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Qualitative and Quantitative Evaluation of a Novel Detergent-Based Method for Decellularization of Peripheral Nerves

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Abstract

Tissue engineering is an emerging strategy for the development of nerve substitutes for peripheral nerve repair. Especially decellularized peripheral nerve allografts are interesting alternatives to replace the gold standard autografts. In this study, a novel decellularization protocol was qualitatively and quantitatively evaluated by histological, biochemical, ultrastructural and mechanical methods and compared to the protocol described by Sondell et al. and a modified version of the protocol described by Hudson et al. Decellularization by the method described by Sondell et al. resulted in a reduction of the cell content, but was accompanied by a loss of essential extracellular matrix (ECM) molecules such as laminin and glycosaminoglycans. This decellularization also caused disruption of the endoneurial tubes and an increased stiffness of the nerves. Decellularization by the adapted method of Hudson et al. did not alter the ECM composition of the nerves, but an efficient cell removal could not be obtained. Finally, decellularization by the method developed in our lab by Roosens et al. led to a successful removal of nuclear material, while maintaining the nerve ultrastructure and ECM composition. In addition, the resulting ECM scaffold was found to be cytocompatible, allowing attachment and proliferation of adipose-derived stem cells. These results show that our decellularization combining Triton X-100, DNase, RNase and trypsin created a promising scaffold for peripheral nerve regeneration.

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References

  1. Brooks, D. N., R. V. Weber, J. D. Chao, B. D. Rinker, J. Zoldos, M. R. Robichaux, S. B. Ruggeri, K. A. Anderson, E. E. Bonatz, S. M. Wisotsky, M. S. Cho, C. Wilson, E. O. Cooper, J. V. Ingari, B. Safa, B. M. Parrett, and G. M. Buncke. Processed nerve allografts for peripheral nerve reconstruction: a multicenter study of utilization and outcomes in sensory, mixed, and motor nerve reconstructions. Microsurgery 32:1–14, 2012.

    Article  PubMed  Google Scholar 

  2. Bueno, F. R., and S. B. Shah. Implications of tensile loading for the tissue engineering of nerves. Tissue Eng. Part B 14:219–233, 2008.

    Article  Google Scholar 

  3. Campos, F., A. B. Bonhome-Espinosa, L. Garcia-Martinez, J. D. Duran, M. T. Lopez-Lopez, M. Alaminos, M. C. Sanchez-Quevedo, and V. Carriel. Ex vivo characterization of a novel tissue-like cross-linked fibrin-agarose hydrogel for tissue engineering applications. Biomed. Mater. 11:055004, 2016.

    Article  CAS  PubMed  Google Scholar 

  4. Campos, F., A. B. Bonhome-Espinosa, G. Vizcaino, I. A. Rodriguez, D. Duran-Herrera, M. T. Lopez-Lopez, I. Sanchez-Montesinos, M. Alaminos, M. C. Sanchez-Quevedo, and V. Carriel. Generation of genipin cross-linked fibrin-agarose hydrogel tissue-like models for tissue engineering applications. Biomed. Mater. 13:025021, 2018.

    Article  PubMed  Google Scholar 

  5. Carbonetto, S., D. Evans, and P. Cochard. Nerve fiber growth in culture on tissue substrata from central and peripheral nervous systems. J. Neurosci. 7:610–620, 1987.

    Article  CAS  PubMed  Google Scholar 

  6. Carriel, V., M. Alaminos, I. Garzon, A. Campos, and M. Cornelissen. Tissue engineering of the peripheral nervous system. Expert Rev. Neurother. 14:301–318, 2014.

    Article  CAS  PubMed  Google Scholar 

  7. Carriel, V. S., J. Aneiros-Fernandez, S. Arias-Santiago, I. J. Garzon, M. Alaminos, and A. Campos. A novel histochemical method for a simultaneous staining of melanin and collagen fibers. J. Histochem. Cytochem. 59:270–277, 2011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Carriel, V., A. Campos, M. Alaminos, S. Raimondo, and S. Geuna. Staining methods for normal and regenerative myelin in the nervous system. Methods Mol. Biol. 1560:207–218, 2017.

