Research on Runx2 gene induced differentiation of human amniotic mesenchymal stem cells into ligament fibroblasts <i>in vitro</i> and promotion of tendon-bone healing in rabbits_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XIE Tao ,  ZHONG Hehe , JIN Ying , LIU Xiuqi , CHEN Fang , XIANG Kuan ,  WU Shuhong

Department of Orthopedics, Affiliated Hospital of Zunyi Medical University, Zunyi Guizhou, 563000, P. R. China;

Corresponding author：

ZHONG Hehe, Email: zhonghehe@126.com; WU Shuhong, Email: 15329112966@163.com

Keywords：

Runx2 gene; human amniotic mesenchymal stem cells; ligament fibroblasts; tenbon-bone healing; ligament tissue engineering

DOI：

10.7507/1002-1892.202306010

Video：

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Objective To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits. Methods hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. Results Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection (P<0.05), Fibronectin significantly increased at 3 days (P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days (P<0.05). CCK-8 detection showed that there was no significant difference (P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. Conclusion Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.

Citation： XIE Tao, ZHONG Hehe, JIN Ying, LIU Xiuqi, CHEN Fang, XIANG Kuan, WU Shuhong. Research on Runx2 gene induced differentiation of human amniotic mesenchymal stem cells into ligament fibroblasts in vitro and promotion of tendon-bone healing in rabbits. Chinese Journal of Reparative and Reconstructive Surgery, 2023, 37(12): 1523-1532. doi: 10.7507/1002-1892.202306010 Copy

1.	Beard DJ, Davies L, Cook JA, et al. Rehabilitation versus surgical reconstruction for non-acute anterior cruciate ligament injury (ACL SNNAP): a pragmatic randomised controlled trial. Lancet, 2022, 400(10352): 605-615.
2.	Yang C, Teng Y, Geng B, et al. Strategies for promoting tendon-bone healing: Current status and prospects. Front Bioeng Biotechnol, 2023, 11: 1118468.
3.	Cottrell JA, Turner JC, Arinzeh TL, et al. The biology of bone and ligament healing. Foot Ankle Clin, 2016, 21(4): 739-761.
4.	Rossetti L, Kuntz LA, Kunold E, et al. The microstructure and micromechanics of the tendon-bone insertion. Nat Mater, 2017, 16(6): 664-670.
5.	熊波涵, 余洋, 卢晓君, 等. 促进前交叉韧带重建腱骨愈合的研究与进展. 中国组织工程研究, 2023, 27(5): 779-786.
6.	Zhang C, Jiang C, Jin J, et al. Cartilage fragments combined with BMSCs-derived exosomes can promote tendon-bone healing after ACL reconstruction. Mater Today Bio, 2023, 23: 100819.
7.	Yao SY, Cao MD, He X, et al. Biological modulations to facilitate graft healing in anterior cruciate ligament reconstruction (ACLR), when and where to apply? A systematic review. J Orthop Translat, 2021, 30: 51-60.
8.	Yamahara K. Cell therapy using amnion-derived mesenchymal stem cells. Rinsho Ketsueki, 2022, 63(10): 1440-1445.
