Preparation of acellular matrix from antler cartilage and its biological compatibility_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

FU Jing ^1,2 , ZHANG Wei ¹ , ZHANG Aiwu ² , MA Lijuan ² ,  CHU Wenhui ¹ ,  LI Chunyi ¹

1. State Key Laboratory for Molecular Biology of Special Animals, Institute of Special Wild Economic Animals and Plants, Chinese Academy of Agricultural Sciences, Changchun Jinlin, 130000, P.R.China;
2. Medicinal Materials College of Jilin Agricultural University, Changchun Jilin, 130118, P.R.China;

Corresponding author：

CHU Wenhui, Email: jake-chu@hotmail.com; LI Chunyi, Email: lichunyi1959@163.com

Keywords：

Deer antler; acellular matrix; cartilage tissue engineering

DOI：

10.7507/1002-1892.201612072

Video：

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ObjectiveTo study the feasibility of acellular matrix materials prepared from deer antler cartilage and its biological compatibility so as to search for a new member of the extracellular matrix family for cartilage regeneration. MethodsThe deer antler mesenchymal (M) layer tissue was harvested and treated through decellular process to prepare M layer acellular matrix; histologic observation and detection of M layer acellular matrix DNA content were carried out. The antler stem cells [antlerogenic periosteum (AP) cells] at 2nd passage were labelled by fluorescent stains and by PKH26. Subsequently, the M layer acellular matrix and the AP cells at 2nd passage were co-cultured for 7 days; then the samples were transplanted into nude mice to study the tissue compatibility of M layer acellular matrix in the living animals. ResultsHE and DAPI staining confirmed that the M layer acellular matrix did not contain nucleus; the DNA content of the M layer acellular matrix was (19.367±5.254) ng/mg, which was significantly lower than that of the normal M layer tissue [(3 805.500±519.119) ng/mg](t=12.630, P=0.000). In vitro co-culture experiments showed that AP cells could adhere to or even embedded in the M layer acellular matrix. Nude mice transplantation experiments showed that the introduced AP cells could proliferate and induce angiogenesis in the M layer acellular matrix. ConclusionThe deer antler cartilage acellular matrix is successfully prepared. The M layer acellular matrix is suitable for adhesion and proliferation of AP cells in vitro and in vivo, and it has the function of stimulating angiogenesis. This model for deer antler cartilage acellular matrix can be applied in cartilage tissue engineering in the future.

Citation： FU Jing, ZHANG Wei, ZHANG Aiwu, MA Lijuan, CHU Wenhui, LI Chunyi. Preparation of acellular matrix from antler cartilage and its biological compatibility. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(6): 723-729. doi: 10.7507/1002-1892.201612072 Copy

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2.	Minas T, Nehrer S. Current concepts in the treatment of articular cartilage defects. Orthopedics, 1997, 20(6): 525-538.
3.	Hunziker EB. Articular cartilage repair: are the intrinsic biological constraints undermining this process insuperable? Osteoarthritis Cartilage, 1999, 7(1): 15-28.
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8.	孟庆阳, 胡晓青, 黄洪杰, 等. 猪腹膜脱细胞基质联合微骨折技术修复兔膝关节软骨缺损. 中国运动医学杂志, 2016, 35(7): 637-641.
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12.	Linsley CS, Wu BM, Tawil B. Mesenchymal stem cell growth on and mechanical properties of fibrin-based biomimetic bone scaffolds. J Biomed Mater Res A, 2016, 104(12): 2945-2953.
13.	Ozkanlar S, Akcay F. Antioxidant vitamins in atherosclerosis——animal experiments and clinical studies. Adv Clin Exp Med, 2012, 21(1): 115-123.
14.	Li C, Waldrup KA, Corson ID, et al. Histogenesis of antlerogenic tissues cultivated in diffusion chambers in vivo in red deer (Cervus elaphus). Journal of Experimental Zoology Part A: Ecological Genetics and Physiology, 1995, 272(5): 345-355.
15.	张伟, 褚文辉, 李春义. 鹿茸成骨过程及其相关调控机制研究进展. 中国农学通报, 2015, 31(8): 6-11.
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19.	Utomo L, Pleumeekers MM, Nimeskern L, et al. Preparation and characterization of a decellularized cartilage scaffold for ear cartilage reconstruction. Biomed Mater, 2015, 10(1): 015010.
20.	Traverse JH. Using biomaterials to improve the efficacy of cell therapy following acute myocardial infarction. J Cardiovasc Transl Res, 2012, 5(1): 67-72.
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22.	员海超, 蒲春晓, 魏强, 等. 组织工程细胞外基质材料研究进展. 中国修复重建外科杂志, 2012, 26(10): 1251-1254.
23.	祁洁, 杨志明. 骨组织工程细胞外基质的研究进展. 中国修复重建外科杂志, 2006, 20(1): 61-64.
24.	于龙, 胡蕴玉, 毕龙, 等. 脱细胞软骨基质三维支架的制备及特性研究. 中国矫形外科杂志, 2010,18(9): 743-747.
25.	Gilbert TW. Strategies for tissue and organ decellularization[J]. Journal of cellular biochemistry, 2012, 113(7): 2217-2222.
26.	Pati F, Song TH, Rijal G, et al. Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration. Biomaterials, 2015, 37: 230-241.
27.	周红星, 杨柳, 李起鸿. 骨组织工程研究中种子细胞、支架材料及其相互关系.中国临床康复, 2004, 8(14): 2706-2707.
28.	Nakamura A, Ikarashi Y, Tsuchiya T, et al. Correlations among chemical constituents, cytotoxicities and tissue responses: in the case of natural rubber latex materials. Biomaterials, 1990, 11: 92-94.

