Effect of Kartogenin combined with adipose-derived stem cells on tendon-bone healing after anterior cruciate ligament reconstruction_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHEN Gang , ZHENG Qian , LIU Mengfei , HE Haiyang ,  JU Xiaochen , JIANG Kan

Department of Joint Surgery, the Sixth Affiliated Hospital of Xinjiang Medical University, Urumqi Xinjiang, 830000, P. R. China;

Corresponding?author：

JU Xiaochen, Email: 31841528@qq.com

Keywords：

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

DOI：

10.7507/1002-1892.202305011

Video：

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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.

Citation： CHEN Gang, ZHENG Qian, LIU Mengfei, HE Haiyang, JU Xiaochen, JIANG Kan. Effect of Kartogenin combined with adipose-derived stem cells on tendon-bone healing after anterior cruciate ligament reconstruction. Chinese Journal of Reparative and Reconstructive Surgery, 2023, 37(8): 1002-1010. doi: 10.7507/1002-1892.202305011 Copy

1.	湯義民, 陶鈞, 董斌, 等. 關節鏡下前交叉韌帶重建術的手術時機對患者膝關節功能恢復的影響. 現代生物醫學進展, 2019, 19(4): 755-758, 788.
2.	Matsumoto T, Takayama K, Hayashi S, et al. Therapeutic potential of vascular stem cells for anterior cruciate ligament reconstruction. Ann Transl Med, 2019, 7(Suppl 8): S286.
3.	Tajima T, Yamaguchi N, Nagasawa M, et al. Early weight-bearing after anterior cruciate ligament reconstruction with hamstring grafts induce femoral bone tunnel enlargement: a prospective clinical and radiographic study. BMC Musculoskelet Disord, 2019, 20(1): 274.
4.	Dini F, Tecame A, Ampollini A, et al. Multiple ACL revision: Failure analysis and clinical outcomes. J Knee Surg, 2021, 34(8): 801-809.
5.	張培, 劉泉, 吳敏, 等. TGF-β₁、bFGF對自體腘繩肌腱重建前交叉韌帶腱-骨愈合的影響. 中華全科醫學, 2020, 18(1): 30-33.
6.	魏鈺, 運行, 李眾利, 等. 前交叉韌帶重建失敗的原因分析. 中國矯形外科雜志, 2021, 29(16): 1461-1465.
7.	孫暢, 郭亭, 李林濤, 等. 前交叉韌帶重建術后腱骨愈合的生物學干預研究進展. 中華骨與關節外科雜志, 2020, 13(10): 863-869.
8.	辜劉偉, 周霖, 庹偉, 等. 促進膝關節前交叉韌帶重建術后腱骨愈合方法的研究進展. 中國現代醫學雜志, 2021, 31(23): 49-56.
9.	Feng W, Jin Q, Ming-Yu Y, et al. MiR-6924-5p-rich exosomes derived from genetically modified Scleraxis-overexpressing PDGFRα(+) BMMSCs as novel nanotherapeutics for treating osteolysis during tendon-bone healing and improving healing strength. Biomaterials, 2021, 279: 121242.
10.	熊波涵, 余洋, 盧曉君, 等. 促進前交叉韌帶重建腱骨愈合的研究與進展. 中國組織工程研究, 2023, 27(5): 779-786.
11.	Mostafa S, Pakvasa M, Coalson E, et al. The wonders of BMP9: From mesenchymal stem cell differentiation, angiogenesis, neurogenesis, tumorigenesis, and metabolism to regenerative medicine. Genes Dis, 2019, 6(3): 201-223.
12.	Ma XF, Ma XB, Qian WJ, et al. Co-culture of adipose-derived stem cells and chondrocytes with transforming growth factor-beta 3 promotes chondrogenic differentiation. J Craniofac Surg, 2020, 31(8): 2355-2359.
