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Collagen-Dependent Neurite Outgrowth and Response to Dynamic Deformation in Three-Dimensional Neuronal Cultures

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Abstract

In vitro models of brain injury that use thick 3-D cultures and control extracellular matrix constituents allow evaluation of cell–matrix interactions in a more physiologically relevant configuration than traditional 2-D cultures. We have developed a 3-D cell culture system consisting of primary rat cortical neurons distributed throughout thick (>500 μm) gels consisting of type IV collagen (Col) conjugated to agarose. Neuronal viability and neurite outgrowth were examined for a range of agarose (AG) percentages (1.0–3.0%) and initial collagen concentrations ([Col]i; 0–600 μg/mL). In unmodified AG, 1.5% gels supported viable cultures with significant neurite outgrowth, which was not found at lower (≤1.0%) concentrations. Varying [Col]i in 1.25% AG revealed the formation of dense, 3-D neurite networks at [Col]i of 300 μg/mL, while neurons in unmodified AG and at higher [Col]i (600 μg/mL) exhibited significantly less neurite outgrowth; although, neuronal survival did not vary with [Col]i. The effect of [Col]i on acute neuronal response following high magnitude, high rate shear deformation (0.50 strain, 30 s−1 strain rate) was evaluated in 1.5% AG for [Col]i of 30, 150, and 300 μg/mL, which supported cultures with similar baseline viability and neurite outgrowth. Conjugation of Col to AG also increased the complex modulus of the hydrogel. Following high rate deformation, neuronal viability significantly decreased with increasing [Col]i, implicating cell–matrix adhesions in acute mechanotransduction events associated with traumatic loading. These results suggest interrelated roles for matrix mechanical properties and receptor-mediated cell–matrix interactions in neuronal viability, neurite outgrowth, and transduction of high rate deformation. This model system may be further exploited for the elucidation of mechanotransduction mechanisms and cellular pathology following mechanical insult.

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References

  1. Adelson P. D., Dixon C. E., Kochanek P. M. (2000) Long-term dysfunction following diffuse traumatic brain injury in the immature rat. J. Neurotrauma 17:273–282

    PubMed  CAS  Google Scholar 

  2. Alenghat F. J., Ingber D. E. (2002) Mechanotransduction: all signals point to cytoskeleton, matrix, and integrins. Sci. STKE 2002:PE6

    PubMed  Google Scholar 

  3. Anderson K. L., Ferreira A. (2004) Alpha1 Integrin activation: a link between beta-amyloid deposition and neuronal death in aging hippocampal neurons. J. Neurosci. Res. 75:688–697

    Article  PubMed  CAS  Google Scholar 

  4. Arregui C. O., Carbonetto S., McKerracher L. (1994) Characterization of neural cell adhesion sites: point contacts are the sites of interaction between integrins and the cytoskeleton in PC12 cells. J. Neurosci. 14:6967–6977

    PubMed  CAS  Google Scholar 

  5. Balgude A. P., Yu X., Szymanski A., Bellamkonda R. V. (2001) Agarose gel stiffness determines rate of DRG neurite extension in 3D cultures. Biomaterials 22:1077–1084

    Article  PubMed  CAS  Google Scholar 

  6. Bellamkonda R., Ranieri J. P., Aebischer P. (1995) Laminin oligopeptide derivatized agarose gels allow three-dimensional neurite extension in vitro. J. Neurosci. Res. 41:501–509

    Article  PubMed  CAS  Google Scholar 

  7. Bellamkonda R., Ranieri J. P., Bouche N., Aebischer P. (1995) Hydrogel-based three-dimensional matrix for neural cells. J. Biomed. Mater. Res. 29:663–671

    Article  PubMed  CAS  Google Scholar 

  8. Bradshaw A. D., McNagny K. M., Gervin D. B., Cann G. M., Graf T., Clegg D. O. (1995) Integrin alpha 2 beta 1 mediates interactions between developing embryonic retinal cells and collagen. Development 121:3593–3602

    PubMed  CAS  Google Scholar 

  9. Carmeliet G., Himpens B., Cassiman J. J. (1994) Selective increase in the binding of the alpha 1 beta 1 integrin for collagen type IV during neurite outgrowth of human neuroblastoma TR 14 cells. J. Cell Sci. 107(Pt 12):3379–3392

    PubMed  CAS  Google Scholar 

  10. Cavalcanti-Adam E. A., Tomakidi P., Bezler M., Spatz J. P. (2005) Geometric organization of the extracellular matrix in the control of integrin-mediated adhesion and cell function in osteoblasts. Prog. Orthod. 6:232–237

    PubMed  Google Scholar 

  11. Cukierman E., Pankov R., Stevens D. R., Yamada K. M. (2001) Taking cell–matrix adhesions to the third dimension. Science 294:1708–1712

