Skip to main content
SpringerLink
Log in

Redirection of Neurite Outgrowth by Coupling Chondroitin Sulfate Proteoglycans to Polymer Membranes

  • Published:
Annals of Biomedical Engineering Aims and scope Submit manuscript

Abstract

Upon nerve injury, the body creates an environment consisting of permissive and non-permissive cues that instruct the function of cells involved in nerve repair. Among other roles, the developing extracellular matrix (ECM) acts as an underlying substrate to guide the union of neurites extending from the proximal stump for bridging the nerve gap. Chondroitin sulfate proteoglycans (CSPGs) are present in the nerve ECM and inhibit axon growth, potentially providing molecular cues to prevent aberrant growth and direct regeneration. In this study, we examined the potential of CSPGs to guide dorsal root ganglia (DRG) neurite outgrowth when freely available in the media or presented from a polymeric membrane. Soluble CSPGs added to the media of DRG explant cultures inhibited neurite outgrowth without spatial bias, caused retraction of axons, and decreased neurite extension in a dose-dependent manner. Poly-l-lactic acid membranes were chemically treated to enhance adsorption of CSPGs to the surface. CSPGs bound to 1,6-hexanediamine-treated membranes directed the orientation of neurite outgrowth, as neurites avoided bound CSPGs and a higher number and percentage grew on treated membranes lacking CSPGs. DRG explants cultured on CSPG-coated membranes without 1,6-hexanediamine-treatment had a smaller number of neurites and decreased neurite outgrowth, suggesting CSPGs were not retained on the membrane and were released into the culture medium. Taken together, these data demonstrate the potential of CSPG presentation to guide axonal growth. This approach offers a strategy to improve upon existing nerve guidance conduits by incorporating axon guidance molecules to direct nerve regeneration.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Aframian, D. J., R. S. Redman, S. Yamano, J. Nikolovski, E. Cukierman, K. M. Yamada, M. F. Kriete, W. D. Swaim, D. J. Mooney, and B. J. Baum. Tissue compatibility of two biodegradable tubular scaffolds implanted adjacent to skin or buccal mucosa in mice. Tissue Eng. 8(4):649–659, 2002.

    Article  CAS  PubMed  Google Scholar 

  2. Ara, J., P. Bannerman, A. Hahn, S. Ramirez, and D. Pleasure. Modulation of sciatic nerve expression of class 3 semaphorins by nerve injury. Neurochem. Res. 29(6):1153–1159, 2004.

    Article  CAS  PubMed  Google Scholar 

  3. Bertelli, J. A., M. Taleb, J. C. Mira, and M. F. Ghizoni. Variation in nerve autograft length increases fibre misdirection and decreases pruning effectiveness: an experimental study in the rat median nerve. Neurol. Res. 27(6):657–665, 2005.

    Article  CAS  PubMed  Google Scholar 

  4. Braunewell, K. H., R. Martini, R. LeBaron, H. Kresse, A. Faissner, B. Schmitz, and M. Schachner. Up-regulation of a chondroitin sulphate epitope during regeneration of mouse sciatic nerve: evidence that the immunoreactive molecules are related to the chondroitin sulphate proteoglycans decorin and versican. Eur. J. Neurosci. 7(4):792–804, 1995.

    Article  CAS  PubMed  Google Scholar 

  5. Ceballos, D., X. Navarro, N. Dubey, G. Wendelschafer-Crabb, W. R. Kennedy, and R. T. Tranquillo. Magnetically aligned collagen gel filling a collagen nerve guide improves peripheral nerve regeneration. Exp. Neurol. 158(2):290–300, 1999.

    Article  CAS  PubMed  Google Scholar 

  6. Chernousov, M. A., and D. J. Carey. Schwann cell extracellular matrix molecules and their receptors. Histol. Histopathol. 15(2):593–601, 2000.

    CAS  PubMed  Google Scholar 

  7. Choi, D., and G. Raisman. After facial nerve damage, regenerating axons become aberrant throughout the length of the nerve and not only at the site of the lesion: an experimental study. Br. J. Neurosurg. 18(1):45–48, 2004.