13.	朱亮, 林享玉, 何晶. 脂肪干細胞在皮膚損傷再生及組織工程中的應用及進展. 解剖學雜志, 2021, 44(5): 433-437.
14.	張棟鑫, 肖麗玲. 脂肪干細胞聯合水凝膠材料在組織工程中的研究進展及前景. 國際生物醫學工程雜志, 2021, 44(4): 323-328.
15.	李慶標, 羅惠慈, 戴冠東. Kartogenin對肩袖止點腱骨愈合的干預作用及機制研究. 心電圖雜志(電子版), 2017, 6(2): 179-182.
16.	Kosaka M, Nakase J, Hayashi K, et al. Adipose-derived regenerative cells promote tendon-bone healing in a rabbit model. Arthroscopy, 2016, 32(5): 851-859.
17.	徐文悅, 趙娜, 王燕, 等. 脂肪干細胞用于肌腱損傷修復研究進展. 動物醫學進展, 2023, 44(3): 99-103.
18.	丁偉, 譚洪波. KGN修復軟骨、促進腱-骨愈合作用的研究進展. 山東醫藥, 2017, 57(16): 102-104.
19.	Zhang J, Wang JH. Kartogenin induces cartilage-like tissue formation in tendon-bone junction. Bone Res, 2014, 2: 14008.
20.	楊德勇, 凱沙爾·百合提亞爾, 姜鈞耀, 等. 局部注射肝細胞生長因子基因修飾的骨髓間充質干細胞對兔前交叉韌帶重建后腱骨愈合的影響. 中國醫藥導報, 2022, 19(35): 28-31.
21.	馬子越, 巨嘯晨, 張磊, 等. 保留與不保留殘端重建前交叉韌帶后移植物腱骨愈合的比較. 中國組織工程研究, 2021, 25(4): 582-587.
22.	舒莉, 柴浩, 巨嘯晨, 等. 鞘內保殘重建術與牽張保殘重建術對兔前交叉韌帶殘端成纖維細胞增殖、凋亡的影響. 山東醫藥, 2019, 59(13): 35-38.
23.	Oh JH, Chung SW, Kim SH, et al. 2013 Neer Award: Effect of the adipose-derived stem cell for the improvement of fatty degeneration and rotator cuff healing in rabbit model. J Shoulder Elbow Surg, 2014, 23(4): 445-455.
24.	賴文濤, 張曉璐, 張春陽, 等. 脂肪干細胞轉染Bmp2后成骨能力變化的研究. 生物骨科材料與臨床研究, 2022, 19(1): 15-19.
25.	張詩晨, 金麗鷗, 李躍, 等. kartogenin對骨關節炎的治療作用機制及其在軟骨組織工程學中應用的研究進展. 吉林大學學報 (醫學版), 2021, 47(2): 519-527.
26.	Ren E, Chen H, Qin Z, et al. Harnessing bifunctional ferritin with Kartogenin loading for mesenchymal stem cell capture and enhancing chondrogenesis in cartilage regeneration. Adv Healthc Mater, 2022, 11(8): e2101715.
27.	Xu X, Shi D, Shen Y, et al. Full-thickness cartilage defects are repaired via a microfracture technique and intraarticular injection of the small-molecule compound kartogenin. Arthritis Res Ther, 2015, 17(1): 20.
28.	Advancements in the treatment options for shoulder problems [Z/OL]. [2023-07-26]. https: //biospectrumindia.com/ article/pdf/21372.
29.	Wang R, Xu B, Xu HG. Up-regulation of TGF-β promotes tendon-to-bone healing after anterior cruciate ligament reconstruction using bone marrow-derived mesenchymal stem cells through the TGF-β/MAPK signaling pathway in a New Zealand white rabbit model. Cell Physiol Biochem, 2017, 41(1): 213-226.
30.	Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res, 2007, 100(9): 1249-1260.
31.	Valencia Mora M, Antu?a Antu?a S, García Arranz M, et al. Application of adipose tissue-derived stem cells in a rat rotator cuff repair model. Injury, 2014, 45 Suppl 4: S22-S27.
32.	Skutek M, van Griensven M, Zeichen J, et al. Cyclic mechanical stretching of human patellar tendon fibroblasts: activation of JNK and modulation of apoptosis. Knee Surg Sports Traumatol Arthrosc, 2003, 11(2): 122-129.

