRF中生长因子，包括血小板衍生生长因子、转化生长因子-β、胰岛素样生长因子、血管内皮生长因子、表皮生长因子和成纤维细胞生长因子的释放；采用活/死细胞双染法及MTT法检测A-PRF对兔BMSCs细胞毒性及增殖情况的影响；采用实时荧光定量聚合酶链反应（qRT-PCR）检测A-PRF对兔BMSCs Ⅱ型胶原蛋白、聚集蛋白聚糖、碱性磷酸酶（ALP）和骨钙素（OCN）基因表达的影响；使用transwell小室测定A-PRF对于兔BMSCs以及软骨细胞迁移能力的影响。建立兔膝关节骨软骨缺损模型，将18只兔随机分为3组：A-PRF组（n=6）在缺损处植入A-PRF；A-PRF+BMSCs组（n=6）植入接种兔BMSCs的A-PRF；对照组（n=6）不进行植入操作。术后12周处死兔，采用苏木精-伊红（H-E）、甲苯胺蓝和番红O-固绿染色进行膝关节标本的组织学观察，并根据膝关节的表面形态学与组织学情况，采用国际软骨修复协会（International Cartilage Repair Society，ICRS）评分系统进行宏观与组织学评分。结果·A-PRF具有松散的网络结构，可以缓慢释放生长因子。加入A-PRF后，未观察到其对兔BMSCs具有细胞毒性；在加入A-PRF后24、48和72 h，BMSCs的增殖能力均明显升高（均P<0.05），成软骨相关基因Ⅱ型胶原蛋白、聚集蛋白聚糖，以及成骨相关基因ALP和OCN均显著上调（均P<0.05）。加入A-PRF后，兔BMSCs与软骨细胞的迁移能力均显著增强（均P<0.05），且兔BMSCs的迁移能力显著高于软骨细胞（P=0.025）。在兔膝关节缺损模型中，观察关节表面形态，可见A-PRF组和A-PRF+BMSCs组缺损均基本恢复，而对照组仅有软组织覆盖。在ICRS宏观评分方面，A-PRF组与A-PRF+BMSCs组的差异无统计学意义，但2组评分均显著高于对照组（均P<0.05）。组织学观察显示，A-PRF组和A-PRF+BMSCs组均产生骨软骨修复，但A-PRF组软骨更加成熟，对照组则形成纤维修复。在ICRS组织学评分方面，A-PRF组与A-PRF+BMSCs组的差异无统计学意义，但2组评分均显著高于对照组（均P<0.05）。结论·自体A-PRF具有良好的生物相容性和促进BMSCs增殖的能力，在体外和体内均可促进软骨和软骨下骨的修复。

关键词: 骨软骨损伤, 改良型富血小板纤维蛋白, 生长因子, 硬组织再生

Abstract:

Objective ·To explore the role of advanced platelet-rich fibrin (A-PRF) in osteochondral regeneration. Methods ·Bone-marrow mesenchymal stem cells (BMSCs) and knee joint chondrocytes were obtained from New Zealand rabbits. A-PRF was obtained by low-speed centrifugation of the heart blood of rabbits. The histological structure of A-PRF was observed by an optical microscope. The release of growth factors in A-PRF was detected by ELISA, including platelet-derived growth factor, transforming growth factor-β, insulin-like growth factor, vascular endothelial growth factor, epidermal growth factor and fibroblast growth factor. A-PRF's cytotoxicity and capability for promoting the proliferation of rabbit BMSCs were detected by live/dead double staining and MTT methods. The effect of A-PRF on the gene expression of type Ⅱ collagen, aggrecan, alkaline phosphatase (ALP) and osteocalcin (OCN) in rabbit BMSCs was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Transwell chambers were used to determine the effect of A-PRF on the migration ability of rabbit BMSCs and the chondrocytes. Rabbit knee osteochondral defect models were established, and 18 rabbits were randomly divided into 3 groups. The A-PRF group (n=6) was implanted with A-PRF in the defect, the A-PRF+BMSCs group (n=6) was implanted with rabbit BMSCs on A-PRF, and the control group (n=6) did not undergo implantation. The rabbits were sacrificed 12 weeks after surgery and the knee joint specimens were stained with hematoxylin-eosin (H-E), toluidine blue and safranin O/fast green. Based on the surface morphology and histology of the knee joints, the International Cartilage Repair Society (ICRS) scoring system was used for macroscopic and histological scoring. Results ·A-PRF had a loose network structure and can slowly release growth factors. No cytotoxicity to rabbit BMSCs was observed after adding A-PRF, and the the capability for promoting the proliferation of rabbit BMSCs was significantly increased at 24, 48 and 72 h after adding A-PRF (all P<0.05). Chondrogenesis-related gene Ⅱ collagen and aggrecan, as well as osteogenesis-related genes ALP and OCN were significantly up-regulated (all P<0.05). After adding A-PRF, the migration abilities of rabbit BMSCs and chondrocytes were significantly enhanced (both P<0.05), and the migration ability of rabbit BMSCs was significantly higher than that of chondrocytes (P=0.025). The joint surface morphology in the rabbit knee joint defect models was observed. It can be seen that the defects in the A-PRF group and the A-PRF+BMSCs group were basically restored, while the the defects in the control group were only covered by soft tissue. In the ICRS macroscopic score, there was no statistical difference between the A-PRF group and the A-PRF+BMSCs group, but the scores of the two groups were all significantly higher than those of the control group (all P<0.05). According to the histological results, both the A-PRF group and the A-PRF+BMSCs group formed osteochondral repair, but the cartilage in the A-PRF group was more mature, while the control group formed fibrous repair. In the ICRS histological score, there was no statistical difference between the A-PRF group and the A-PRF+BMSCs group, but the scores of both the groups were significantly higher than those of the control group (both P<0.05). Conclusion ·Autologous A-PRF has good biocompatibility and the capability for promoting the proliferation of BMSCs. It can promote the repair of cartilage and subchondral bone both in vitro and in vivo.