    Article  CAS  PubMed  Google Scholar 

  9. Carriel, V., F. Campos, J. Aneiros-Fernandez, and J. A. Kiernan. Tissue Fixation and Processing for the Histological Identification of Lipids. Methods Mol. Biol. 1560:197–206, 2017.

    Article  CAS  PubMed  Google Scholar 

  10. Carriel, V., J. Garrido-Gomez, P. Hernandez-Cortes, I. Garzon, S. Garcia-Garcia, J. A. Saez-Moreno, M. Del Carmen Sanchez-Quevedo, A. Campos, and M. Alaminos. Combination of fibrin-agarose hydrogels and adipose-derived mesenchymal stem cells for peripheral nerve regeneration. J. Neural Eng. 10:026022, 2013.

    Article  PubMed  Google Scholar 

  11. Carriel, V., I. Garzon, M. Alaminos, and A. Campos. Evaluation of myelin sheath and collagen reorganization pattern in a model of peripheral nerve regeneration using an integrated histochemical approach. Histochem. Cell Biol. 136:709–717, 2011.

    Article  CAS  PubMed  Google Scholar 

  12. Carriel, V., I. Garzon, A. Campos, M. Cornelissen, and M. Alaminos. Differential expression of GAP-43 and neurofilament during peripheral nerve regeneration through bio-artificial conduits. J Tissue Eng Regen. Med. 11:553–563, 2014.

    Article  CAS  PubMed  Google Scholar 

  13. Carriel, V., G. Scionti, F. Campos, O. Roda, B. Castro, M. Cornelissen, I. Garzon, and M. Alaminos. In vitro characterization of a nanostructured fibrin agarose bio-artificial nerve substitute. J. Tissue Eng. Regen. Med. 11:1412–1426, 2017.

    Article  CAS  PubMed  Google Scholar 

  14. Cebotari, S., I. Tudorache, T. Jaekel, A. Hilfiker, S. Dorfman, W. Ternes, A. Haverich, and A. Lichtenberg. Detergent decellularization of heart valves for tissue engineering: toxicological effects of residual detergents on human endothelial cells. Artif. Organs 34:206–210, 2010.

    Article  PubMed  Google Scholar 

  15. Chernousov, M. A., W. M. Yu, Z. L. Chen, D. J. Carey, and S. Strickland. Regulation of Schwann cell function by the extracellular matrix. Glia 56:1498–1507, 2008.

    Article  PubMed  Google Scholar 

  16. Crapo, P. M., T. W. Gilbert, and S. F. Badylak. An overview of tissue and whole organ decellularization processes. Biomaterials 32:3233–3243, 2011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Dahlin, L. B. Techniques of peripheral nerve repair. Scand. J. Surg. 97:310–316, 2008.

    Article  CAS  PubMed  Google Scholar 

  18. Daly, W., L. Yao, D. Zeugolis, A. Windebank, and A. Pandit. A biomaterials approach to peripheral nerve regeneration: bridging the peripheral nerve gap and enhancing functional recovery. J. R. Soc. Interface 9:202–221, 2012.

    Article  CAS  PubMed  Google Scholar 

  19. Díaz-Moreno, E., D. Durand-Herrera, V. Carriel, M.-Á. Martín-Piedra, M. D. C. Sánchez-Quevedo, I. Garzón, A. Campos, R. Fernández-Valadés, and M. Alaminos. Evaluation of freeze-drying and cryopreservation protocols for long-term storage of biomaterials based on decellularized intestine. J. Biomed. Mater. Res. Part B 106:488–500, 2018.

    Article  CAS  Google Scholar 

  20. Evans, P. J., S. E. Mackinnon, A. D. Levi, J. A. Wade, D. A. Hunter, Y. Nakao, and R. Midha. Cold preserved nerve allografts: changes in basement membrane, viability, immunogenicity, and regeneration. Muscle Nerve 21:1507–1522, 1998.

    Article  CAS  PubMed  Google Scholar 

  21. Garcia-Martinez, L., F. Campos, C. Godoy-Guzman, M. Del Carmen Sanchez-Quevedo, I. Garzon, M. Alaminos, A. Campos, and V. Carriel. Encapsulation of human elastic cartilage-derived chondrocytes in nanostructured fibrin-agarose hydrogels. Histochem. Cell Biol. 147:83–95, 2017.