    Article  PubMed  CAS  Google Scholar 

  12. Cukierman E., Pankov R., Yamada K. M. (2002) Cell interactions with three-dimensional matrices. Curr. Opin. Cell Biol. 14:633–639

    Article  PubMed  CAS  Google Scholar 

  13. Danen E. H., Sonnenberg A. (2003) Integrins in regulation of tissue development and function. J. Pathol. 201:632–641

    Article  PubMed  CAS  Google Scholar 

  14. Dash P. K., Mach S. A., Moore A. N. (2002) The role of extracellular signal-regulated kinase in cognitive and motor deficits following experimental traumatic brain injury. Neuroscience 114:755–767

    Article  PubMed  CAS  Google Scholar 

  15. De Arcangelis A., Georges-Labouesse E. (2000) Integrin and ECM functions: roles in vertebrate development. Trends Genet. 16:389–395

    Article  PubMed  Google Scholar 

  16. Dillon G. P., Yu X., Sridharan A., Ranieri J. P., Bellamkonda R. V. (1998) The influence of physical structure and charge on neurite extension in a 3D hydrogel scaffold. J. Biomater. Sci. Polym. Ed 9:1049–1069

    PubMed  CAS  Google Scholar 

  17. Dodla M. C., Bellamkonda R. V. (2006) Anisotropic scaffolds facilitate enhanced neurite extension in vitro. J. Biomed. Mater. Res. A. 78:213–221

    PubMed  Google Scholar 

  18. Dolz R., Engel J., Kuhn K. (1988) Folding of collagen IV. Eur. J. Biochem. 178:357–366

    Article  PubMed  CAS  Google Scholar 

  19. Ellis E. F., McKinney J. S., Willoughby K. A., Liang S., Povlishock J. T. (1995) A new model for rapid stretch-induced injury of cells in culture: characterization of the model using astrocytes. J. Neurotrauma 12:325–339

    PubMed  CAS  Google Scholar 

  20. Fawcett J. W., Barker R. A., Dunnett S. B. (1995) Dopaminergic neuronal survival and the effects of bFGF in explant, three dimensional and monolayer cultures of embryonic rat ventral mesencephalon. Exp. Brain Res. 106:275–282

    Article  PubMed  CAS  Google Scholar 

  21. Fitzpatrick M. O., Dewar D., Teasdale G. M., Graham D. I. (1998) The neuronal cytoskeleton in acute brain injury. Br. J. Neurosurg. 12:313–317

    Article  PubMed  CAS  Google Scholar 

  22. Flanagan L. A., Ju Y. E., Marg B., Osterfield M., Janmey P. A. (2002) Neurite branching on deformable substrates. Neuroreport 13:2411–2415

    Article  PubMed  Google Scholar 

  23. Geddes D. M., Cargill R. S. II. (2001) An in vitro model of neural trauma: device characterization and calcium response to mechanical stretch. J. Biomech. Eng. 123:247–255

    Article  PubMed  CAS  Google Scholar 

  24. Geddes D. M., Cargill R. S. II, LaPlaca M. C. (2003) Mechanical stretch to neurons results in a strain rate and magnitude-dependent increase in plasma membrane permeability. J. Neurotrauma 20:1039–1049

    Article  PubMed  Google Scholar 

  25. Gefen A., Gefen N., Zhu Q., Raghupathi R., Margulies S. S. (2003) Age-dependent changes in material properties of the brain and braincase of the rat. J. Neurotrauma 20:1163–1677

    Article  PubMed  Google Scholar 

  26. Genis L., Chen Y., Shohami E., Michaelson D. M. (2000) Tau hyperphosphorylation in apolipoprotein E-deficient and control mice after closed head injury. J. Neurosci. Res. 60:559–564

    Article  PubMed  CAS  Google Scholar 

  27. Granet C., Laroche N., Vico L., Alexandre C., Lafage-Proust M. H. (1998) Rotating-wall vessels, promising bioreactors for osteoblastic cell culture: comparison with other 3D conditions. Med. Biol. Eng. Comput. 36:513–519

    Article  PubMed  CAS  Google Scholar 

  28. Gumbiner B. M., Yamada K. M. (1995) Cell-to-cell contact and extracellular matrix. Curr. Opin. Cell Biol. 7:615–618

    Article  PubMed  CAS  Google Scholar 

  29. Hamill O. P., Martinac B. (2001) Molecular basis of mechanotransduction in living cells. Physiol. Rev. 81:685–740

    PubMed  CAS  Google Scholar 

  30. Hoffman R. M. (1993) To do tissue culture in two or three dimensions? That is the question. Stem. Cells 11:105–111

    Article  PubMed  CAS  Google Scholar 

  31. Huh J. W., Laurer H. L., Raghupathi R., Helfaer M. A., Saatman K. E. (2002) Rapid loss and partial recovery of neurofilament immunostaining following focal brain injury in mice. Exp. Neurol. 175:198–208

