Study on anti-adhesion effect and mechanism of dynamic and static stress stimulation during early healing process of rat Achilles tendon injury_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WU Jiani ^1,2 , JIANG Yingzi ¹ , WANG Guanyu ¹ , WANG Liliao ² ,  BAO Jie ² ,  WANG Jun ¹

1. Department of Rehabilitation Medicine, Wuxi Ninth People’s Hospital, Wuxi Jiangsu, 214000, P. R. China;
2. School of Physical Education, Soochow University, Suzhou Jiangsu, 215000, P. R. China;

Corresponding author：

BAO Jie, Email: Baojie@suda.edu.cn; WANG Jun, Email: Wangjun2115@suda.edu.cn

Keywords：

Dynamic and static stress stimulation; Achilles tendon cells; rat; early healing stage; anti-adhesion

DOI：

10.7507/1002-1892.202405090

Video：

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Objective To investigate the anti-adhesive effect and underlying mechanism of dynamic and static stress stimulation on the early healing process of rat Achilles tendon injury. Methods Achilles tendon tissues of 15 male Sprague Dawley (SD) rats aged 4-6 weeks were isolated and cultured by enzyme digestion method. Rat Achilles tendon cells were treated with tumor necrosis factor α to construct the Achilles tendon injury cell model, and dynamic stress stimulation (dynamic group) and static stress stimulation (static group) were applied respectively, while the control group was not treated. Live/dead cell double staining was used to detect cell activity, ELISA assay was used to detect the expression of α smooth muscle actin (α-SMA), and real-time fluorescence quantitative PCR was used to detect the mRNA expression of collagen type Ⅰ (COL1A1), collagen type Ⅲ (COL3A1), and Scleraxis (SCX). Thirty male SD rats aged 4-6 weeks underwent Achilles tendon suture and were randomly divided into dynamic group (treated by dynamic stress stimulation), static group (treated by static stress stimulation), and control group (untreated), with 10 rats in each group. HE staining and scoring were performed to evaluate the healing of Achilles tendon at 8 days after operation. COL1A1 and COL3A1 protein expressions were detected by immunohistochemical staining, α-SMA and SCX protein expressions were detected by Western blot, and maximum tendon breaking force and tendon stiffness were detected by biomechanical stretching test. Results In vitro cell experiment, when compared to the static group, the number of living cells in the dynamic group was higher, the expression of α-SMA protein was decreased, the relative expression of COL3A1 mRNA was decreased, and the relative expression of SCX mRNA was increased, and the differences were all significant (P<0.05). In the in vivo animal experiment, when compared to the static group, the tendon healing in the dynamic group was better, the HE staining score was lower, the expression of COL1A1 protein was increased, the expression of COL3A1 protein was decreased, the relative expression of SCX protein was increased, the relative expression of α-SMA protein was decreased, and the tendon stiffness was increased, the differences were all significant (P<0.05). Conclusion Compared with static stress stimulation, the dynamic stress stimulation improves the fibrosis of the scar tissue of the rat Achilles tendon, promote the recovery of the biomechanical property of the Achilles tendon, and has obvious anti-adhesion effect.

Citation： WU Jiani, JIANG Yingzi, WANG Guanyu, WANG Liliao, BAO Jie, WANG Jun. Study on anti-adhesion effect and mechanism of dynamic and static stress stimulation during early healing process of rat Achilles tendon injury. Chinese Journal of Reparative and Reconstructive Surgery, 2024, 38(11): 1391-1398. doi: 10.7507/1002-1892.202405090 Copy