9.	Li Y, Liu Z, Jin Y, et al. Differentiation of human amniotic mesenchymal stem cells into human anterior cruciate ligament fibroblast cells by in vitro coculture. Biomed Res Int, 2017, 2017: 7360354.
10.	Abbasi-Kangevari M, Ghamari SH, Safaeinejad F, et al. Potential therapeutic features of human amniotic mesenchymal stem cells in multiple sclerosis: immunomodulation, inflammation suppression, angiogenesis promotion, oxidative stress inhibition, neurogenesis induction, MMPs regulation, and remyelination stimulation. Front Immunol, 2019, 10: 238.
11.	薛玲玲, 陈锦阳, 庄盼, 等. 人羊膜上皮细胞和人羊膜间充质干细胞的研究进展. 中华细胞与干细胞杂志(电子版), 2021, 11(3): 184-188.
12.	Liu DD, Zhang CY, Liu Y, et al. RUNX2 regulates osteoblast differentiation via the BMP4 signaling pathway. J Dent Res, 2022, 101(10): 1227-1237.
13.	Xu J, Li Z, Hou Y, et al. Potential mechanisms underlying the Runx2 induced osteogenesis of bone marrow mesenchymal stem cells. Am J Transl Res, 2015, 7(12): 2527-2535.
14.	Zhang J, Liu Z, Tang J, et al. Fibroblast growth factor 2-induced human amniotic mesenchymal stem cells combined with autologous platelet rich plasma augmented tendon-to-bone healing. J Orthop Translat, 2020, 24: 155-165.
15.	朱喜忠, 刘子铭, 刘毅, 等. 低氧诱导因子1α联合Scleraxis修饰人羊膜间充质干细胞促进腱骨愈合的体外实验. 中国组织工程研究, 2018, 22(1): 113-118.
16.	朱喜忠, 刘子铭, 吴术红, 等. 骨形态发生蛋白9基因修饰人羊膜间充质干细胞体外向韧带成纤维细胞分化研究. 中国运动医学杂志, 2018, 37(6): 496-502.
17.	Wang J, Xu J, Wang X, et al. Magnesium-pretreated periosteum for promoting bone-tendon healing after anterior cruciate ligament reconstruction. Biomaterials, 2021, 268: 120576.
18.	Özdemir E, Karaguven D, Turhan E, et al. Biological augmentation strategies in rotator cuff repair. Med Glas (Zenica), 2021, 18(1): 186-191.
19.	邹刚, 徐志, 刘子铭, 等. 人脱细胞羊膜支架促进Scleraxis修饰人羊膜间充质干细胞体外成韧带分化. 中国组织工程研究, 2021, 25(7): 1037-1044.
20.	Hojo H, Ohba S. Runt-related transcription factors and gene regulatory mechanisms in skeletal development and diseases. Curr Osteoporos Rep, 2023, 21(5): 485-492.
21.	Xu HJ, Liu XZ, Yang L, et al. Runx2 overexpression promotes bone repair of osteonecrosis of the femoral head (ONFH). Mol Biol Rep, 2023, 50(6): 4769-4779.
22.	Byers BA, Guldberg RE, Hutmacher DW, et al. Effects of Runx2 genetic engineering and in vitro maturation of tissue-engineered constructs on the repair of critical size bone defects. J Biomed Mater Res A, 2006, 76(3): 646-655.
23.	Hijlkema A, Roozenboom C, Mensink M, et al. The impact of nutrition on tendon health and tendinopathy: a systematic review. J Int Soc Sports Nutr, 2022, 19(1): 474-504.
24.	Giacomini F, Baião Barata D, Suk Rho H, et al. Microfluidically aligned collagen to maintain the phenotype of tenocytes in vitro. Adv Healthc Mater, 2023: e2303672.
25.	López De Padilla CM, Coenen MJ, Tovar A, et al. Picrosirius red staining: revisiting its application to the qualitative and quantitative assessment of collagen type Ⅰ and type Ⅲ in tendon. J Histochem Cytochem, 2021, 69(10): 633-643.
26.	Hasegawa M, Yoshida T, Sudo A. Tenascin-C in osteoarthritis and rheumatoid arthritis. Front Immunol, 2020, 11: 577015.

