The biocompatibility and immunogenicity study of decellularized tracheal matrix_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

PAN Shu ^1,2 , LIU Xingchen ³ ,  SHI Hongcan ¹

1. Department of Cardiothoracic Surgery, Medical College of Yangzhou University, Yangzhou Jiangsu, 225001, P.R.China;
2. Department of Cardiothoracic Surgery, the Second Xiangya Hospital of Central South University, Changsha Hunan, 410011, P.R.China;
3. Department of Cardiothoracic Surgery, Yancheng Third People’s Hospital, Yancheng Jiangsu, 224000, P.R.China;

Corresponding author：

SHI Hongcan, Email: shihongcan@hotmail.com

Keywords：

Tissue engineered trachea; decellularized material; biocompatibility; immunogenicity; rabbit

DOI：

10.7507/1002-1892.201710007

Video：

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ObjectiveTo investigate the biocompatibility and immunogenicity of the tracheal matrix decellularized by sodium perchlorate (NaClO₄).MethodsBone marrow mesenchymal stem cells (BMSCs) were divided from 2-month-old New Zealand white rabbits. The trachea of 6-month-old New Zealand white rabbits were trimmed to a length of 1.5 cm and randomly divided into control group (group A₁, n=5, just stripped the loose connective tissue outside the trachea) and experimental group (group B₁, n=5, decellularized by improved NaClO₄ immersion method). The cytotoxicity of the scaffold leaching solution was detected by MTT assay, and the major histocompatibility complex (MHC) expression was detected by immunohistochemical method. The 4th generation of BMSCs were seeded onto the scaffold of 2 groups, and the cell activity around the material was observed by inverted microscope after Giemsa staining at 48 hours, while the cells states on the scaffold were observed at 7 and 14 days after culturing by scanning electron microscope. Another 10 6-month-old New Zealand white rabbits were randomly divided into control group (group A₂, n=5) and experimental group (group B₂, n=5), which implanted the native trachea and decellularized tracheal matrix into the subcutaneous sac of the back neck, respectively. The serum immunoglobulin IgM and IgG contents were analysed at 5, 10, 15, 20, 25, and 30 days after operation, and HE staining observation was performed at 30 days after operation.ResultsMTT assay showed that the proliferation activity of BMSCs cultured in the leach liquor of group B₁ was well, showing no significant difference when compared with group A₁ and negative control group with pure culture medium (P>0.05). The immunohistochemical staining showed that the decellularized process could significantly reducing the antigenicity of matrix materials. Giemsa staining showed that BMSCs grew well around the two tracheal matrixs (groups A₁ and B₁) in vitro. Scanning electron microscope observation showed that the cells were attached to the outer wall of the tracheal material in group A₁, which present a flat, round, oval shaped, tightly arranged cells and cluster distribution; and in group B₁, the cells formed a single lamellar sheet cover the outer wall of the tracheal material, whose morphology was similar to that in group A₁, and the growth trend was better. In vivo experimental results showed that the rejection of group B₂ was lower than that of group A₂. The contens of IgM and IgG in group A₂ were significantly higher than those in group B₂ at each time point after operation (P<0.05). HE staining showed no signs of rejection, macrophagocyte, or lymphocyte infiltration occurred, and the collagen fibers maintained their integrity in group B₂.ConclusionThe decellularized matrix treated by NaClO₄ has a fine biocompatibility, while its immunogenicity decreased, and it is suitable for the scaffold material for constructing of tissue engineered trachea.

