EXPERIMENTAL STUDY ON CO-CULTURE OF HUMAN FIBROBLASTS ON DECELLULARIZED Achilles TENDON_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANG Zhibing , ZHANG Xia , GUO Xinyu , QIN Chuan.

Department of Orthopedics, Xinqiao Hospital, the Third Military Medical University, Chongqing, 400037, P.R.China. Corresponding author: ZHANG Xia, E-mail: zhangsw199254330@163.com;

Corresponding author：

Keywords：

Tissue engineered ligament; Decellularized; Achilles tendon; Human fibroblasts; Cytocompati-bility; Rabbit

DOI：

10.7507/1002-1892.20130177

Video：

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Objective To investigate the preparation of decellularized Achilles tendons and the effect of co-culture of human fibroblasts on the scaffold so as to provide a scaffold for the tissue engineered ligament reconstruction. Methods Achilles tendons of both hind limbs were harvested from 10 male New Zealand white rabbits (5-month-old; weighing, 4-5 kg). The Achilles tendons were decellularized using trypsin, Triton X-100, and sodium dodecyl sulfate (SDS), and then gross observation, histological examination, and scanning electron microscope (SEM) observation were performed; the human fibroblasts were seeded on the decellularized Achilles tendon, and then cytocompatibility was tested using the cell counting kit 8 method at 1, 3, 5, 7, and 9 days after co-culture. At 4 weeks after co-culture, SEM, HE staining, and biomechanical test were performed for observing cell-scaffold composite, and a comparison was made with before and after decellularization. ResultsAfter decellularization, the tendons had integrated aponeurosis and enlarged volume with soft texture and good toughness; there was no loose connective tissue and tendon cells between tendon bundles, the collagen fibers arranged loosely with three-dimensional network structure and more pores between tendon bundles; and it had good cytocompatibility. At 4 weeks after co-culture, cells migrated into the pores, and three-dimensional network structure disappeared. By biomechanical test, the tensile strength and Young’s elastic modulus of the decellularized Achilles tendon group decreased significantly when compared with normal Achilles tendons group and cell-scaffold composite group (P lt; 0.05), but no significant difference was found between normal Achilles tendons group and cell-scaffold composite group (P gt; 0.05). There was no significant difference in elongation at break among 3 groups (P gt; 0.05). ConclusionThe decellularized Achilles tendon is biocompatible to fibroblasts. It is suit for the scaffold for tissue engineered ligament reconstruction.

Citation： WANG Zhibing,ZHANG Xia,GUO Xinyu,QIN Chuan.. EXPERIMENTAL STUDY ON CO-CULTURE OF HUMAN FIBROBLASTS ON DECELLULARIZED Achilles TENDON. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(7): 805-809. doi: 10.7507/1002-1892.20130177 Copy