    Article  PubMed  CAS  Google Scholar 

  32. Ingber D. E. (1997) Tensegrity: the architectural basis of cellular mechanotransduction. Annu. Rev. Physiol. 59:575–599

    Article  PubMed  CAS  Google Scholar 

  33. Jin H., Varner J. (2004) Integrins: roles in cancer development and as treatment targets. Br. J. Cancer 90:561–565

    Article  PubMed  CAS  Google Scholar 

  34. Kamm R. D., Kaazempur-Mofrad M. R. (2004) On the molecular basis for mechanotransduction. Mech. Chem. Biosyst. 1:201–209

    PubMed  Google Scholar 

  35. Kleinman H. K., McGarvey M. L., Hassell J. R., Star V. L., Cannon F. B., Laurie G. W., Martin G. R. (1986) Basement membrane complexes with biological activity. Biochemistry 25:312–318

    Article  PubMed  CAS  Google Scholar 

  36. Ko K. S., McCulloch C. A. (2001) Intercellular mechanotransduction: cellular circuits that coordinate tissue responses to mechanical loading. Biochem. Biophys. Res. Commun. 285:1077–1083

    Article  PubMed  CAS  Google Scholar 

  37. Krewson C., Chung S., Dai W., Saltzman W. (1994) Cell-aggregation and neurite growth in gels of extracellular-matrix molecules. Biotechnol. Bioeng. 43:555–562

    Article  CAS  PubMed  Google Scholar 

  38. Langlois, J., W. Rutland-Brown and K. Thomas. Traumatic brain injury in the United States: emergency department visits, hospitalizations, and deaths, 2004

  39. LaPlaca M. C., Cullen D. K., McLoughlin J. J., Cargill R. S. II (2005) High rate shear strain of three-dimensional neural cell Cultures: a new in vitro traumatic brain injury model. J. Biomech. 38:1093–1105

    Article  PubMed  Google Scholar 

  40. LaPlaca M. C., Lee V. M., Thibault L. E. (1997) An in vitro model of traumatic neuronal injury: loading rate-dependent changes in acute cytosolic calcium and lactate dehydrogenase release. J. Neurotrauma 14:355–368

    PubMed  CAS  Google Scholar 

  41. Li B. S., Zhang L., Gu J., Amin N. D., Pant H. C. (2000) Integrin alpha(1) beta(1)-mediated activation of cyclin-dependent kinase 5 activity is involved in neurite outgrowth and human neurofilament protein H Lys-Ser-Pro tail domain phosphorylation. J. Neurosci. 20:6055–6062

    PubMed  CAS  Google Scholar 

  42. Margulies S. S., Thibault L. E. (1992) A proposed tolerance criterion for diffuse axonal injury in man. J. Biomech. 25:917–923

    Article  PubMed  CAS  Google Scholar 

  43. Masi L., Franchi A., Santucci M., Danielli D., Arganini L., Giannone V., Formigli L., Benvenuti S., Tanini A., Beghe F., et al. (1992) Adhesion, growth, and matrix production by osteoblasts on collagen substrata. Calcif. Tissue Int. 51:202–212

    Article  PubMed  CAS  Google Scholar 

  44. Mori T., Wang X., Jung J. C., Sumii T., Singhal A. B., Fini M. E., Dixon C. E., Alessandrini A., Lo E. H. (2002) Mitogen-activated protein kinase inhibition in traumatic brain injury: in vitro and in vivo effects. J. Cereb. Blood Flow Metab. 22:444–452

    Article  PubMed  CAS  Google Scholar 

  45. Nakayama Y., Aoki Y., Niitsu H. (2001) Studies on the mechanisms responsible for the formation of focal swellings on neuronal processes using a novel in vitro model of axonal injury. J. Neurotrauma 18:545–554

    Article  PubMed  CAS  Google Scholar 

  46. O’Connor S. M., Andreadis J. D., Shaffer K. M., Ma W., Pancrazio J. J., Stenger D. A. (2000) Immobilization of neural cells in three-dimensional matrices for biosensor applications. Biosens. Bioelectron 14:871–881

    Article  PubMed  CAS  Google Scholar 

  47. O’Connor S. M., Stenger D. A., Shaffer K. M., Ma W. (2001) Survival and neurite outgrowth of rat cortical neurons in three-dimensional agarose and collagen gel matrices. Neurosci. Lett. 304:189–193

    Article  PubMed  CAS  Google Scholar 

  48. Povlishock J. T., Katz D. I. (2005) Update of neuropathology and neurological recovery after traumatic brain injury. J. Head Trauma Rehabil. 20:76–94