Citation： PAN Shu, LIU Xingchen, SHI Hongcan. The biocompatibility and immunogenicity study of decellularized tracheal matrix. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(4): 441-447. doi: 10.7507/1002-1892.201710007 Copy

1.	Baiguera S, D’Innocenzo B, Macchiarini P. Current status of regenerative replacement of the airway. Expert Rev Respir Med, 2011, 5(4): 487-494.
2.	Haag JC, Jungebluth P, Macchiarini P. Tracheal replacement for primary tracheal cancer. Curr Opin Otolaryngol Head Neck Surg, 2013, 21(2): 171-177.
3.	Jungebluth P, Moll G, Baiguera S, et al. Tissue-engineered airway: a regenerative solution. Clin Pharmacol Ther, 2012, 91(1): 81-93.
4.	Kojima K, Vacanti CA. Tissue engineering in the trachea. Anat Rec (Hoboken), 2014, 297(1): 44-50.
5.	Jungebluth P, Macchiarini P. Airway transplantation. Thorac Surg Clin, 2014, 24(1): 97-106.
6.	Ma R, Li M, Luo J, et al. Structural integrity, ECM components and immunogenicity of decellularized laryngeal scaffold with preserved cartilage. Biomaterials, 2013, 34(7): 1790-1798.
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8.	Kisilevskiĭ MV, Anisimova NIu, Lebedinskaia OV, et al. Heterotopic transplantation of non-immunogenic trachea populated with recipient bone marrow stromal cells. Morfologiia, 2012, 141(1): 66-70.
9.	Zhang W, Zhang F, Shi H, et al. Comparisons of rabbit bone marrow mesenchymal stem cell isolation and culture methods in vitro. PLoS One, 2014, 9(2): e88794.
10.	潘枢, 孙飞, 史宏灿, 等. 保留软骨的兔脱细胞气管支架的性能. 中华实验外科杂志, 2014, 31(8): 1659-1661.
11.	Zang M, Zhang Q, Chang EI, et al. Decellularized tracheal matrix scaffold for tissue engineering. Plast Reconstr Surg, 2012, 130(3): 532-540.
12.	Kalathur M, Baiguera S, Macchiarini P. Translating tissue-engineered tracheal replacement from bench to bedside. Cell Mol Life Sci, 2010, 67(24): 4185-4196.
13.	Luo X, Liu Y, Zhang Z, et al. Long-term functional reconstruction of segmental tracheal defect by pedicled tissue-engineered trachea in rabbits. Biomaterials, 2013, 34(13): 3336-3344.
14.	Baiguera S, Jungebluth P, Burns A, et al. Tissue engineered human tracheas for in vivo implantation. Biomaterials, 2010, 31(34): 8931-8938.
15.	Jungebluth P, Go T, Asnaghi A, et al. Structural and morphologic evaluation of a novel detergent-enzymatic tissue-engineered tracheal tubular matrix. J Thorac Cardiovasc Surg, 2009, 138(3): 586-593.
16.	Jungebluth P, Bader A, Baiguera S, et al. The concept of in vivo airway tissue engineering. Biomaterials, 2012, 33(17): 4319-4326.
17.	van Kasteren SI, Overkleeft H, Ovaa H, et al. Chemical biology of antigen presentation by MHC molecules. Curr Opin Immunol, 2014, 26: 21-31.
18.	Alonso Arias R, López-Vázquez A, López-Larrea C. Immunology and the challenge of transplantation. Adv Exp Med Biol, 2012, 741: 27-43.
19.	Figueiredo C, Wedekind D, Müller T, et al. MHC universal cells survive in an allogeneic environment after incompatible transplantation. Biomed Res Int, 2013, 2013: 796046.
20.	Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials, 2011, 32(12): 3233-3243.
21.	Partington L, Mordan NJ, Mason C, et al. Biochemical changes caused by decellularization may compromise mechanical integrity of tracheal scaffolds. Acta Biomater, 2013, 9(2): 5251-5261.
22.	Delaere P, Vranckx J, Verleden G, et al. Tracheal allotransplantation after withdrawal of immunosuppressive therapy. N Engl J Med, 2010, 362(2): 138-145.

