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Decellularized Ear Tissues as Scaffolds for Stem Cell Differentiation

  • Peter A. SantiEmail author
  • Shane B. Johnson
Research Article

Abstract

Permanent sensorineural hearing loss is a major medical problem and is due to the loss of hair cells and subsequently spiral ganglion neurons in the cochlea. Since these cells lack the capacity of renewal in mammals, their regeneration would be an optimal solution to reverse hearing loss. In other tissues, decellularized extracellular matrix (ECM) has been used as a mechanical and biochemical scaffold for the induction of stem and other cells toward a target tissue phenotype. Such induced cells have been used for tissue and organ transplants in preclinical animal and human clinical applications. This paper reports for the first time the decellularization of the cochlea and identification of remaining laminin and collagen type IV as a first step in preparing an ECM scaffold for directing stem cells toward an auditory lineage. Fresh ear tissues were removed from euthanized mice, a rat and a human and processed for decellularization using two different detergent extraction methods. Cochleas were imaged with scanning thin-sheet laser imaging microscopy (sTSLIM) and brightfield microscopy. Detergent treatment of fresh tissue removed all cells as evidenced by lack of H&E and DNA staining of the membranous labyrinth while preserving components of the ECM. The organ of Corti was completely removed, as were spiral ganglion neurons, which appeared as hollow sheaths and tubes of basal lamina (BL) material. Cells of the stria vascularis were removed and its only vestige left was its laterally linking network of capillary BL that appeared to “float” in the endolymphatic space. Laminin and type IV collagen were detected in the ECM after decellularization and were localized in vascular, neural and epithelial BL. Further work is necessary to attempt to seed neural and other stem cells into the decellularized ECM to hopefully induce differentiation and subsequent in vivo engraftment into damaged cochleas.

Keywords

tissue engineering decellularization extracellular matrix cochlea vestibular stem cells 

Notes

Acknowledgments

The authors acknowledge the assistance by Sebahattin Cureoglu, Monika Schachern, and Meredith Adams for human temporal bone processing. Funding was provided to PAS by the NIDCD (RO1DC007588-04), an ARRA supplement (RO1DC007588-03S1), human temporal bone processing by NIDCD (U24DC011968-01), the Capita Foundation, and the Lions Hearing Foundation.

Supplementary material

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Supplementary material 1 (MOV 6554 KB)
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Supplementary material 2 (MOV 17259 KB)