    PubMed  Google Scholar 

  49. Raghupathi R. (2004) Cell death mechanisms following traumatic brain injury. Brain Pathol. 14:215–222

    Article  PubMed  Google Scholar 

  50. Roskelley C. D., Desprez P. Y., Bissell M. J. (1994) Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction. Proc. Natl. Acad. Sci. USA 91:12378–12382

    Article  PubMed  CAS  Google Scholar 

  51. Schense J. C., Hubbell J. A. (2000) Three-dimensional migration of neurites is mediated by adhesion site density and affinity. J. Biol. Chem. 275:6813–6818

    Article  PubMed  CAS  Google Scholar 

  52. Schmeichel K. L., Bissell M. J. (2003) Modeling tissue-specific signaling and organ function in three dimensions. J. Cell Sci. 116:2377–2388

    Article  PubMed  CAS  Google Scholar 

  53. Sjaastad M. D., Angres B., Lewis R. S., Nelson W. J. (1994) Feedback regulation of cell-substratum adhesion by integrin-mediated intracellular Ca2+ signaling. Proc. Natl. Acad. Sci. USA 91:8214–8218

    Article  PubMed  CAS  Google Scholar 

  54. Smith D. H., Wolf J. A., Lusardi T. A., Lee V. M., Meaney D. F. (1999) High tolerance and delayed elastic response of cultured axons to dynamic stretch injury. J. Neurosci. 19:4263–4269

    PubMed  CAS  Google Scholar 

  55. Thurman D. J., Alverson C., Dunn K. A., Guerrero J., Sniezek J. E. (1999) Traumatic brain injury in the United States: a public health perspective. J. Head Trauma Rehabil. 14:602–615

    Article  PubMed  CAS  Google Scholar 

  56. Tomaselli K. J. (1991) Beta 1-integrin-mediated neuronal responses to extracellular matrix proteins. Ann. NY Acad. Sci. 633:100–104

    Article  PubMed  CAS  Google Scholar 

  57. Volbracht C., Chua B. T., Ng C. P., Bahr B. A., Hong W., Li P. (2005) The critical role of calpain versus caspase activation in excitotoxic injury induced by nitric oxide. J. Neurochem. 93:1280–1292

    Article  PubMed  CAS  Google Scholar 

  58. Wehrle-Haller B., Imhof B. A. (2003) Integrin-dependent pathologies. J. Pathol. 200:481–487

    Article  PubMed  CAS  Google Scholar 

  59. Willits R. K., Skornia S. L. (2004) Effect of collagen gel stiffness on neurite extension. J. Biomater. Sci. Polym. Ed. 15:1521–1531

    Article  PubMed  CAS  Google Scholar 

  60. Woerly S., Plant G. W., Harvey A. R. (1996) Cultured rat neuronal and glial cells entrapped within hydrogel polymer matrices: a potential tool for neural tissue replacement. Neurosci. Lett. 205:197–201

    Article  PubMed  CAS  Google Scholar 

  61. Yamada K. M., Pankov R., Cukierman E. (2003) Dimensions and dynamics in integrin function. Braz J. Med. Biol. Res. 36:959–966

    Article  PubMed  CAS  Google Scholar 

  62. Yu T. T., Shoichet M. S. (2005) Guided cell adhesion and outgrowth in peptide-modified channels for neural tissue engineering.Biomaterials 26:1507–1514

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was partially supported by NSF (CAREER Award BES-0093830), NIH/NIBIB (EB001014), NSF (EEC-9731643), and the Southern Consortium for Injury Biomechanics at the University of Alabama Birmingham-Injury Control Research Center, through a grant from the National Center for Injury Prevention and Control, Centers for Disease Control and Prevention, Award R49/CE000191 and Cooperative Agreement TNH22-01-H-07551 with the National Highway Traffic Safety Administration. This work made use of shared facilities from the Georgia Tech/Emory Center (GTEC) for the Engineering of Living Tissues, an ERC supported under Award Number EEC-9731643. The authors would like to thank A. Makhmalbaf and M. Wolfson for their technical support with viability and neurite extension measurements and Dr. M. Levenston, C. Wilson, and S. Stabenfeldt for their technical support with the rheological assessments.

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  1. GT/Emory Coulter Department of Biomedical Engineering, Georgia Institute of Technology, 313 Ferst Dr., Atlanta, GA, 30332-0535, USA

    D. Kacy Cullen, M. Christian Lessing & Michelle C. LaPlaca

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  1. D. Kacy Cullen
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Correspondence to Michelle C. LaPlaca.

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Cullen, D.K., Lessing, M.C. & LaPlaca, M.C. Collagen-Dependent Neurite Outgrowth and Response to Dynamic Deformation in Three-Dimensional Neuronal Cultures. Ann Biomed Eng 35, 835–846 (2007). https://doi.org/10.1007/s10439-007-9292-z

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