1. Baiguera S, D’Innocenzo B, Macchiarini P. Current status of regenerative replacement of the airway. Expert Rev Respir Med, 2011, 5(4): 487-494.
2. Haag JC, Jungebluth P, Macchiarini P. Tracheal replacement for primary tracheal cancer. Curr Opin Otolaryngol Head Neck Surg, 2013, 21(2): 171-177.
3. Jungebluth P, Moll G, Baiguera S, et al. Tissue-engineered airway: a regenerative solution. Clin Pharmacol Ther, 2012, 91(1): 81-93.
4. Kojima K, Vacanti CA. Tissue engineering in the trachea. Anat Rec (Hoboken), 2014, 297(1): 44-50.
5. Jungebluth P, Macchiarini P. Airway transplantation. Thorac Surg Clin, 2014, 24(1): 97-106.
6. Ma R, Li M, Luo J, et al. Structural integrity, ECM components and immunogenicity of decellularized laryngeal scaffold with preserved cartilage. Biomaterials, 2013, 34(7): 1790-1798.
7. Kiselevsky MV, Anisimova NY, Lebedinskaya OV, et al. Optimization of a method for preparation and repopulation of the tracheal matrix for allogenic transplantation. Bull Exp Biol Med, 2011, 151(1): 107-113.
8. Kisilevskiĭ MV, Anisimova NIu, Lebedinskaia OV, et al. Heterotopic transplantation of non-immunogenic trachea populated with recipient bone marrow stromal cells. Morfologiia, 2012, 141(1): 66-70.
9. Zhang W, Zhang F, Shi H, et al. Comparisons of rabbit bone marrow mesenchymal stem cell isolation and culture methods in vitro. PLoS One, 2014, 9(2): e88794.
10. 潘枢, 孙飞, 史宏灿, 等. 保留软骨的兔脱细胞气管支架的性能. 中华实验外科杂志, 2014, 31(8): 1659-1661.
11. Zang M, Zhang Q, Chang EI, et al. Decellularized tracheal matrix scaffold for tissue engineering. Plast Reconstr Surg, 2012, 130(3): 532-540.
12. Kalathur M, Baiguera S, Macchiarini P. Translating tissue-engineered tracheal replacement from bench to bedside. Cell Mol Life Sci, 2010, 67(24): 4185-4196.
13. Luo X, Liu Y, Zhang Z, et al. Long-term functional reconstruction of segmental tracheal defect by pedicled tissue-engineered trachea in rabbits. Biomaterials, 2013, 34(13): 3336-3344.
14. Baiguera S, Jungebluth P, Burns A, et al. Tissue engineered human tracheas for in vivo implantation. Biomaterials, 2010, 31(34): 8931-8938.
15. Jungebluth P, Go T, Asnaghi A, et al. Structural and morphologic evaluation of a novel detergent-enzymatic tissue-engineered tracheal tubular matrix. J Thorac Cardiovasc Surg, 2009, 138(3): 586-593.
16. Jungebluth P, Bader A, Baiguera S, et al. The concept of in vivo airway tissue engineering. Biomaterials, 2012, 33(17): 4319-4326.
17. van Kasteren SI, Overkleeft H, Ovaa H, et al. Chemical biology of antigen presentation by MHC molecules. Curr Opin Immunol, 2014, 26: 21-31.
18. Alonso Arias R, López-Vázquez A, López-Larrea C. Immunology and the challenge of transplantation. Adv Exp Med Biol, 2012, 741: 27-43.
19. Figueiredo C, Wedekind D, Müller T, et al. MHC universal cells survive in an allogeneic environment after incompatible transplantation. Biomed Res Int, 2013, 2013: 796046.
20. Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials, 2011, 32(12): 3233-3243.
21. Partington L, Mordan NJ, Mason C, et al. Biochemical changes caused by decellularization may compromise mechanical integrity of tracheal scaffolds. Acta Biomater, 2013, 9(2): 5251-5261.
22. Delaere P, Vranckx J, Verleden G, et al. Tracheal allotransplantation after withdrawal of immunosuppressive therapy. N Engl J Med, 2010, 362(2): 138-145.

Chinese Journal of Reparative and Reconstructive Surgery

The biocompatibility and immunogenicity study of decellularized tracheal matrix

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