Preliminary study on preparation of decellularized nerve grafts from GGTA1 gene-edited pigs and their immune rejection in xenotransplantation_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIU Yuli ¹ , ZHAO Jinjuan ² , SONG Xiangyu ² , JIA Zhibo ² , LI Chaochao ² , ZHANG Tieyuan ² , LI Xiangling ² , YAN Shi ² , HE Ruichao ² ,  PENG Jiang ²

1. Graduate School, Guizhou Medical University, Guiyang Guizhou, 550005, P. R. China;
2. Institute of Orthopedics, General Hospital of Chinese PLA, Beijing, 100853, P. R. China;

Corresponding author：

PENG Jiang, Email: pengjiang301@126.com

Keywords：

GGTA1 gene-edited pig; peripheral nerve; repair; decellularized matrix; xenotransplantation

DOI：

10.7507/1002-1892.202408052

Video：

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Objective To prepare decellularized nerve grafts from alpha-1, 3-galactosyltransferase (GGTA1) gene-edited pigs and explore their biocompatibility for xenotransplantation. Methods The sciatic nerves from wild-type pigs and GGTA1 gene-edited pigs were obtained and underwent decellularization. The alpha-galactosidase (α-gal) content in the sciatic nerves of GGTA1 gene-edited pigs was detected by using IB4 fluorescence staining and ELISA method to verify the knockout status of the GGTA1 gene, and using human sciatic nerve as a control. HE staining and scanning electron microscopy observation were used to observe the structure of the nerve samples. Immunofluorescence staining and DNA content determination were used to evaluate the degree of decellularization of the nerve samples. Fourteen nude mice were taken, and subcutaneous capsules were prepared on both sides of the spine. Decellularized nerve samples of wild-type pigs (n=7) and GGTA1 gene-edited pigs (n=7) were randomly implanted in the subcutaneous capsules. Blood was drawn at 1, 3, 5, and 7 days after implantation to detect neutrophil counting. Results IB4 fluorescence staining and ELISA detection showed that GGTA1 gene was successfully knocked out in the nerves of GGTA1 gene-edited pigs. HE staining showed that the structure of the decellularized nerve from GGTA1 gene-edited pigs was well preserved; the nerve basement membrane tube structure was visible under scanning electron microscopy; no cell nuclei was observed, and the extracellular matrix components was retained in the nerve grafts by immunofluorescence staining; and the DNA content was significantly reduced when compared with the normal nerves (P<0.05). In vivo experiments showed that the number of neutrophils in the two groups were similar at 1, 3, and 7 days after implantation, with no significant difference (P>0.05); only at 5 days, the number of neutrophils was significantly lower in the GGTA1 gene-edited pigs than in the wild-type pigs (P<0.05). Conclusion The decellularized nerve grafts from GGTA1 gene-edited pigs have well-preserved nerve structure, complete decellularization, retain the natural nerve basement membrane tube structure and components, and low immune response after xenotransplantation through in vitro experiments.

Citation： LIU Yuli, ZHAO Jinjuan, SONG Xiangyu, JIA Zhibo, LI Chaochao, ZHANG Tieyuan, LI Xiangling, YAN Shi, HE Ruichao, PENG Jiang. Preliminary study on preparation of decellularized nerve grafts from GGTA1 gene-edited pigs and their immune rejection in xenotransplantation. Chinese Journal of Reparative and Reconstructive Surgery, 2025, 39(2): 224-229. doi: 10.7507/1002-1892.202408052 Copy

1.	Johnson EO, Zoubos AB, Soucacos PN. Regeneration and repair of peripheral nerves. Injury, 2005, 36 Suppl 4: S24-S29. doi: 10.1016/j.injury.2005.10.012.
2.	Gu X, Ding F, Yang Y, et al. Construction of tissue engineered nerve grafts and their application in peripheral nerve regeneration. Prog Neurobiol, 2011, 93(2): 204-230.
3.	Alluin O, Wittmann C, Marqueste T, et al. Functional recovery after peripheral nerve injury and implantation of a collagen guide. Biomaterials, 2009, 30(3): 363-373.
4.	Hudson TW, Liu SY, Schmidt CE. Engineering an improved acellular nerve graft via optimized chemical processing. Tissue Eng, 2004, 10(9-10): 1346-1358.
5.	Levi-Montalcini R, Skaper SD, Dal Toso R, et al. Nerve growth factor: from neurotrophin to neurokine. Trends Neurosci, 1996, 19(11): 514-520.
6.	Porrett PM, Orandi BJ, Kumar V, et al. First clinical-grade porcine kidney xenotransplant using a human decedent model. Am J Transplant, 2022, 22(4): 1037-1053.
7.	Ekser B, Rigotti P, Gridelli B, et al. Xenotransplantation of solid organs in the pig-to-primate model. Transpl Immunol, 2009, 21(2): 87-92.
8.	Stahl EC, Bonvillain RW, Skillen CD, et al. Evaluation of the host immune response to decellularized lung scaffolds derived from α-Gal knockout pigs in a non-human primate model. Biomaterials, 2018, 187: 93-104.
9.	刘梅珍, 王立人, 李咏梅, 等. 利用CRISPR/Cas9技术构建基因编辑大鼠模型. 遗传, 2023, 45(1): 78-88.
10.	Griffith BP, Goerlich CE, Singh AK, et al. Genetically modified porcine-to-human cardiac xenotransplantation. N Engl J Med, 2022, 387(1): 35-44.
11.	Montgomery RA, Stern JM, Lonze BE, et al. Results of two cases of pig-to-human kidney xenotransplantation. N Engl J Med, 2022, 386(20): 1889-1898.
12.	Battiston B, Titolo P, Ciclamini D, et al. Peripheral nerve defects: overviews of practice in Europe. Hand Clin, 2017, 33(3): 545-550.
13.	Moffat D, Ye K, Jin S. Decellularization for the retention of tissue niches. J Tissue Eng, 2022, 13: 20417314221101151. doi: 10.1177/20417314221101151.
14.	Cooper DK, Keogh AM. The potential role of xenotransplantation in treating endstage cardiac disease: a summary of the report of the Xenotransplantation Advisory Committee of the International Society for Heart and Lung Transplantation. Curr Opin Cardiol, 2001, 16(2): 105-109.
15.	Tearle RG, Tange MJ, Zannettino ZL, et al. The alpha-1, 3-galactosyltransferase knockout mouse. Implications for xenotransplantation. Transplantation, 1996, 61(1): 13-19.
16.	Galili U, Rachmilewitz EA, Peleg A, et al. A unique natural human IgG antibody with anti-alpha-galactosyl specificity. J Exp Med, 1984, 160(5): 1519-1531.
17.	Raeder RH, Badylak SF, Sheehan C, et al. Natural anti-galactose alpha1, 3 galactose antibodies delay, but do not prevent the acceptance of extracellular matrix xenografts. Transpl Immunol, 2002, 10(1): 15-24.
18.	Serrano M C, Nardecchia S, García-Rama C, et al. Chondroitin sulphate-based 3D scaffolds containing MWCNTs for nervous tissue repair. Biomaterials, 2014, 35(5): 1543-1551.
19.	Kang SW, Rahman MS, Kim AN, et al. Comparative study of the quality characteristics of defatted soy flour treated by supercritical carbon dioxide and organic solvent. J Food Sci Technol, 2017, 54(8): 2485-2493.