1. Beard DJ, Davies L, Cook JA, et al. Rehabilitation versus surgical reconstruction for non-acute anterior cruciate ligament injury (ACL SNNAP): a pragmatic randomised controlled trial. Lancet, 2022, 400(10352): 605-615.
2. Yang C, Teng Y, Geng B, et al. Strategies for promoting tendon-bone healing: Current status and prospects. Front Bioeng Biotechnol, 2023, 11: 1118468.
3. Cottrell JA, Turner JC, Arinzeh TL, et al. The biology of bone and ligament healing. Foot Ankle Clin, 2016, 21(4): 739-761.
4. Rossetti L, Kuntz LA, Kunold E, et al. The microstructure and micromechanics of the tendon-bone insertion. Nat Mater, 2017, 16(6): 664-670.
5. 熊波涵, 余洋, 卢晓君, 等. 促进前交叉韧带重建腱骨愈合的研究与进展. 中国组织工程研究, 2023, 27(5): 779-786.
6. Zhang C, Jiang C, Jin J, et al. Cartilage fragments combined with BMSCs-derived exosomes can promote tendon-bone healing after ACL reconstruction. Mater Today Bio, 2023, 23: 100819.
7. Yao SY, Cao MD, He X, et al. Biological modulations to facilitate graft healing in anterior cruciate ligament reconstruction (ACLR), when and where to apply? A systematic review. J Orthop Translat, 2021, 30: 51-60.
8. Yamahara K. Cell therapy using amnion-derived mesenchymal stem cells. Rinsho Ketsueki, 2022, 63(10): 1440-1445.
9. Li Y, Liu Z, Jin Y, et al. Differentiation of human amniotic mesenchymal stem cells into human anterior cruciate ligament fibroblast cells by in vitro coculture. Biomed Res Int, 2017, 2017: 7360354.
10. Abbasi-Kangevari M, Ghamari SH, Safaeinejad F, et al. Potential therapeutic features of human amniotic mesenchymal stem cells in multiple sclerosis: immunomodulation, inflammation suppression, angiogenesis promotion, oxidative stress inhibition, neurogenesis induction, MMPs regulation, and remyelination stimulation. Front Immunol, 2019, 10: 238.
11. 薛玲玲, 陈锦阳, 庄盼, 等. 人羊膜上皮细胞和人羊膜间充质干细胞的研究进展. 中华细胞与干细胞杂志(电子版), 2021, 11(3): 184-188.
12. Liu DD, Zhang CY, Liu Y, et al. RUNX2 regulates osteoblast differentiation via the BMP4 signaling pathway. J Dent Res, 2022, 101(10): 1227-1237.
13. Xu J, Li Z, Hou Y, et al. Potential mechanisms underlying the Runx2 induced osteogenesis of bone marrow mesenchymal stem cells. Am J Transl Res, 2015, 7(12): 2527-2535.
14. Zhang J, Liu Z, Tang J, et al. Fibroblast growth factor 2-induced human amniotic mesenchymal stem cells combined with autologous platelet rich plasma augmented tendon-to-bone healing. J Orthop Translat, 2020, 24: 155-165.
15. 朱喜忠, 刘子铭, 刘毅, 等. 低氧诱导因子1α联合Scleraxis修饰人羊膜间充质干细胞促进腱骨愈合的体外实验. 中国组织工程研究, 2018, 22(1): 113-118.
16. 朱喜忠, 刘子铭, 吴术红, 等. 骨形态发生蛋白9基因修饰人羊膜间充质干细胞体外向韧带成纤维细胞分化研究. 中国运动医学杂志, 2018, 37(6): 496-502.
17. Wang J, Xu J, Wang X, et al. Magnesium-pretreated periosteum for promoting bone-tendon healing after anterior cruciate ligament reconstruction. Biomaterials, 2021, 268: 120576.
18. Özdemir E, Karaguven D, Turhan E, et al. Biological augmentation strategies in rotator cuff repair. Med Glas (Zenica), 2021, 18(1): 186-191.
19. 邹刚, 徐志, 刘子铭, 等. 人脱细胞羊膜支架促进Scleraxis修饰人羊膜间充质干细胞体外成韧带分化. 中国组织工程研究, 2021, 25(7): 1037-1044.
20. Hojo H, Ohba S. Runt-related transcription factors and gene regulatory mechanisms in skeletal development and diseases. Curr Osteoporos Rep, 2023, 21(5): 485-492.
21. Xu HJ, Liu XZ, Yang L, et al. Runx2 overexpression promotes bone repair of osteonecrosis of the femoral head (ONFH). Mol Biol Rep, 2023, 50(6): 4769-4779.
22. Byers BA, Guldberg RE, Hutmacher DW, et al. Effects of Runx2 genetic engineering and in vitro maturation of tissue-engineered constructs on the repair of critical size bone defects. J Biomed Mater Res A, 2006, 76(3): 646-655.
23. Hijlkema A, Roozenboom C, Mensink M, et al. The impact of nutrition on tendon health and tendinopathy: a systematic review. J Int Soc Sports Nutr, 2022, 19(1): 474-504.
24. Giacomini F, Baião Barata D, Suk Rho H, et al. Microfluidically aligned collagen to maintain the phenotype of tenocytes in vitro. Adv Healthc Mater, 2023: e2303672.
25. López De Padilla CM, Coenen MJ, Tovar A, et al. Picrosirius red staining: revisiting its application to the qualitative and quantitative assessment of collagen type Ⅰ and type Ⅲ in tendon. J Histochem Cytochem, 2021, 69(10): 633-643.
26. Hasegawa M, Yoshida T, Sudo A. Tenascin-C in osteoarthritis and rheumatoid arthritis. Front Immunol, 2020, 11: 577015.

Chinese Journal of Reparative and Reconstructive Surgery

Research on Runx2 gene induced differentiation of human amniotic mesenchymal stem cells into ligament fibroblasts in vitro and promotion of tendon-bone healing in rabbits

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