    Article  CAS  PubMed  Google Scholar 

  22. Gulati, A. K. Evaluation of acellular and cellular nerve grafts in repair of rat peripheral nerve. J. Neurosurg. 68:117–123, 1988.

    Article  CAS  PubMed  Google Scholar 

  23. Haastert-Talini, K., S. Geuna, L. B. Dahlin, C. Meyer, L. Stenberg, T. Freier, C. Heimann, C. Barwig, L. F. Pinto, S. Raimondo, G. Gambarotta, S. R. Samy, N. Sousa, A. J. Salgado, A. Ratzka, S. Wrobel, and C. Grothe. Chitosan tubes of varying degrees of acetylation for bridging peripheral nerve defects. Biomaterials 34:9886–9904, 2013.

    Article  CAS  PubMed  Google Scholar 

  24. Hess, J. R., M. J. Brenner, I. K. Fox, C. M. Nichols, T. M. Myckatyn, D. A. Hunter, S. R. Rickman, and S. E. Mackinnon. Use of cold-preserved allografts seeded with autologous Schwann cells in the treatment of a long-gap peripheral nerve injury. Plast. Reconstr. Surg. 119:246–259, 2007.

    Article  CAS  PubMed  Google Scholar 

  25. Hudson, T. W., S. Y. Liu, and C. E. Schmidt. Engineering an improved acellular nerve graft via optimized chemical processing. Tissue Eng. 10:1346–1358, 2004.

    Article  CAS  PubMed  Google Scholar 

  26. Ide, C., T. Osawa, and K. Tohyama. Nerve regeneration through allogeneic nerve grafts, with special reference to the role of the Schwann cell basal lamina. Prog. Neurobiol. 34:1–38, 1990.

    Article  CAS  PubMed  Google Scholar 

  27. Ide, C., K. Tohyama, R. Yokota, T. Nitatori, and S. Onodera. Schwann cell basal lamina and nerve regeneration. Brain Res. 288:61–75, 1983.

    Article  CAS  PubMed  Google Scholar 

  28. Kehoe, S., X. F. Zhang, and D. Boyd. FDA approved guidance conduits and wraps for peripheral nerve injury: a review of materials and efficacy. Injury 43:553–572, 2012.

    Article  CAS  PubMed  Google Scholar 

  29. Kvist, M., M. Sondell, M. Kanje, and L. B. Dahlin. Regeneration in, and properties of, extracted peripheral nerve allografts and xenografts. J. Plast. Surg. Hand. Surg. 45:122–128, 2011.

    Article  PubMed  Google Scholar 

  30. Meek, M. F., and J. H. Coert. US Food and Drug Administration/Conformit Europe-approved absorbable nerve conduits for clinical repair of peripheral and cranial nerves. Ann. Plast. Surg. 60:466–472, 2008.

    Article  PubMed  Google Scholar 

  31. Moore, A. M., R. Kasukurthi, C. K. Magill, H. F. Farhadi, G. H. Borschel, and S. E. Mackinnon. Limitations of conduits in peripheral nerve repairs. Hand 4:180–186, 2009.

    Article  PubMed  Google Scholar 

  32. Moore, A. M., M. MacEwan, K. B. Santosa, K. E. Chenard, W. Z. Ray, D. A. Hunter, S. E. Mackinnon, and P. J. Johnson. Acellular nerve allografts in peripheral nerve regeneration: a comparative study. Muscle Nerve 44:221–234, 2011.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Muir D. F. Method for decellularization of tissue grafts. Google Patents, 2014.

  34. Noble, J., C. A. Munro, V. S. Prasad, and R. Midha. Analysis of upper and lower extremity peripheral nerve injuries in a population of patients with multiple injuries. J. Trauma 45:116–122, 1998.

    Article  CAS  PubMed  Google Scholar 

  35. Oliveira, A. C., I. Garzon, A. M. Ionescu, V. Carriel, J. Cardona, M. Gonzalez-Andrades, M. del Perez, M. Alaminos, and A. Campos. Evaluation of small intestine grafts decellularization methods for corneal tissue engineering. PLoS ONE 8:e66538, 2013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Philips, C., R. Cornelissen, and V. Carriel. Evaluation methods as quality control in the generation of decellularized peripheral nerve allografts. J. Neural Eng. 15:021003, 2018.