1.	Howell K, Chien C, Bell R, et al. Novel model of tendon regeneration reveals distinct cell mechanisms underlying regenerative and fibrotic tendon healing. Sci Rep, 2017, 7: 45238. doi: 10.1038/srep45238.
2.	Graham JG, Wang ML, Rivlin M, et al. Biologic and mechanical aspects of tendon fibrosis after injury and repair. Connect Tissue Res, 2019, 60(1): 10-20.
3.	Al Makhzoomi AK, Kirk TB, Allison GT. A multiscale study of morphological changes in tendons following repeated cyclic loading. J Biomech, 2021, 128: 110790. doi: 10.1016/j.jbiomech.2021.110790.
4.	Choi WJ, Park MS, Park KH, et al. Comparative analysis of gene expression in normal and degenerative human tendon cells: effects of cyclic strain. Foot Ankle Int, 2014, 35(10): 1045-1056.
5.	Herchenhan A, Dietrich-Zagonel F, Schjerling P, et al. Early growth response genes increases rapidly after mechanical overloading and unloading in tendon constructs. J Orthop Res, 2020, 38(1): 173-181.
6.	Zhang J, Wang JH. The effects of mechanical loading on tendons—an in vivo and in vitro model study. PLoS One, 2013, 8(8): e71740. doi: 10.1371/journal.pone.0071740.
7.	Huisman E, Lu A, McCormack RG, et al. Enhanced collagen type Ⅰ synthesis by human tenocytes subjected to periodic in vitro mechanical stimulation. BMC Musculoskelet Disord, 2014, 15: 386. doi: 10.1186/1471-2474-15-386.
8.	Khayyeri H, Hammerman M, Turunen MJ, et al. Diminishing effects of mechanical loading over time during rat Achilles tendon healing. PLoS One, 2020, 15(12): e0236681. doi: 10.1371/journal.pone.0236681.
9.	Longo UG, Franceschi F, Ruzzini L, et al. Characteristics at haematoxylin and eosin staining of ruptures of the long head of the biceps tendon. Br J Sports Med, 2009, 43(8): 603-607.
10.	Li S, Gong F, Zhou Z, et al. Combined verapamil-polydopamine nanoformulation inhibits adhesion formation in Achilles tendon injury using rat model. Int J Nanomedicine, 2023, 18: 115-126.
11.	Metzler SA, Waller SC, Warnock JN. Quantitative characterization of aortic valve endothelial cell viability and morphology in situ under cyclic stretch. Cardiovasc Eng Technol, 2019, 10(1): 173-180.
12.	Chen Z, Chen P, Zheng M, et al. Challenges and perspectives of tendon-derived cell therapy for tendinopathy: from bench to bedside. Stem Cell Res Ther, 2022, 13(1): 444. doi: 10.1186/s13287-022-03113-6.
13.	Hall C, Law JP, Reyat JS, et al. Chronic activation of human cardiac fibroblasts in vitro attenuates the reversibility of the myofibroblast phenotype. Sci Rep, 2023, 13(1): 12137. doi: 10.1038/s41598-023-39369-y.
14.	Nichols AEC, Muscat SN, Miller SE, et al. Impact of isolation method on cellular activation and presence of specific tendon cell subpopulations during in vitro culture. FASEB J, 2021, 35(7): e21733. doi: 10.1096/fj.202100405R.
15.	Best KT, Nichols AEC, Knapp E, et al. NF-κB activation persists into the remodeling phase of tendon healing and promotes myofibroblast survival. Sci Signal, 2020, 13(658): eabb7209. doi: 10.1126/scisignal.abb7209.
16.	Koga M, Kuramochi M, Karim MR, et al. Immunohistochemical characterization of myofibroblasts appearing in isoproterenol-induced rat myocardial fibrosis. J Vet Med Sci, 2019, 81(1): 127-133.
17.	Cui HS, Hong AR, Kim JB, et al. Extracorporeal shock wave terapy alters the expression of fibrosis-related molecules in fibroblast derived from human hypertrophic scar. Int J Mol Sci, 2018, 19(1): 124. doi: 10.3390/ijms19010124.
18.	Skalli O, Schürch W, Seemayer T, et al. Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins. Lab Invest, 1989, 60(2): 275-85.
19.	Zhou J, Zhao Y, Yang W, et al. Use of mechanical stretching to treat skin graft contracture. J Burn Care Res, 2020, 41(4): 892-899.
20.	Hortobagyi D, Grossmann T, Tschernitz M, et al. In vitro mechanical vibration down-regulates pro-inflammatory and pro-fibrotic signaling in human vocal fold fibroblasts. PLoS One, 2020, 15(11): e0241901. doi: 10.1371/journal.pone.0241901.
21.	Benage LG, Sweeney JD, Giers MB, et al. Dynamic load model systems of tendon inflammation and mechanobiology. Front Bioeng Biotechnol, 2022, 10: 896336. doi: 10.3389/fbioe.2022.896336.
22.	Gonçalves RM, Saggese T, Yong Z, et al. Interleukin-1β more than mechanical loading induces a degenerative phenotype in human annulus fibrosus cells, partially impaired by anti-proteolytic activity of mesenchymal stem cell secretome. Front Bioeng Biotechnol, 2022, 9: 802789. doi: 10.3389/fbioe.2021.802789.
23.	Min J, Li B, Liu C, et al. Extracellular matrix metabolism disorder induced by mechanical strain on human parametrial ligament fibroblasts. Mol Med Rep, 2017, 15(5): 3278-3284.
24.	Saito T, Nakamichi R, Yoshida A, et al. The effect of mechanical stress on enthesis homeostasis in a rat Achilles enthesis organ culture model. J Orthop Res, 2022, 40(8): 1872-1882.
25.	Kubo Y, Hoffmann B, Goltz K, et al. Different frequency of cyclic tensile strain relates to anabolic/catabolic conditions consistent with immunohistochemical staining intensity in Ttenocytes. Int J Mol Sci, 2020, 21(3): 1082. doi: 10.3390/ijms21031082.
26.	Scott A, Danielson P, Abraham T, et al. Mechanical force modulates scleraxis expression in bioartificial tendons. J Musculoskelet Neuronal Interact, 2011, 11(2): 124-32.
27.	Wang A, Cao S, Stowe JC, et al. Substrate stiffness and stretch regulate profibrotic mechanosignaling in pulmonary arterial adventitial fibroblasts. Cells, 2021, 10(5): 1000. doi: 10.3390/cells10051000.
28.	Liu X, Zhou M, Tan J, et al. Inhibition of CX3CL1 by treadmill training prevents osteoclast-induced fibrocartilage complex resorption during TBI healing. Front Immunol, 2024, 14: 1295163. doi: 10.3389/fimmu.2023.1295163.
29.	Guo D, Yang J, Liu D, et al. Human umbilical cord mesenchymal stem cells overexpressing RUNX1 promote tendon-bone healing by inhibiting osteolysis, enhancing osteogenesis and promoting angiogenesis. Genes Genomics, 2024, 46(4): 461-473.
30.	Bontemps B, Gruet M, Louis J, et al. Patellar tendon adaptations to downhill running training and their relationships with changes in mechanical stress and loading history. J Strength Cond Res, 2024, 38(1): 21-29.
31.	He P, Ruan D, Huang Z, et al. Comparison of tendon development versus tendon healing and regeneration. Front Cell Dev Biol, 2022, 10: 821667. doi: 10.3389/fcell.2022.821667.

