A dual-crosslinked injectable hydrogel derived from muscular decellularized matrix promoting myoblasts proliferation and myogenic differentiation_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHAO Shaohua ¹ , HAO Xiaoliang ² , JIAN Yanpeng ³ , WANG Yigong ³ , LIU Weijie ³ , SHAO Xinwei ³ , FAN Jun ³ ,  XU Songshan ³

1. Department of Plastic Surgery, Xuchang Central Hospital Affiliated to Henan University of Science and Technology, Xuchang Henan, 461000, P. R. China;
2. Department of Thyroid and Breast Diagnosis and Treatment Center, Weifang Hospital of Traditional Chinese Medicine, Weifang Shandong, 261000, P. R. China;
3. Department of Spine and Spinal Cord Surgery, Xuchang Central Hospital Affiliated to Henan University of Science and Technology, Xuchang Henan, 461000, P. R. China;

Corresponding?author：

XU Songshan, Email: 906698125@qq.com

Keywords：

Hydrogel; acellular musclar matrix; cell proliferation; myogenic differentiation; muscle tissue engineering

DOI：

10.7507/1002-1892.202306054

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Objective To investigate the feasibility of a dual-crosslinked injectable hydrogel derived from acellular musclar matrix (AMM) for promoting myoblasts proliferation and myogenic differentiation. Methods Firstly, hyaluronic acid was oxidized with NaIO₄ and methylated to prepare methacrylamidated oxidized hyaluronic acid (MOHA). Then, AMM obtained by washing enzymatically treated muscle tissue was aminolyzed to prepare aminated AMM (AAMM). MOHA hydrogel and AAMM were crosslinked using Schiff based reaction and UV radiation to prepare a dual-crosslinked MOHA/AAMM injectable hydrogel. Fourier transform infrared spectroscopy (FTIR) was used to characterize MOHA, AAMM, and MOHA/AAMM hydrogels. The injectability of MOHA/AAMM hydrogel were evaluated by manual injection, and the gelation performance was assessed by UV crosslinking. The rheological properties and Young’s modulus of the hydrogel were examined through mechanical tests. The degradation rate of the hydrogel was assessed by immersing it in PBS. The active components of the hydrogel were verified using immunofluorescence staining and ELISA assay kits. The promotion of cell proliferation by the hydrogel was tested using live/dead staining and cell counting kit 8 (CCK-8) assays after co-culturing with C2C12 myoblasts for 9 days. The effect of the hydrogel on myogenic differentiation was evaluated by immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR). Results FTIR spectra confirmed the successful preparation of MOHA/AAMM hydrogel. The hydrogel exhibited good injectability and gelation ability. Compared to MOHA hydrogel, MOHA/AAMM hydrogel exhibited higher viscosity and Young’s modulus, a reduced degradation rate, and contained a higher amount of collagen (including collagen type Ⅰ and collagen type Ⅲ) as well as bioactive factors (including epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, and insulin-like growth factor 1). The live/dead cell staining and CCK-8 assay indicated that with prolonged incubation time, there was a significant increase in viable cells and a decrease in dead cells in the C2C12 myoblasts within the MOHA/AAMM hydrogel. Compared with MOHA hydrogel, the difference was significant at each time point (P<0.05). Immunofluorescence staining and RT-qPCR analysis demonstrated that the deposition of IGF-1 and expression levels of myogenic-related genes (including Myogenin, Troponin T, and myosin heavy chain) in the MOHA/AAMM group were significantly higher than those in the MOHA group (P<0.05). Conclusion The MOHA/AAMM hydrogel prepared based on AMM can promote myoblasts proliferation and myogenic differentiation, providing a novel dual-crosslinked injectable hydrogel for muscle tissue engineering.

Citation： ZHAO Shaohua, HAO Xiaoliang, JIAN Yanpeng, WANG Yigong, LIU Weijie, SHAO Xinwei, FAN Jun, XU Songshan. A dual-crosslinked injectable hydrogel derived from muscular decellularized matrix promoting myoblasts proliferation and myogenic differentiation. Chinese Journal of Reparative and Reconstructive Surgery, 2023, 37(12): 1514-1522. doi: 10.7507/1002-1892.202306054 Copy

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14.	Yoshida T, Delafontaine P. Mechanisms of IGF-1-mediated regulation of skeletal muscle hypertrophy and atrophy. Cells, 2020, 9(9): 1970.
15.	Smith LR, Kok HJ, Zhang B, et al. Matrix metalloproteinase 13 from satellite cells is required for efficient muscle growth and regeneration. Cell Physiol Biochem, 2020, 54(3): 333-353.
16.	Nuge T, Liu Z, Liu X, et al. Recent advances in scaffolding from natural-based polymers for volumetric muscle injury. Molecules (Basel, Switzerland), 2021, 26(3): 699.
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19.	李丹丹, 莫秀梅. 基于席夫堿反應的氧化葡聚糖/胺化羧甲基殼聚糖雙組分水凝膠粘合劑. 中國組織工程研究, 2018, 22(22): 6.
20.	Xu Y, Wang Z, Hua Y, et al. Photocrosslinked natural hydrogel composed of hyaluronic acid and gelatin enhances cartilage regeneration of decellularized trachea matrix. Mater Sci Eng C Mater Biol Appl, 2021, 120: 111628.
21.	Yu T, Hu Y, He W, et al. An injectable and self-healing hydrogel with dual physical crosslinking for in-situ bone formation. Mat Today Bio, 2023, 19: 100558.
22.	Urciuolo A, Urbani L, Perin S, et al. Decellularised skeletal muscles allow functional muscle regeneration by promoting host cell migration. Scientific reports, 2018, 8(1): 8398.
23.	李昱輝, 黃國友, 徐峰, 等. 基于水凝膠的細胞力學微環境構建及在肌肉組織再生中的應用. 十二屆全國生物力學學術會議暨第十四屆全國生物流變學學術會議會議論文摘要匯編, 2018.