    Article  CAS  PubMed  Google Scholar 

  8. Clement, A. M., K. Sugahara, and A. Faissner. Chondroitin sulfate E promotes neurite outgrowth of rat embryonic day 18 hippocampal neurons. Neurosci. Lett. 269(3):125–128, 1999.

    Article  CAS  PubMed  Google Scholar 

  9. Davies, J. E., X. Tang, J. W. Denning, S. J. Archibald, and S. J. Davies. Decorin suppresses neurocan, brevican, phosphacan and NG2 expression and promotes axon growth across adult rat spinal cord injuries. Eur. J. Neurosci. 19(5):1226–1242, 2004.

    Article  PubMed  Google Scholar 

  10. de Ruiter, G. C., M. J. Malessy, A. O. Alaid, R. J. Spinner, J. K. Engelstad, E. J. Sorenson, K. R. Kaufman, P. J. Dyck, and A. J. Windebank. Misdirection of regenerating motor axons after nerve injury and repair in the rat sciatic nerve model. Exp. Neurol. 211(2):339–350, 2008.

    Article  PubMed Central  PubMed  Google Scholar 

  11. Dubey, N., P. C. Letourneau, and R. T. Tranquillo. Neuronal contact guidance in magnetically aligned fibrin gels: effect of variation in gel mechano-structural properties. Biomaterials 22(10):1065–1075, 2001.

    Article  CAS  PubMed  Google Scholar 

  12. Evans, G. R., K. Brandt, S. Katz, P. Chauvin, L. Otto, M. Bogle, B. Wang, R. K. Meszlenyi, L. Lu, A. G. Mikos, et al. Bioactive poly(l-lactic acid) conduits seeded with Schwann cells for peripheral nerve regeneration. Biomaterials 23(3):841–848, 2002.

    Article  CAS  PubMed  Google Scholar 

  13. Evans, G. R., K. Brandt, M. S. Widmer, L. Lu, R. K. Meszlenyi, P. K. Gupta, A. G. Mikos, J. Hodges, J. Williams, A. Gurlek, et al. In vivo evaluation of poly(l-lactic acid) porous conduits for peripheral nerve regeneration. Biomaterials 20(12):1109–1115, 1999.

    Article  CAS  PubMed  Google Scholar 

  14. Fernaud-Espinosa, I., M. Nieto-Sampedro, and P. Bovolenta. Differential effects of glycosaminoglycans on neurite outgrowth from hippocampal and thalamic neurones. J. Cell Sci. 107(Pt 6):1437–1448, 1994.

    CAS  PubMed  Google Scholar 

  15. Funakoshi, H., J. Frisen, G. Barbany, T. Timmusk, O. Zachrisson, V. M. Verge, and H. Persson. Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve. J. Cell Biol. 123(2):455–465, 1993.

    Article  CAS  PubMed  Google Scholar 

  16. Giger, R. J., J. F. Cloutier, A. Sahay, R. K. Prinjha, D. V. Levengood, S. E. Moore, S. Pickering, D. Simmons, S. Rastan, F. S. Walsh, et al. Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins. Neuron 25(1):29–41, 2000.

    Article  CAS  PubMed  Google Scholar 

  17. Hammarberg, H., F. Piehl, S. Cullheim, J. Fjell, T. Hokfelt, and K. Fried. GDNF mRNA in Schwann cells and DRG satellite cells after chronic sciatic nerve injury. NeuroReport 7(4):857–860, 1996.

    Article  CAS  PubMed  Google Scholar 

  18. Hawkins, R. L., and N. W. Seeds. Protease inhibitors influence the direction of neurite outgrowth. Brain Res. Dev. Brain Res. 45(2):203–209, 1989.

    Article  CAS  PubMed  Google Scholar 

  19. He, Z., and M. Tessier-Lavigne. Neuropilin is a receptor for the axonal chemorepellent Semaphorin III. Cell 90(4):739–751, 1997.

