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.
4.	李海鵬, 劉玉杰. 關節軟骨損傷治療的最新進展. 中國矯形外科雜志, 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.
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8.	孟慶陽, 胡曉青, 黃洪杰, 等. 豬腹膜脫細胞基質聯合微骨折技術修復兔膝關節軟骨缺損. 中國運動醫學雜志, 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.
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13.	Ozkanlar S, Akcay F. Antioxidant vitamins in atherosclerosis——animal experiments and clinical studies. Adv Clin Exp Med, 2012, 21(1): 115-123.
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15.	張偉, 褚文輝, 李春義. 鹿茸成骨過程及其相關調控機制研究進展. 中國農學通報, 2015, 31(8): 6-11.
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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.
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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.	員海超, 蒲春曉, 魏強, 等. 組織工程細胞外基質材料研究進展. 中國修復重建外科雜志, 2012, 26(10): 1251-1254.
23.	祁潔, 楊志明. 骨組織工程細胞外基質的研究進展. 中國修復重建外科雜志, 2006, 20(1): 61-64.
24.	于龍, 胡蘊玉, 畢龍, 等. 脫細胞軟骨基質三維支架的制備及特性研究. 中國矯形外科雜志, 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.	周紅星, 楊柳, 李起鴻. 骨組織工程研究中種子細胞、支架材料及其相互關系.中國臨床康復, 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. 李海鵬, 劉玉杰. 關節軟骨損傷治療的最新進展. 中國矯形外科雜志, 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. 孟慶陽, 胡曉青, 黃洪杰, 等. 豬腹膜脫細胞基質聯合微骨折技術修復兔膝關節軟骨缺損. 中國運動醫學雜志, 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 Bola?os 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. 張偉, 褚文輝, 李春義. 鹿茸成骨過程及其相關調控機制研究進展. 中國農學通報, 2015, 31(8): 6-11.
16. 李光鳳, 趙麗紅, 郭斌, 等. 梅花鹿鹿茸生長頂端的組織結構. 中國畜牧獸醫學會動物解剖學及組織胚胎學分會第十五次學術研討會論文集. 北京: 中國畜牧獸醫學會, 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. 員海超, 蒲春曉, 魏強, 等. 組織工程細胞外基質材料研究進展. 中國修復重建外科雜志, 2012, 26(10): 1251-1254.
23. 祁潔, 楊志明. 骨組織工程細胞外基質的研究進展. 中國修復重建外科雜志, 2006, 20(1): 61-64.
24. 于龍, 胡蘊玉, 畢龍, 等. 脫細胞軟骨基質三維支架的制備及特性研究. 中國矯形外科雜志, 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. 周紅星, 楊柳, 李起鴻. 骨組織工程研究中種子細胞、支架材料及其相互關系.中國臨床康復, 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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