1. Johnson EO, Zoubos AB, Soucacos PN. Regeneration and repair of peripheral nerves. Injury, 2005, 36 Suppl 4: S24-S29. doi: 10.1016/j.injury.2005.10.012.
2. Gu X, Ding F, Yang Y, et al. Construction of tissue engineered nerve grafts and their application in peripheral nerve regeneration. Prog Neurobiol, 2011, 93(2): 204-230.
3. Alluin O, Wittmann C, Marqueste T, et al. Functional recovery after peripheral nerve injury and implantation of a collagen guide. Biomaterials, 2009, 30(3): 363-373.
4. Hudson TW, Liu SY, Schmidt CE. Engineering an improved acellular nerve graft via optimized chemical processing. Tissue Eng, 2004, 10(9-10): 1346-1358.
5. Levi-Montalcini R, Skaper SD, Dal Toso R, et al. Nerve growth factor: from neurotrophin to neurokine. Trends Neurosci, 1996, 19(11): 514-520.
6. Porrett PM, Orandi BJ, Kumar V, et al. First clinical-grade porcine kidney xenotransplant using a human decedent model. Am J Transplant, 2022, 22(4): 1037-1053.
7. Ekser B, Rigotti P, Gridelli B, et al. Xenotransplantation of solid organs in the pig-to-primate model. Transpl Immunol, 2009, 21(2): 87-92.
8. Stahl EC, Bonvillain RW, Skillen CD, et al. Evaluation of the host immune response to decellularized lung scaffolds derived from α-Gal knockout pigs in a non-human primate model. Biomaterials, 2018, 187: 93-104.
9. 刘梅珍, 王立人, 李咏梅, 等. 利用CRISPR/Cas9技术构建基因编辑大鼠模型. 遗传, 2023, 45(1): 78-88.
10. Griffith BP, Goerlich CE, Singh AK, et al. Genetically modified porcine-to-human cardiac xenotransplantation. N Engl J Med, 2022, 387(1): 35-44.
11. Montgomery RA, Stern JM, Lonze BE, et al. Results of two cases of pig-to-human kidney xenotransplantation. N Engl J Med, 2022, 386(20): 1889-1898.
12. Battiston B, Titolo P, Ciclamini D, et al. Peripheral nerve defects: overviews of practice in Europe. Hand Clin, 2017, 33(3): 545-550.
13. Moffat D, Ye K, Jin S. Decellularization for the retention of tissue niches. J Tissue Eng, 2022, 13: 20417314221101151. doi: 10.1177/20417314221101151.
14. Cooper DK, Keogh AM. The potential role of xenotransplantation in treating endstage cardiac disease: a summary of the report of the Xenotransplantation Advisory Committee of the International Society for Heart and Lung Transplantation. Curr Opin Cardiol, 2001, 16(2): 105-109.
15. Tearle RG, Tange MJ, Zannettino ZL, et al. The alpha-1, 3-galactosyltransferase knockout mouse. Implications for xenotransplantation. Transplantation, 1996, 61(1): 13-19.
16. Galili U, Rachmilewitz EA, Peleg A, et al. A unique natural human IgG antibody with anti-alpha-galactosyl specificity. J Exp Med, 1984, 160(5): 1519-1531.
17. Raeder RH, Badylak SF, Sheehan C, et al. Natural anti-galactose alpha1, 3 galactose antibodies delay, but do not prevent the acceptance of extracellular matrix xenografts. Transpl Immunol, 2002, 10(1): 15-24.
18. Serrano M C, Nardecchia S, García-Rama C, et al. Chondroitin sulphate-based 3D scaffolds containing MWCNTs for nervous tissue repair. Biomaterials, 2014, 35(5): 1543-1551.
19. Kang SW, Rahman MS, Kim AN, et al. Comparative study of the quality characteristics of defatted soy flour treated by supercritical carbon dioxide and organic solvent. J Food Sci Technol, 2017, 54(8): 2485-2493.

Chinese Journal of Reparative and Reconstructive Surgery

Preliminary study on preparation of decellularized nerve grafts from GGTA1 gene-edited pigs and their immune rejection in xenotransplantation

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