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14.	Elder BD, Eleswarapu SV, Athanasiou KA. Extraction techniques for the decellularization of tissue engineered articular cartilage constructs. Biomaterials, 2009, 30(22): 3749-3756．.
15.	Ott HC, Matthiesen TS, Goh SK, et al. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med, 2008, 14(2): 213-221.
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1. Liu W, Chen B, Deng D, et al. Repair of tendon defect with dermal fibroblast engineered tendon in a porcine model. Tissue Eng, 2006, 12(4): 775-788.
2. Cao D, Liu W, Wei X, et al. In vitro tendon engineering with avian tenocytes and polyglycolic acids: a preliminary report. Tissue Eng, 2006, 12(5): 1369-1377.
3. Whitlock PW, Smith TL, Poehling GG, et al. A naturally derived, cytocompatible, and architecturally optimized scaffold for tendon and ligament regeneration. Biomaterials, 2007, 28(29): 4321-4329.
4. Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials, 2011, 32(12): 3233-3243.
5. Brown BN, Valentin JE, Stewart-Akers AM, et al. Macrophage phenotype and remodeling outcomes in response to biologic scaffolds with and without a cellular component. Biomaterials, 2009, 30(8): 1482-1491.
6. 解传飚, 林月秋, 阮默, 等. EDC 交联改性的脱细胞异种(猪)肌腱生物学特性研究. 中国临床解剖学杂志, 2010, 28(4): 425-429.
7. 冷元曦, 林月秋, 阮默, 等. 肌腱移植材料的研究进展. 中国矫形外科杂志, 2009, 17(22): 1704-1706.
8. Yang B, Zhang Y, Zhou L, et al. Development of a porcine bladder acellular matrix with well-preserved extracellular bioactive factors for tissue engineering. Tissue Eng Part C Methods, 2010, 16(5): 1201-1211.
9. 胡成栋, 刘曦, 张伯勋, 等. 脱细胞鸡肌腱的制备与体外生物力学测定的实验研究. 河北医药, 2011, 33(18): 2761-2763.
10. 梁黎明, 柴家科, 杨红明, 等. 脱细胞肌腱制备的实验研究. 中国美容医学, 2006, 15(3): 239-240.
11. Meyer SR, Chiu B, Churchill TA, et al. Comparison of aortic valve allograft decellularization techniques in the rat. J Biomed Mater Res A, 2006, 79(2): 254-262.
12. Cebotari S, Tudorache I, Jaekel T, et al. Detergent decellularization of heart valves for tissue engineering: toxicological effects of residual detergents on human endothelial cells. Artif Organs, 2010, 34(3): 206-210.
13. Feil G, Christ-Adler M, Maurer S, et al. Investigations of urothelial cells seeded on commercially available small intestine submucosa. Eur Urol, 2006, 50(6): 1330-1337.
14. Elder BD, Eleswarapu SV, Athanasiou KA. Extraction techniques for the decellularization of tissue engineered articular cartilage constructs. Biomaterials, 2009, 30(22): 3749-3756．.
15. Ott HC, Matthiesen TS, Goh SK, et al. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med, 2008, 14(2): 213-221.
16. Uygun BE, Soto-Gutierrez A, Yagi H, et al. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med, 2010, 16(7): 814-820.
17. Petersen TH, Calle EA, Zhao L, et al. Tissue-engineered lungs for in vivo implantation. Science, 2010, 329(5991): 538-541.
18. Nakayama KH, Batchelder CA, Lee CI, et al. Decellularized rhesus monkey kidney as a three-dimensional scaffold for renal tissue engineering. Tissue Eng Part A, 2010, 16(7): 2207-2216.
19. Ni M, Rui YF, Tan Q, et al. Engineered scaffold-free tendon tissue produced by tendon-derived stem cells. Biomaterials, 2013, 34(8): 2024-2037.
20. Zhang J, Li B, Wang JH. The role of engineered tendon matrix in the stemness of tendon stem cells in vitro and the promotion of tendon-like tissue formation in vivo. Biomaterials, 2011, 32(29): 6972-6981.
21. Hashimoto T, Sun YL, An KN, et al. The effect of tendon surface treatment on cell attachment for potential enhancement of tendon graft healing: an ex vivo model. Med Eng Phys, 2012, 34(10): 1387-1393.
22. Kew SJ, Gwynne JH, Enea D, et al. Regeneration and repair of tendon and ligament tissue using collagen fibre biomaterials. Acta Biomater, 2011, 7(9): 3237-3247.
23. Ikeda J, Zhao C, Chen Q, et al. Compressive properties of cd-HA-gelatin modified intrasynovial tendon allograft in canine model in vivo. Biomechanics, 2011, 44(9): 1793-1796.
24. Wang JH, Guo Q, Li B. Tendon biomechanics and mechanobiology—a minireview of basic concepts and recent advancements. J Hand Ther, 2012, 25(2): 133-141.
25. Garvin J, Qi J, Maloney M, et al. Novel system for engineering bioartificial tendons and application of mechanical load. Tissue Eng, 2003, 9(5): 967-979.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL STUDY ON CO-CULTURE OF HUMAN FIBROBLASTS ON DECELLULARIZED Achilles TENDON

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