    Article  CAS  PubMed  Google Scholar 

  20. Heumann, R., D. Lindholm, C. Bandtlow, M. Meyer, M. J. Radeke, T. P. Misko, E. Shooter, and H. Thoenen. Differential regulation of mRNA encoding nerve growth factor and its receptor in rat sciatic nerve during development, degeneration, and regeneration: role of macrophages. Proc. Natl. Acad. Sci. USA 84(23):8735–8739, 1987.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Hoke, A., C. Cheng, and D. W. Zochodne. Expression of glial cell line-derived neurotrophic factor family of growth factors in peripheral nerve injury in rats. NeuroReport 11(8):1651–1654, 2000.

    Article  CAS  PubMed  Google Scholar 

  22. Jones, L. L., D. Sajed, and M. H. Tuszynski. Axonal regeneration through regions of chondroitin sulfate proteoglycan deposition after spinal cord injury: a balance of permissiveness and inhibition. J. Neurosci. 23(28):9276–9288, 2003.

    CAS  PubMed  Google Scholar 

  23. Kofron, C. M., V. J. Fong, and D. Hoffman-Kim. Neurite outgrowth at the interface of 2D and 3D growth environments. J. Neural Eng. 6(1):016002, 2009.

    Article  PubMed  Google Scholar 

  24. Lindholm, T., M. K. Skold, A. Suneson, T. Carlstedt, S. Cullheim, and M. Risling. Semaphorin and neuropilin expression in motoneurons after intraspinal motoneuron axotomy. NeuroReport 15(4):649–654, 2004.

    Article  CAS  PubMed  Google Scholar 

  25. Lubinska, L. Patterns of Wallerian degeneration of myelinated fibres in short and long peripheral stumps and in isolated segments of rat phrenic nerve. Interpretation of the role of axoplasmic flow of the trophic factor. Brain Res. 233(2):227–240, 1982.

    Article  CAS  PubMed  Google Scholar 

  26. Mahoney, M. J., R. R. Chen, J. Tan, and W. M. Saltzman. The influence of microchannels on neurite growth and architecture. Biomaterials 26(7):771–778, 2005.

    Article  CAS  PubMed  Google Scholar 

  27. McGeachie, A. B., K. Koishi, T. Imamura, and I. S. McLennan. Fibroblast growth factor-5 is expressed in Schwann cells and is not essential for motoneurone survival. Neuroscience 104(3):891–899, 2001.

    Article  CAS  PubMed  Google Scholar 

  28. McKeon, R. J., R. C. Schreiber, J. S. Rudge, and J. Silver. Reduction of neurite outgrowth in a model of glial scarring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes. J. Neurosci. 11(11):3398–3411, 1991.

    CAS  PubMed  Google Scholar 

  29. Meijering, E., M. Jacob, J. C. Sarria, P. Steiner, H. Hirling, and M. Unser. Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. Cytometry A 58(2):167–176, 2004.

    Article  CAS  PubMed  Google Scholar 

  30. Meyer, M., I. Matsuoka, C. Wetmore, L. Olson, and H. Thoenen. Enhanced synthesis of brain-derived neurotrophic factor in the lesioned peripheral nerve: different mechanisms are responsible for the regulation of BDNF and NGF mRNA. J. Cell Biol. 119(1):45–54, 1992.

    Article  CAS  PubMed  Google Scholar 

  31. Mizuno, H., H. Warita, M. Aoki, and Y. Itoyama. Accumulation of chondroitin sulfate proteoglycans in the microenvironment of spinal motor neurons in amyotrophic lateral sclerosis transgenic rats. J. Neurosci. Res. 86(11):2512–2523, 2008.

    Article  CAS  PubMed  Google Scholar 

  32. Morrison, S., L. S. Mitchell, M. S. Ecob-Prince, I. R. Griffiths, C. E. Thomson, J. A. Barrie, and D. Kirkham. P0 gene expression in cultured Schwann cells. J. Neurocytol. 20(9):769–780, 1991.

    Article  CAS  PubMed  Google Scholar 

  33. National Center for Health Statistics based on Classification of Diseases tR. Clinical modification for the following categories: ICD-9 CM code: 04.3, 04.5, 04.6, 04.7.

  34. Naveilhan, P., W. M. ElShamy, and P. Ernfors. Differential regulation of mRNAs for GDNF and its receptors Ret and GDNFR alpha after sciatic nerve lesion in the mouse. Eur. J. Neurosci. 9(7):1450–1460, 1997.