    Article  PubMed  Google Scholar 

  37. Roosens, A., P. Somers, F. De Somer, V. Carriel, G. Van Nooten, and R. Cornelissen. Impact of detergent-based decellularization methods on porcine tissues for heart valve engineering. Ann. Biomed. Eng. 44:2827–2839, 2016.

    Article  PubMed  Google Scholar 

  38. Sandrock, Jr., A. W., and W. D. Matthew. Identification of a peripheral nerve neurite growth-promoting activity by development and use of an in vitro bioassay. Proc. Natl. Acad. Sci. USA 84:6934–6938, 1987.

    Article  CAS  PubMed  Google Scholar 

  39. Siemionow, M., and G. Brzezicki. Chapter 8 current techniques and concepts in peripheral nerve repair. In: International Review of Neurobiology, edited by S. Geuna, P. Tos, and B. Battiston. Cambridge: Academic Press, 2009, pp. 141–172.

    Google Scholar 

  40. Sondell, M., G. Lundborg, and M. Kanje. Regeneration of the rat sciatic nerve into allografts made acellular through chemical extraction. Brain Res. 795:44–54, 1998.

    Article  CAS  PubMed  Google Scholar 

  41. Sridharan, R., R. B. Reilly, and C. T. Buckley. Decellularized grafts with axially aligned channels for peripheral nerve regeneration. J. Mech. Behav. Biomed. Mater. 41:124–135, 2015.

    Article  PubMed  Google Scholar 

  42. Stocum, D. L. Regenerative Biology and Medicine. Boston: Elsevier Academic Press, 2006.

    Book  Google Scholar 

  43. Walsh, S., J. Biernaskie, S. W. Kemp, and R. Midha. Supplementation of acellular nerve grafts with skin derived precursor cells promotes peripheral nerve regeneration. Neuroscience 164:1097–1107, 2009.

    Article  CAS  PubMed  Google Scholar 

  44. Wang, Q., C. Zhang, L. Zhang, W. Guo, G. Feng, S. Zhou, Y. Zhang, T. Tian, Z. Li, and F. Huang. The preparation and comparison of decellularized nerve scaffold of tissue engineering. J. Biomed. Mater. Res. A 102:4301–4308, 2014.

    PubMed  Google Scholar 

  45. Whitlock, E. L., S. H. Tuffaha, J. P. Luciano, Y. Yan, D. A. Hunter, C. K. Magill, A. M. Moore, A. Y. Tong, S. E. Mackinnon, and G. H. Borschel. Processed allografts and type I collagen conduits for repair of peripheral nerve gaps. Muscle Nerve 39:787–799, 2009.

    Article  CAS  PubMed  Google Scholar 

  46. Zhao, Z., Y. Wang, J. Peng, Z. Ren, L. Zhang, Q. Guo, W. Xu, and S. Lu. Improvement in nerve regeneration through a decellularized nerve graft by supplementation with bone marrow stromal cells in fibrin. Cell Transplant. 23:97–110, 2014.

    Article  PubMed  Google Scholar 

  47. Zilic, L., S. P. Wilshaw, and J. W. Haycock. Decellularisation and histological characterisation of porcine peripheral nerves. Biotechnol. Bioeng. 113:2041–2053, 2016.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This study was supported by the Grant FIS PI14/1343 and FIS PI17/0393 of the Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica from the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (co-financed by FEDER funds, European Union) and by the Special Research Fund (BOF 14/IOP/045) from Ghent University, Belgium. The authors would like to thank Dr. Víctor Domingo Roa, Amalia de la Rosa and Concepción Villegas (Experimental Unit of the University Hospital Virgen de las Nieves, Granada) for their assistance with the laboratory animals, Leen Pieters (Ghent University) for the technical assistance with the TEM and Lisa Van Vlaenderen (Ghent University) for the assistance with the ex vivo cytocompatibility.

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Correspondence to Charlot Philips.

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Associate Editor Emmanuel Opara oversaw the review of this article.

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Philips, C., Campos, F., Roosens, A. et al. Qualitative and Quantitative Evaluation of a Novel Detergent-Based Method for Decellularization of Peripheral Nerves. Ann Biomed Eng 46, 1921–1937 (2018). https://doi.org/10.1007/s10439-018-2082-y

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