1. 湯義民, 陶鈞, 董斌, 等. 關節鏡下前交叉韌帶重建術的手術時機對患者膝關節功能恢復的影響. 現代生物醫學進展, 2019, 19(4): 755-758, 788.
2. Matsumoto T, Takayama K, Hayashi S, et al. Therapeutic potential of vascular stem cells for anterior cruciate ligament reconstruction. Ann Transl Med, 2019, 7(Suppl 8): S286.
3. Tajima T, Yamaguchi N, Nagasawa M, et al. Early weight-bearing after anterior cruciate ligament reconstruction with hamstring grafts induce femoral bone tunnel enlargement: a prospective clinical and radiographic study. BMC Musculoskelet Disord, 2019, 20(1): 274.
4. Dini F, Tecame A, Ampollini A, et al. Multiple ACL revision: Failure analysis and clinical outcomes. J Knee Surg, 2021, 34(8): 801-809.
5. 張培, 劉泉, 吳敏, 等. TGF-β₁、bFGF對自體腘繩肌腱重建前交叉韌帶腱-骨愈合的影響. 中華全科醫學, 2020, 18(1): 30-33.
6. 魏鈺, 運行, 李眾利, 等. 前交叉韌帶重建失敗的原因分析. 中國矯形外科雜志, 2021, 29(16): 1461-1465.
7. 孫暢, 郭亭, 李林濤, 等. 前交叉韌帶重建術后腱骨愈合的生物學干預研究進展. 中華骨與關節外科雜志, 2020, 13(10): 863-869.
8. 辜劉偉, 周霖, 庹偉, 等. 促進膝關節前交叉韌帶重建術后腱骨愈合方法的研究進展. 中國現代醫學雜志, 2021, 31(23): 49-56.
9. Feng W, Jin Q, Ming-Yu Y, et al. MiR-6924-5p-rich exosomes derived from genetically modified Scleraxis-overexpressing PDGFRα(+) BMMSCs as novel nanotherapeutics for treating osteolysis during tendon-bone healing and improving healing strength. Biomaterials, 2021, 279: 121242.
10. 熊波涵, 余洋, 盧曉君, 等. 促進前交叉韌帶重建腱骨愈合的研究與進展. 中國組織工程研究, 2023, 27(5): 779-786.
11. Mostafa S, Pakvasa M, Coalson E, et al. The wonders of BMP9: From mesenchymal stem cell differentiation, angiogenesis, neurogenesis, tumorigenesis, and metabolism to regenerative medicine. Genes Dis, 2019, 6(3): 201-223.
12. Ma XF, Ma XB, Qian WJ, et al. Co-culture of adipose-derived stem cells and chondrocytes with transforming growth factor-beta 3 promotes chondrogenic differentiation. J Craniofac Surg, 2020, 31(8): 2355-2359.
13. 朱亮, 林享玉, 何晶. 脂肪干細胞在皮膚損傷再生及組織工程中的應用及進展. 解剖學雜志, 2021, 44(5): 433-437.
14. 張棟鑫, 肖麗玲. 脂肪干細胞聯合水凝膠材料在組織工程中的研究進展及前景. 國際生物醫學工程雜志, 2021, 44(4): 323-328.
15. 李慶標, 羅惠慈, 戴冠東. Kartogenin對肩袖止點腱骨愈合的干預作用及機制研究. 心電圖雜志(電子版), 2017, 6(2): 179-182.
16. Kosaka M, Nakase J, Hayashi K, et al. Adipose-derived regenerative cells promote tendon-bone healing in a rabbit model. Arthroscopy, 2016, 32(5): 851-859.
17. 徐文悅, 趙娜, 王燕, 等. 脂肪干細胞用于肌腱損傷修復研究進展. 動物醫學進展, 2023, 44(3): 99-103.
18. 丁偉, 譚洪波. KGN修復軟骨、促進腱-骨愈合作用的研究進展. 山東醫藥, 2017, 57(16): 102-104.
19. Zhang J, Wang JH. Kartogenin induces cartilage-like tissue formation in tendon-bone junction. Bone Res, 2014, 2: 14008.
20. 楊德勇, 凱沙爾·百合提亞爾, 姜鈞耀, 等. 局部注射肝細胞生長因子基因修飾的骨髓間充質干細胞對兔前交叉韌帶重建后腱骨愈合的影響. 中國醫藥導報, 2022, 19(35): 28-31.
21. 馬子越, 巨嘯晨, 張磊, 等. 保留與不保留殘端重建前交叉韌帶后移植物腱骨愈合的比較. 中國組織工程研究, 2021, 25(4): 582-587.
22. 舒莉, 柴浩, 巨嘯晨, 等. 鞘內保殘重建術與牽張保殘重建術對兔前交叉韌帶殘端成纖維細胞增殖、凋亡的影響. 山東醫藥, 2019, 59(13): 35-38.
23. Oh JH, Chung SW, Kim SH, et al. 2013 Neer Award: Effect of the adipose-derived stem cell for the improvement of fatty degeneration and rotator cuff healing in rabbit model. J Shoulder Elbow Surg, 2014, 23(4): 445-455.
24. 賴文濤, 張曉璐, 張春陽, 等. 脂肪干細胞轉染Bmp2后成骨能力變化的研究. 生物骨科材料與臨床研究, 2022, 19(1): 15-19.
25. 張詩晨, 金麗鷗, 李躍, 等. kartogenin對骨關節炎的治療作用機制及其在軟骨組織工程學中應用的研究進展. 吉林大學學報 (醫學版), 2021, 47(2): 519-527.
26. Ren E, Chen H, Qin Z, et al. Harnessing bifunctional ferritin with Kartogenin loading for mesenchymal stem cell capture and enhancing chondrogenesis in cartilage regeneration. Adv Healthc Mater, 2022, 11(8): e2101715.
27. Xu X, Shi D, Shen Y, et al. Full-thickness cartilage defects are repaired via a microfracture technique and intraarticular injection of the small-molecule compound kartogenin. Arthritis Res Ther, 2015, 17(1): 20.
28. Advancements in the treatment options for shoulder problems [Z/OL]. [2023-07-26]. https: //biospectrumindia.com/ article/pdf/21372.
29. Wang R, Xu B, Xu HG. Up-regulation of TGF-β promotes tendon-to-bone healing after anterior cruciate ligament reconstruction using bone marrow-derived mesenchymal stem cells through the TGF-β/MAPK signaling pathway in a New Zealand white rabbit model. Cell Physiol Biochem, 2017, 41(1): 213-226.
30. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res, 2007, 100(9): 1249-1260.
31. Valencia Mora M, Antu?a Antu?a S, García Arranz M, et al. Application of adipose tissue-derived stem cells in a rat rotator cuff repair model. Injury, 2014, 45 Suppl 4: S22-S27.
32. Skutek M, van Griensven M, Zeichen J, et al. Cyclic mechanical stretching of human patellar tendon fibroblasts: activation of JNK and modulation of apoptosis. Knee Surg Sports Traumatol Arthrosc, 2003, 11(2): 122-129.

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

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