References

  1. Baiguera S, Jungebluth P, Burns A, Mavilia C, Haag J, De Coppi P, Macchiarini P (2010) Tissue engineered human tracheas for in vivo implantation. Biomaterials 33:780–789Google Scholar
  2. Corrales CE, Pan L, Li H, Liberman MC, Heller S, Edge ASB (2006) Engraftment and differentiation of embryonic stem cell-derived neural progenitor cells in the cochlear nerve trunk: growth of processes into the organ of Corti. J Neurobio 66:1489–1500CrossRefGoogle Scholar
  3. Crapo PM, Gilbert TW, Badylak SF (2011) An overview of tissue and whole organ decellularization processes. Biomaterials 32:3233–3243PubMedCrossRefGoogle Scholar
  4. Erdbrügger W, Konertz W, Dohmen PM, Posner S, Ellerbrok H, Brodde OE, Robenek H, Modersohn D, Pruss A, Holinski S, Stein-Konertz M, Pauli G (2006) Decellularized xenogenic heart valves reveal remodeling and growth potential in vivo. Tissue Eng 12:2059–2068PubMedCrossRefGoogle Scholar
  5. Evans A, Euteneuer S, Chavez E, Mullen L, Hui E, Bhatia S, Ryan A (2007) Laminin and fibronectin modulate inner ear spiral ganglion neurite outgrowth in an in vitro alternate choice assay. Bev Neurobiol 67:1721–1730CrossRefGoogle Scholar
  6. Gilbert TW, Sellaro TL, Badylak SF (2006) Decellularization of tissues and organs. Biomaterials 27:3675–3683PubMedGoogle Scholar
  7. Hori R, Nakagawa T, Sakamoto T, Matsuoka Y, Takebayashi S, Ito J (2007) Pharmacological inhibition of Notch signaling in the mature guinea pig cochlea. Neuroreport 18:1911–1914PubMedCrossRefGoogle Scholar
  8. Hynes RO (2009) The extracellular matrix: not just pretty fibrils. Science 326:1219–1261CrossRefGoogle Scholar
  9. Iguchi F, Nakagawa T, Tateya I, Kim TS, Endo T, Taniguchi Z, Naito Y, Ito J (2003) Trophic support of mouse inner ear by neural stem cell transplantation. Neuroreport 14:77–80PubMedCrossRefGoogle Scholar
  10. Izumikawa M, Minoda R, Kawamoto K, Abrashkin KA, Swiderski DL, Dolan DF, Brough DE, Raphael Y (2005) Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals. Nat Med 11:271–276PubMedCrossRefGoogle Scholar
  11. Kesser BW, Lalwani AK (2009) Gene therapy and stem cell transplantation: strategies for hearing restoration. Adv Otorhionlaryngol 66:64–86Google Scholar
  12. Khan SA, Nelson MS et al (2008) Endogenous heparan sulfate and heparin modulate bone morphogenetic protein-4 signaling and activity. Am J Physiol Cell Physiol 294:C1387–C1397PubMedCrossRefGoogle Scholar
  13. Lang H, Schulte BA, Goddard JC, Hedrick M, Schulte JB, Wei L, Schmiedt RA (2008) Transplantation of mouse embryonic stem cells into the cochlea of an auditory-neuropathy animal model: effects of timing after injury. JARO 9:225–240PubMedCrossRefGoogle Scholar
  14. Löwenheim H, Furness DN, Kil J, Zinn C, Gültig K, Fero ML, Frost D, Gummer AW, Roberts JM, Rubel EW, Hackney CM, Zenner HP (1999) Gene disruption of p27K(Kip1) allows cell proliferation in the postnatal and adult organ of Corti. PNAS 96:4048–4088CrossRefGoogle Scholar
  15. Martinez-Monedero R, Corrales CE, Cuajungco MP, Heller S, Edge AS (2006) Reinnervation of hair cells by auditory neurons after selective removal of spiral ganglion neurons. J Neurobiol 66:319–331PubMedCrossRefGoogle Scholar
  16. Nagao RJ, Lundy S, Khaing ZZ, Schmidt CE (2011) Functional characterization of optimized acellular peripheral nerve graft in a rat sciatic nerve injury mode.l. Neurol Res 33:600–608PubMedCrossRefGoogle Scholar
  17. Nayagam B, Minter RL (2011) A comparison of in vitro treatments for directing the differentiation of stem cells towards a sensory neural fate. Am J Otol 33:37–46CrossRefGoogle Scholar
  18. Oshima K, Shin K, Diensthuber M, Peng AW, Ricci AJ, Heller S (2010) Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells. Cell 141:704–716PubMedCrossRefGoogle Scholar
  19. Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI, Taylor DA (2008) Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med 14:213–221PubMedCrossRefGoogle Scholar
  20. Petersen TH, Calle EA, Zhao L, Lee EJ, Gui L, Raredon MB, Gavrilov K, Yi T, Zhuang ZW, Breuer C, Herzog E, Niklason LE (2010) Tissue-engineered lungs for in vivo implantation. Science 329:538–541PubMedCrossRefGoogle Scholar
  21. Ross E, Williams M, Hamazaki T, Terada N, Clapp W, Adin C, Ellison G, Jorgensen M, Batich C (2009) Embryonic stem cells proliferate and differentiate when seeded into kidney scaffolds. J Am Soc Nephrol 20:2338–2347PubMedCrossRefGoogle Scholar
  22. Santi PA, Larson JT, Furcht LT, Economou TS (1989) Immunohistochemical localization of fibronectin in the chinchilla cochlea. Hear Res 39:91–101PubMedCrossRefGoogle Scholar
  23. Santi PA, Johnson SB, Hillenbrand M, GrandPre PZ, Glass TJ, Leger JR (2009) Thin-sheet laser imaging microscopy for optical sectioning of thick tissues. Biotechniques 46:287–294PubMedGoogle Scholar
  24. Schröter TJ, Johnson SB, John K, Santi PA (2012) Scanning thin-sheet laser imaging microscopy (sTSLIM) with structured illumination and HiLo background rejection. Biomedical Opt Express 1:170–177CrossRefGoogle Scholar
  25. Tsuprun V, Santi PA (1999) Ultrastructure and immunohistochemical identification of the extracellular matrix of the chinchilla cochlea. Hear Res 129:35–49PubMedCrossRefGoogle Scholar

Copyright information

© Association for Research in Otolaryngology 2012

Authors and Affiliations

  1. 1.Department of OtolaryngologyUniversity of MinnesotaMinneapolisUSA

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