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. 2023 Aug 15;37(8):1002-1010.
doi: 10.7507/1002-1892.202305011.

[Effect of Kartogenin combined with adipose-derived stem cells on tendon-bone healing after anterior cruciate ligament reconstruction]

[Article in Chinese]
Gang Chen  1 , Qian Zheng  1 , Mengfei Liu  1 , Haiyang He  1 , Xiaochen Ju  1 , Kan Jiang  1
Affiliations

[Effect of Kartogenin combined with adipose-derived stem cells on tendon-bone healing after anterior cruciate ligament reconstruction]

[Article in Chinese]
Gang Chen et al. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. .

Abstract
in English, Chinese

Objective: To investigate the effect of Kartogenin (KGN) combined with adipose-derived stem cells (ADSCs) on tendon-bone healing after anterior cruciate ligament (ACL) reconstruction in rabbits.

Methods: After the primary ADSCs were cultured by passaging, the 3rd generation cells were cultured with 10 μmol/L KGN solution for 72 hours. The supernatant of KGN-ADSCs was harvested and mixed with fibrin glue at a ratio of 1∶1; the 3rd generation ADSCs were mixed with fibrin glue as a control. Eighty adult New Zealand white rabbits were taken and randomly divided into 4 groups: saline group (group A), ADSCs group (group B), KGN-ADSCs group (group C), and sham-operated group (group D). After the ACL reconstruction model was prepared in groups A-C, the saline, the mixture of ADSCs and fibrin glue, and the mixture of supernatant of KGN-ADSCs and fibrin glue were injected into the tendon-bone interface and tendon gap, respectively. ACL was only exposed without other treatment in group D. The general conditions of the animals were observed after operation. At 6 and 12 weeks, the tendon-bone interface tissues and ACL specimens were taken and the tendon-bone healing was observed by HE staining, c-Jun N-terminal kinase (JNK) immunohistochemical staining, and TUNEL apoptosis assay. The fibroblasts were counted, and the positive expression rate of JNK protein and apoptosis index (AI) were measured. At the same time point, the tensile strength test was performed to measure the maximum load and the maximum tensile distance to observe the biomechanical properties.

Results: Twenty-eight rabbits were excluded from the study due to incision infection or death, and finally 12, 12, 12, and 16 rabbits in groups A-D were included in the study, respectively. After operation, the tendon-bone interface of groups A and B healed poorly, while group C healed well. At 6 and 12 weeks, the number of fibroblasts and positive expression rate of JNK protein in group C were significantly higher than those of groups A, B, and D (P<0.05). Compared with 6 weeks, the number of fibroblasts gradually decreased and the positive expression rate of JNK protein and AI decreased in group C at 12 weeks after operation, with significant differences (P<0.05). Biomechanical tests showed that the maximum loads at 6 and 12 weeks after operation in group C were higher than in groups A and B, but lower than those in group D, while the maximum tensile distance results were opposite, but the differences between groups were significant (P<0.05).

Conclusion: After ACL reconstruction, local injection of a mixture of KGN-ADSCs and fibrin glue can promote the tendon-bone healing and enhance the mechanical strength and tensile resistance of the tendon-bone interface.

目的: 探讨Kartogenin（KGN）联合脂肪干细胞（adipose-derived stem cells，ADSCs）对兔前交叉韧带（anterior cruciate ligament，ACL）重建术后腱骨愈合的影响。.

方法: 取原代ADSCs传代培养，将第3代细胞与10 μmol/L KGN溶液培养72 h后，取KGN-ADSCs上清液与纤维蛋白胶以1∶1比例混合备用；另取第3代ADSCs与纤维蛋白胶混合作为对照。取成年新西兰大白兔80只，随机分为4组，分别为生理盐水组（A组）、ADSCs组（B组）、KGN-ADSCs组（C组）、假手术组（D组）。A～C组制备ACL重建模型后，分别于腱骨界面和肌腱间隙中注射生理盐水、ADSCs与纤维蛋白胶混合液、KGN-ADSCs上清液与纤维蛋白胶混合液；D组仅暴露ACL，不作其他处理。术后观察动物一般情况，于6、12周取腱骨界面组织及ACL标本，通过HE染色、c-Jun氨基末端激酶（c-Jun N-terminal kinase，JNK）免疫组织化学染色、TUNEL细胞凋亡检测观察腱骨愈合情况，计数成纤维细胞，测算JNK蛋白阳性表达率及细胞凋亡指数（apotosis index，AI）；同时行抗拉力强度测试，测量最大载荷及最大拉伸距离，观察生物力学性能。.

结果: 术后28只动物因切口感染或死亡排除研究，最终A～D组分别12、12、12、16只纳入研究。术后A、B组腱骨界面愈合欠佳，C组愈合良好；6、12周时C组成纤维细胞计数、JNK蛋白表达率均高于A、B、D组，差异均有统计学意义（P<0.05）；与6周比较，C组术后12周成纤维细胞计数减少、AI及JNK蛋白表达率下降，差异均有统计学意义（P<0.05）。生物力学测试示，术后6、12周， C组最大载荷高于A、B组、但低于D组，而最大拉伸距离结果相反，但组间差异均有统计学意义（P<0.05）。.

结论: 兔ACL重建后局部注射KGN-ADSCs可促进腱骨愈合，增强腱骨界面机械强度和抗拉力性。.

Keywords: Kartogenin; adipose-derived stem cells; anterior cruciate ligament reconstruction; rabbit; tendon-bone healing.

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Conflict of interest statement

利益冲突 在课题研究和文章撰写过程中不存在利益冲突；经费支持没有影响文章观点和对研究数据客观结果的统计分析及其报道

Figures

图 1
图 1
Schematic diagram of the animal model preparation 动物模型制备示意图
图 2
图 2
HE staining observation of the tendon-bone interface in each group (×40) 各组腱骨界面HE染色观察（×40）
图 3
图 3
HE staining observation of ACL in each group (×40) 各组ACL HE染色观察（×40）
图 4
图 4
Apoptosis observation in each group (×40) 各组细胞凋亡观察（×40）
图 5
图 5
JNK immunohistochemical staining observation in each group (×10) 各组JNK免疫组织化学染色观察（×10）
See this image and copyright information in PMC

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