    Article  CAS  PubMed  Google Scholar 

  35. Omura, T., M. Sano, K. Omura, T. Hasegawa, M. Doi, T. Sawada, and A. Nagano. Different expressions of BDNF, NT3, and NT4 in muscle and nerve after various types of peripheral nerve injuries. J. Peripher. Nerv. Syst. 10(3):293–300, 2005.

    Article  CAS  PubMed  Google Scholar 

  36. Pierucci, A., E. A. de Duek, and A. L. de Oliveira. Peripheral nerve regeneration through biodegradable conduits prepared using solvent evaporation. Tissue Eng. Part A 14(5):595–606, 2008.

    Article  CAS  PubMed  Google Scholar 

  37. Sango, K., A. Oohira, K. Ajiki, A. Tokashiki, M. Horie, and H. Kawano. Phosphacan and neurocan are repulsive substrata for adhesion and neurite extension of adult rat dorsal root ganglion neurons in vitro. Exp. Neurol. 182(1):1–11, 2003.

    Article  CAS  PubMed  Google Scholar 

  38. Scarlato, M., J. Ara, P. Bannerman, S. Scherer, and D. Pleasure. Induction of neuropilins-1 and -2 and their ligands, Sema3A, Sema3F, and VEGF, during Wallerian degeneration in the peripheral nervous system. Exp. Neurol. 183(2):489–498, 2003.

    Article  CAS  PubMed  Google Scholar 

  39. Scarlato, M., T. Xu, P. Bannerman, J. Beesley, U. R. Reddy, A. Rostami, S. S. Scherer, and D. Pleasure. Axon-Schwann cell interactions regulate the expression of fibroblast growth factor-5 (FGF-5). J. Neurosci. Res. 66(1):16–22, 2001.

    Article  CAS  PubMed  Google Scholar 

  40. Schlaepfer, W. W. Structural alterations of peripheral nerve induced by the calcium ionophore A23187. Brain Res. 136(1):1–9, 1977.

    Article  CAS  PubMed  Google Scholar 

  41. Snow, D. M., E. M. Brown, and P. C. Letourneau. Growth cone behavior in the presence of soluble chondroitin sulfate proteoglycan (CSPG), compared to behavior on CSPG bound to laminin or fibronectin. Int. J. Dev. Neurosci. 14(3):331–349, 1996.

    Article  CAS  PubMed  Google Scholar 

  42. Snow, D. M., and P. C. Letourneau. Neurite outgrowth on a step gradient of chondroitin sulfate proteoglycan (CS-PG). J. Neurobiol. 23(3):322–336, 1992.

    Article  CAS  PubMed  Google Scholar 

  43. Stewart, H. J., P. A. Eccleston, K. R. Jessen, and R. Mirsky. Interaction between cAMP elevation, identified growth factors, and serum components in regulating Schwann cell growth. J. Neurosci. Res. 30(2):346–352, 1991.

    Article  CAS  PubMed  Google Scholar 

  44. Tansey, K. E., J. L. Seifert, B. Botterman, M. R. Delgado, and M. I. Romero. Peripheral nerve repair through multi-luminal biosynthetic implants. Ann. Biomed. Eng. 39(6):1815–1828, 2011.

    Article  CAS  PubMed  Google Scholar 

  45. Tomita, K., T. Kubo, K. Matsuda, R. Hattori, T. Fujiwara, K. Yano, and K. Hosokawa. Effect of conduit repair on aberrant motor axon growth within the nerve graft in rats. Microsurgery 27(5):500–509, 2007.

    Article  PubMed  Google Scholar 

  46. Trupp, M., M. Ryden, H. Jornvall, H. Funakoshi, T. Timmusk, E. Arenas, and C. F. Ibanez. Peripheral expression and biological activities of GDNF, a new neurotrophic factor for avian and mammalian peripheral neurons. J. Cell Biol. 130(1):137–148, 1995.