1. 宋達疆, 李贊, 章一新, 等. 胸背血管前鋸肌支在胸壁缺損修復中的應用. 中國修復重建外科雜志, 2022, 36(8): 1021-1025.
2. 李艾元, 施心雨, 岳萬福. 絲素支架在肌肉骨骼組織工程的應用及研究進展. 絲綢, 2021, 58(11): 18-22.
3. Zhe M, Wu X, Yu P, et al. Recent advances in decellularized extracellular matrix-based bioinks for 3D gioprinting in tissue engineering. Materials (Basel, Switzerland), 2023, 16(8): 3197.
4. Philips C, Terrie L, Thorrez L. Decellularized skeletal muscle: A versatile biomaterial in tissue engineering and regenerative medicine. Biomaterials, 2022, 283: 121436.
5. 鄧斌斌, 鄧雪強, 劉帥剛, 等. 關節鏡下肱二頭肌長頭肌腱轉位治療不可修復巨大肩袖撕裂的研究進展. 中國修復重建外科雜志, 2022, 36(2): 249-253.
6. 李艾元, 施心雨, 劉夢斐, 等. 基于絲素蛋白的可注射水凝膠在肌肉再生工程的應用. 絲綢, 2023, 60(2): 42-48.
7. Davoudi S, Chin CY, Cooke MJ, et al. Muscle stem cell intramuscular delivery within hyaluronan methylcellulose improves engraftment efficiency and dispersion. Biomaterials, 2018, 173: 34-46.
8. Abaci A, Guvendiren M. Designing decellularized extracellular matrix-based bioinks for 3D bioprinting. Advanced healthcare materials, 2020, 9(24): e2000734.
9. Wang YH, Wang DR, Guo YC, et al. The application of bone marrow mesenchymal stem cells and biomaterials in skeletal muscle regeneration. Regenerative therapy, 2020, 15: 285-294.
10. Alarcin E, Bal-?ztürk A, Avci H, et al. Current strategies for the regeneration of skeletal muscle tissue. Int J Mol Sci, 2021, 22(11): 5929.
11. Qazi TH, Mooney DJ, Pumberger M, et al. Biomaterials based strategies for skeletal muscle tissue engineering: existing technologies and future trends. Biomaterials, 2015, 53: 502-521.
12. Hosoyama K, Ahumada M, Goel K, et al. Electroconductive materials as biomimetic platforms for tissue regeneration. Biotechnology advances, 2019, 37(3): 444-458.
13. Mostafavi E, Medina-Cruz D, Kalantari K, et al. Electroconductive nanobiomaterials for tissue engineering and regenerative medicine. Bioelectricity, 2020, 2(2): 120-149.
14. Yoshida T, Delafontaine P. Mechanisms of IGF-1-mediated regulation of skeletal muscle hypertrophy and atrophy. Cells, 2020, 9(9): 1970.
15. Smith LR, Kok HJ, Zhang B, et al. Matrix metalloproteinase 13 from satellite cells is required for efficient muscle growth and regeneration. Cell Physiol Biochem, 2020, 54(3): 333-353.
16. Nuge T, Liu Z, Liu X, et al. Recent advances in scaffolding from natural-based polymers for volumetric muscle injury. Molecules (Basel, Switzerland), 2021, 26(3): 699.
17. Langridge B, Griffin M, Butler PE. Regenerative medicine for skeletal muscle loss: a review of current tissue engineering approaches. J Mater Scie Mater Med, 2021, 32(1): 15.
18. Carnes ME, Pins GD. Skeletal muscle tissue engineering: Biomaterials-based strategies for the treatment of volumetric muscle loss. Bioengineering (Basel, Switzerland), 2020, 7(3): 85.
19. 李丹丹, 莫秀梅. 基于席夫堿反應的氧化葡聚糖/胺化羧甲基殼聚糖雙組分水凝膠粘合劑. 中國組織工程研究, 2018, 22(22): 6.
20. Xu Y, Wang Z, Hua Y, et al. Photocrosslinked natural hydrogel composed of hyaluronic acid and gelatin enhances cartilage regeneration of decellularized trachea matrix. Mater Sci Eng C Mater Biol Appl, 2021, 120: 111628.
21. Yu T, Hu Y, He W, et al. An injectable and self-healing hydrogel with dual physical crosslinking for in-situ bone formation. Mat Today Bio, 2023, 19: 100558.
22. Urciuolo A, Urbani L, Perin S, et al. Decellularised skeletal muscles allow functional muscle regeneration by promoting host cell migration. Scientific reports, 2018, 8(1): 8398.
23. 李昱輝, 黃國友, 徐峰, 等. 基于水凝膠的細胞力學微環境構建及在肌肉組織再生中的應用. 十二屆全國生物力學學術會議暨第十四屆全國生物流變學學術會議會議論文摘要匯編, 2018.

Chinese Journal of Reparative and Reconstructive Surgery

A dual-crosslinked injectable hydrogel derived from muscular decellularized matrix promoting myoblasts proliferation and myogenic differentiation

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