1. Mierisch CM, Wilson HA, Turner MA, et al. Chondrocyte transplantation into articular cartilage defects with use of calcium alginate: the fate of the cells. J Bone Joint Surg (Am), 2003, 85-A(9): 1757-1767.
2. Minas T, Nehrer S. Current concepts in the treatment of articular cartilage defects. Orthopedics, 1997, 20(6): 525-538.
3. Hunziker EB. Articular cartilage repair: are the intrinsic biological constraints undermining this process insuperable? Osteoarthritis Cartilage, 1999, 7(1): 15-28.
4. 李海鹏, 刘玉杰. 关节软骨损伤治疗的最新进展. 中国矫形外科杂志, 2006, 14(14): 1076-1078.
5. Shao X, Goh JC, Hutmacher DW, et al. Repair of large articular osteochondral defects using hybrid scaffolds and bone marrow-derived mesenchymal stem cells in a rabbit model. Tissue Eng, 2006, 12(6): 1539-1551.
6. Dragoo JL, Carlson G, McCormick F, et al. Healing full-thickness cartilage defects using adipose-derived stem cells. Tissue Eng, 2007, 13(7): 1615-1621.
7. Sutherland AJ, Converse GL, Hopkins RA, et al. The bioactivity of cartilage extracellular matrix in articular cartilage regeneration. Adv Healthc Mater, 2015, 4(1): 29-39.
8. 孟庆阳, 胡晓青, 黄洪杰, 等. 猪腹膜脱细胞基质联合微骨折技术修复兔膝关节软骨缺损. 中国运动医学杂志, 2016, 35(7): 637-641.
9. Novak T, Fites Gilliland K, Xu X, et al. In Vivo Cellular Infiltration and Remodeling in a Decellularized Ovine Osteochondral Allograft. Tissue Eng Part A, 2016, 22(21-22): 1274-1285.
10. Vindas Bolaños RA, Cokelaere SM, McDermott Estrada JM, et al. The use of a cartilage decellularized matrix scaffold for the repair of osteochondral defects: the importance of long-term studies in a large animal model. Osteoarthritis and Cartilage, 2017, 25(3): 413-420.
11. Amiel GE, Komura M, Shapira OZ, et al. Engineering of blood vessels from acellular collagen matrices coated with human endothelial cells. Tissue Eng, 2006, 12(8): 2355-2365.
12. Linsley CS, Wu BM, Tawil B. Mesenchymal stem cell growth on and mechanical properties of fibrin-based biomimetic bone scaffolds. J Biomed Mater Res A, 2016, 104(12): 2945-2953.
13. Ozkanlar S, Akcay F. Antioxidant vitamins in atherosclerosis——animal experiments and clinical studies. Adv Clin Exp Med, 2012, 21(1): 115-123.
14. Li C, Waldrup KA, Corson ID, et al. Histogenesis of antlerogenic tissues cultivated in diffusion chambers in vivo in red deer (Cervus elaphus). Journal of Experimental Zoology Part A: Ecological Genetics and Physiology, 1995, 272(5): 345-355.
15. 张伟, 褚文辉, 李春义. 鹿茸成骨过程及其相关调控机制研究进展. 中国农学通报, 2015, 31(8): 6-11.
16. 李光凤, 赵丽红, 郭斌, 等. 梅花鹿鹿茸生长顶端的组织结构. 中国畜牧兽医学会动物解剖学及组织胚胎学分会第十五次学术研讨会论文集. 北京: 中国畜牧兽医学会, 2008.
17. Berg DK, Li C, Asher G, et al. Red deer cloned from antler stem cells and their differentiated progeny. Biol Reprod, 2007, 77(3): 384-394.
18. Li C, Clark DE, Lord EA, et al. Sampling technique to discriminate the different tissue layers of growing antler tips for gene discovery. Anat Rec, 2002, 268(2): 125-130.
19. Utomo L, Pleumeekers MM, Nimeskern L, et al. Preparation and characterization of a decellularized cartilage scaffold for ear cartilage reconstruction. Biomed Mater, 2015, 10(1): 015010.
20. Traverse JH. Using biomaterials to improve the efficacy of cell therapy following acute myocardial infarction. J Cardiovasc Transl Res, 2012, 5(1): 67-72.
21. Badylak SF, Taylor D, Uygun K. Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds. Annu Rev Biomed Eng, 2011, 13: 27-53.
22. 员海超, 蒲春晓, 魏强, 等. 组织工程细胞外基质材料研究进展. 中国修复重建外科杂志, 2012, 26(10): 1251-1254.
23. 祁洁, 杨志明. 骨组织工程细胞外基质的研究进展. 中国修复重建外科杂志, 2006, 20(1): 61-64.
24. 于龙, 胡蕴玉, 毕龙, 等. 脱细胞软骨基质三维支架的制备及特性研究. 中国矫形外科杂志, 2010,18(9): 743-747.
25. Gilbert TW. Strategies for tissue and organ decellularization[J]. Journal of cellular biochemistry, 2012, 113(7): 2217-2222.
26. Pati F, Song TH, Rijal G, et al. Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration. Biomaterials, 2015, 37: 230-241.
27. 周红星, 杨柳, 李起鸿. 骨组织工程研究中种子细胞、支架材料及其相互关系.中国临床康复, 2004, 8(14): 2706-2707.
28. Nakamura A, Ikarashi Y, Tsuchiya T, et al. Correlations among chemical constituents, cytotoxicities and tissue responses: in the case of natural rubber latex materials. Biomaterials, 1990, 11: 92-94.

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