1. Howell K, Chien C, Bell R, et al. Novel model of tendon regeneration reveals distinct cell mechanisms underlying regenerative and fibrotic tendon healing. Sci Rep, 2017, 7: 45238. doi: 10.1038/srep45238.
2. Graham JG, Wang ML, Rivlin M, et al. Biologic and mechanical aspects of tendon fibrosis after injury and repair. Connect Tissue Res, 2019, 60(1): 10-20.
3. Al Makhzoomi AK, Kirk TB, Allison GT. A multiscale study of morphological changes in tendons following repeated cyclic loading. J Biomech, 2021, 128: 110790. doi: 10.1016/j.jbiomech.2021.110790.
4. Choi WJ, Park MS, Park KH, et al. Comparative analysis of gene expression in normal and degenerative human tendon cells: effects of cyclic strain. Foot Ankle Int, 2014, 35(10): 1045-1056.
5. Herchenhan A, Dietrich-Zagonel F, Schjerling P, et al. Early growth response genes increases rapidly after mechanical overloading and unloading in tendon constructs. J Orthop Res, 2020, 38(1): 173-181.
6. Zhang J, Wang JH. The effects of mechanical loading on tendons—an in vivo and in vitro model study. PLoS One, 2013, 8(8): e71740. doi: 10.1371/journal.pone.0071740.
7. Huisman E, Lu A, McCormack RG, et al. Enhanced collagen type Ⅰ synthesis by human tenocytes subjected to periodic in vitro mechanical stimulation. BMC Musculoskelet Disord, 2014, 15: 386. doi: 10.1186/1471-2474-15-386.
8. Khayyeri H, Hammerman M, Turunen MJ, et al. Diminishing effects of mechanical loading over time during rat Achilles tendon healing. PLoS One, 2020, 15(12): e0236681. doi: 10.1371/journal.pone.0236681.
9. Longo UG, Franceschi F, Ruzzini L, et al. Characteristics at haematoxylin and eosin staining of ruptures of the long head of the biceps tendon. Br J Sports Med, 2009, 43(8): 603-607.
10. Li S, Gong F, Zhou Z, et al. Combined verapamil-polydopamine nanoformulation inhibits adhesion formation in Achilles tendon injury using rat model. Int J Nanomedicine, 2023, 18: 115-126.
11. Metzler SA, Waller SC, Warnock JN. Quantitative characterization of aortic valve endothelial cell viability and morphology in situ under cyclic stretch. Cardiovasc Eng Technol, 2019, 10(1): 173-180.
12. Chen Z, Chen P, Zheng M, et al. Challenges and perspectives of tendon-derived cell therapy for tendinopathy: from bench to bedside. Stem Cell Res Ther, 2022, 13(1): 444. doi: 10.1186/s13287-022-03113-6.
13. Hall C, Law JP, Reyat JS, et al. Chronic activation of human cardiac fibroblasts in vitro attenuates the reversibility of the myofibroblast phenotype. Sci Rep, 2023, 13(1): 12137. doi: 10.1038/s41598-023-39369-y.
14. Nichols AEC, Muscat SN, Miller SE, et al. Impact of isolation method on cellular activation and presence of specific tendon cell subpopulations during in vitro culture. FASEB J, 2021, 35(7): e21733. doi: 10.1096/fj.202100405R.
15. Best KT, Nichols AEC, Knapp E, et al. NF-κB activation persists into the remodeling phase of tendon healing and promotes myofibroblast survival. Sci Signal, 2020, 13(658): eabb7209. doi: 10.1126/scisignal.abb7209.
16. Koga M, Kuramochi M, Karim MR, et al. Immunohistochemical characterization of myofibroblasts appearing in isoproterenol-induced rat myocardial fibrosis. J Vet Med Sci, 2019, 81(1): 127-133.