Key words: osteochondral defect, advanced platelet-rich fibrin (A-PRF), growth factor, hard tissue regeneration

中图分类号:

R681.3

朱泽宇, 吕成奇, 刘旭凌, 陈昱璐, 邹德荣, 陆家瑜. A-PRF促进兔膝关节骨软骨损伤愈合的观察[J]. 上海交通大学学报（医学版）, 2024, 44(1): 13-22.

ZHU Zeyu, LÜ Chengqi, LIU Xuling, CHEN Yulu, ZOU Derong, LU Jiayu. Observation on A-PRF promoting regeneration of osteochondral defects in rabbit knee joints[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(1): 13-22.

图/表 8

表1 qRT-PCR引物

Tab 1 Primer sequences for qRT-PCR

Gene	Sequence
Gene	Forward (5′→3′)	Reverse (5′→3′)
Type Ⅱ collagen	TCCTGTGCGACGACATAAT	CCTTTGGTCCTGGTTTCC
Aggrecan	CCCGAGAATCAAATGG	TAGTTGGGCAGCGAGA
ALP	CGTGGCAACTCCATCTT	AGGGTTTCTTGTCCGTGT
OCN	ACTCTTGTCGCCCTGCTG	TCGCTGCCCTCCCTCT

图1 A-PRF组织结构的光镜观察及生长因子释放Note： A. H-E staining of A-PRF. Sparse fibrous reticular structures were shown, which trapped white blood cells (red circles) and platelets (blue circles). Scale bar=50 μm. B. Sustained release of growth factors of A-PRF in PBS.

Fig 1 Observation of tissue structure under light microscope and growth factor release of A-PRF

图2 A-PRF的细胞毒性与促增殖能力检测Note： A. Live/dead double staining. Scale bar=100 μm. B.The results of proliferative capacity test. ①P=0.015, ②P=0.000, ③P=0.005, compared with the control group.

Fig 2 Cytotoxicity and proliferative capacity of A-PRF

图3 经A-PRF处理后兔BMSCs体外成软骨与成骨基因表达分析(n=3)Note： ①P=0.032, ②P=0.000, ③P=0.007, compared with the control group.

Fig 3 Gene expression analysis of in vitro chondrogenesis and osteogenesis of rabbit BMSCs treated by A-PRF (n=3)

图4 A-PRF对兔BMSCs与软骨细胞迁移能力的影响Note： ①P=0.962, ②P=0.025, ③P=0.006, ④P=0.000.

Fig 4 Effect of A-PRF on the migration ability of rabbit BMSCs and chondrocytes

图5 膝关节标本的大体观察

Fig 5 General observation of knee joint specimens

图6 A-PRF和 BMSCs在骨软骨修复体内实验中的定量评估(n=6)Note： A. ICRS macroscopic score. B. ICRS histological score. ①P=0.003, ②P=0.011, ③P=0.002, ④P=0.019, compared with the control group. ⑤P=0.767, ⑥P=0.534, compared with the A-PRF+BMSCs group.

Fig 6 In vivo-quantitative assessment of A-PRF and BMSCs in osteochondral repair(n=6)

图7 膝关节标本的组织学观察Note： A. Scale bar=1 mm. B. Scale bar=50 μm.

Fig 7 Histological observation of knee joint specimens

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A-PRF促进兔膝关节骨软骨损伤愈合的观察

Observation on A-PRF promoting regeneration of osteochondral defects in rabbit knee joints

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