    Article  CAS  PubMed  Google Scholar 

  47. Vial, J. D. The early changes in the axoplasm during Wallerian degeneration. J. Biophys. Biochem. Cytol. 4(5):551–555, 1958.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  48. Wood, M. D., M. R. MacEwan, A. R. French, A. M. Moore, D. A. Hunter, S. E. Mackinnon, D. W. Moran, G. H. Borschel, and S. E. Sakiyama-Elbert. Fibrin matrices with affinity-based delivery systems and neurotrophic factors promote functional nerve regeneration. Biotechnol. Bioeng. 106(6):970–979, 2010.

    Article  CAS  PubMed  Google Scholar 

  49. Wood, M. D., A. M. Moore, D. A. Hunter, S. Tuffaha, G. H. Borschel, S. E. Mackinnon, and S. E. Sakiyama-Elbert. Affinity-based release of glial-derived neurotrophic factor from fibrin matrices enhances sciatic nerve regeneration. Acta Biomater. 5(4):959–968, 2009.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  50. Yao, L., S. Wang, W. Cui, R. Sherlock, C. O’Connell, G. Damodaran, A. Gorman, A. Windebank, and A. Pandit. Effect of functionalized micropatterned PLGA on guided neurite growth. Acta Biomater. 5(2):580–588, 2009.

    Article  CAS  PubMed  Google Scholar 

  51. Yi, J. H., Y. Katagiri, B. Susarla, D. Figge, A. J. Symes, and H. M. Geller. Alterations in sulfated chondroitin glycosaminoglycans following controlled cortical impact injury in mice. J. Comp. Neurol. 520(15):3295–3313, 2012.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  52. Yu, X., and R. V. Bellamkonda. Dorsal root ganglia neurite extension is inhibited by mechanical and chondroitin sulfate-rich interfaces. J. Neurosci. Res. 66(2):303–310, 2001.

    Article  CAS  PubMed  Google Scholar 

  53. Zhu, Y., C. Gao, Y. Liu, and J. Shen. Endothelial cell functions in vitro cultured on poly(l-lactic acid) membranes modified with different methods. J. Biomed. Mater. Res. A 69(3):436–443, 2004.

    PubMed  Google Scholar 

  54. Zuo, J., Y. J. Hernandez, and D. Muir. Chondroitin sulfate proteoglycan with neurite-inhibiting activity is up-regulated following peripheral nerve injury. J. Neurobiol. 34(1):41–54, 1998.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, University of California, Davis, 451 Health Sciences Dr., GBSF 2303, Davis, CA, 95616, USA

    Alan J. Man & J. Kent Leach

  2. Institute for Pediatric Regenerative Medicine, Shriners Hospital for Children Northern California, 2425 Stockton Blvd., Sacramento, CA, 95817, USA

    Alan J. Man & Peter Bannerman

  3. Department of Cell Biology, UC Davis School of Medicine, UC Davis Medical Center, Sacramento, CA, 95817, USA

    Peter Bannerman

Authors
  1. Alan J. Man
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. Kent Leach
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peter Bannerman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to J. Kent Leach or Peter Bannerman.

Additional information

Associate Editor Scott I. Simon oversaw the review of this article.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig. 1. Soluble CSPG inhibits neurite extension. Time-lapse phase contrast images of DRG explants cultured in basal media with a CSPG concentration of 25 μg/mL were taken at 1 h intervals for 48 h. DRG explants were cultured in normal growth media for 24 h prior to exchange with CSPG media. Images at 10× magnification. Supplementary material 1 (MOV 2105 kb)

Supplementary Fig. 2. DRG explants cultured in basal media exhibited normal neurite extension. Time-lapse phase contrast images of DRG explants cultured in basal media were taken at 1 h intervals for 48 h. DRG explants were cultured in normal growth media for 24 h prior to exchange with basal media. Images at 10× magnification. Supplementary material 2 (MOV 1555 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Man, A.J., Leach, J.K. & Bannerman, P. Redirection of Neurite Outgrowth by Coupling Chondroitin Sulfate Proteoglycans to Polymer Membranes. Ann Biomed Eng 42, 1271–1281 (2014). https://doi.org/10.1007/s10439-014-0991-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10439-014-0991-y

Keywords

Search

Navigation