17. Cui HS, Hong AR, Kim JB, et al. Extracorporeal shock wave terapy alters the expression of fibrosis-related molecules in fibroblast derived from human hypertrophic scar. Int J Mol Sci, 2018, 19(1): 124. doi: 10.3390/ijms19010124.
18. Skalli O, Schürch W, Seemayer T, et al. Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins. Lab Invest, 1989, 60(2): 275-85.
19. Zhou J, Zhao Y, Yang W, et al. Use of mechanical stretching to treat skin graft contracture. J Burn Care Res, 2020, 41(4): 892-899.
20. Hortobagyi D, Grossmann T, Tschernitz M, et al. In vitro mechanical vibration down-regulates pro-inflammatory and pro-fibrotic signaling in human vocal fold fibroblasts. PLoS One, 2020, 15(11): e0241901. doi: 10.1371/journal.pone.0241901.
21. Benage LG, Sweeney JD, Giers MB, et al. Dynamic load model systems of tendon inflammation and mechanobiology. Front Bioeng Biotechnol, 2022, 10: 896336. doi: 10.3389/fbioe.2022.896336.
22. Gonçalves RM, Saggese T, Yong Z, et al. Interleukin-1β more than mechanical loading induces a degenerative phenotype in human annulus fibrosus cells, partially impaired by anti-proteolytic activity of mesenchymal stem cell secretome. Front Bioeng Biotechnol, 2022, 9: 802789. doi: 10.3389/fbioe.2021.802789.
23. Min J, Li B, Liu C, et al. Extracellular matrix metabolism disorder induced by mechanical strain on human parametrial ligament fibroblasts. Mol Med Rep, 2017, 15(5): 3278-3284.
24. Saito T, Nakamichi R, Yoshida A, et al. The effect of mechanical stress on enthesis homeostasis in a rat Achilles enthesis organ culture model. J Orthop Res, 2022, 40(8): 1872-1882.
25. Kubo Y, Hoffmann B, Goltz K, et al. Different frequency of cyclic tensile strain relates to anabolic/catabolic conditions consistent with immunohistochemical staining intensity in Ttenocytes. Int J Mol Sci, 2020, 21(3): 1082. doi: 10.3390/ijms21031082.
26. Scott A, Danielson P, Abraham T, et al. Mechanical force modulates scleraxis expression in bioartificial tendons. J Musculoskelet Neuronal Interact, 2011, 11(2): 124-32.
27. Wang A, Cao S, Stowe JC, et al. Substrate stiffness and stretch regulate profibrotic mechanosignaling in pulmonary arterial adventitial fibroblasts. Cells, 2021, 10(5): 1000. doi: 10.3390/cells10051000.
28. Liu X, Zhou M, Tan J, et al. Inhibition of CX3CL1 by treadmill training prevents osteoclast-induced fibrocartilage complex resorption during TBI healing. Front Immunol, 2024, 14: 1295163. doi: 10.3389/fimmu.2023.1295163.
29. Guo D, Yang J, Liu D, et al. Human umbilical cord mesenchymal stem cells overexpressing RUNX1 promote tendon-bone healing by inhibiting osteolysis, enhancing osteogenesis and promoting angiogenesis. Genes Genomics, 2024, 46(4): 461-473.
30. Bontemps B, Gruet M, Louis J, et al. Patellar tendon adaptations to downhill running training and their relationships with changes in mechanical stress and loading history. J Strength Cond Res, 2024, 38(1): 21-29.
31. He P, Ruan D, Huang Z, et al. Comparison of tendon development versus tendon healing and regeneration. Front Cell Dev Biol, 2022, 10: 821667. doi: 10.3389/fcell.2022.821667.

Chinese Journal of Reparative and Reconstructive Surgery

Study on anti-adhesion effect and mechanism of dynamic and static stress stimulation during early healing process of rat Achilles tendon injury

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