BONE MARROW DERIVED CELLS PROMOTING PRE-DEGENERATION OF SCIATIC NERVE IN VITRO /_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WU Min , WANG Xiaopan , XIAO Yuzhou , ZHOU Jiansheng.

Department of Orthopedics, the First Affiliated Hospital of Bengbu Medical College, Laboratory of Tissue and Transplant in Anhui Province, Bengbu Anhui, 233003, P.R.China. Corresponding author: WU Min, E-mail: wumin200207@163.com;

Corresponding author：

Keywords：

Sciatic nerve; Schwann cells; Bone marrow derived cells; Pre-degeneration in vitro; Mouse

DOI：

10.7507/1002-1892.20130123

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Objective To explore a new method for the pre-degeneration of peripheral nerve in vitro for obtaining many effective Schwann cells so as to provide a large number of seed cells for the research and application of tissue engineered nerves. Methods The bone marrow derived cells (BMDCs) from transgenic green fluorescent protein C57BL/6 mouse and the sciatic nerve segments from the C57BL/6 mouse were co-cultured to prepare the pre-degeneration of sciatic nerve in vitro (experimental group, group A), and only sciatic nerve was cultured (control group, group B). At 7 days after culture, whether BMDCs can permeate into the sciatic nerve in vitro for pre-degeneration was observed by gross and immunohistofluorescence staining. And then Schwann cells were obtained from the sciatic nerves by enzymic digestion and cultured. The cell number was counted, and then the purity of primary Schwann cells was determined using immunohistofluorescence staining and flow cytometer analysis. Results At 7 days after pre-degeneration, gross observation showed that enlargement was observed at nerve stumps, and neuroma-like structure formed; the group A was more obvious than group B. Immunohistofluorescence staining showed many BMDCs permeated into the nerve segments, with positive F4/80 staining in group A. After culture, the yield of Schwann cells was (5.59 ± 0.19) × 104 /mg in group A and (3.20 ± 0.21) × 104/mg in group B, showing significant difference (t=2.14, P=0.03). At 48 hours after inoculation, the cells had blue bipolar or tripolar cell nuclei with small size and red soma by immunohistofluorescence staining; fibroblasts were flat polygonal with clear nucleus and nucleolus, showing negative p75NTR staining; and there were few of fibroblasts in group A. The purity of Schwann cells was 88.4% ± 5.8% in group A and 76.1% ± 3.7% in group B, showing significant difference (t=2.38, P=0.04). And the flow cytometer analysis showed that the purity was 89.6% in group A and 74.9% in group B. Conclusion BMDCs can promote the pre-degeneration of peripheral nerve in vitro, and it is a new method to effectively obtain Schwann cells for tissue engineered nerve.

Citation： WU Min,WANG Xiaopan,XIAO Yuzhou,ZHOU Jiansheng.. BONE MARROW DERIVED CELLS PROMOTING PRE-DEGENERATION OF SCIATIC NERVE IN VITRO /. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(5): 554-558. doi: 10.7507/1002-1892.20130123 Copy

1.	Barrette B, Hebert MA, Filali M, et al. Requirement of myeloid cells for axon regeneration. J Neurosci, 2008, 28(38): 9363-9376.
2.	Lee H, Jo EK, Choi SY, et al. Necrotic neuronal cells induce inflammatory Schwann cell activation via TLR2 and TLR3: implication in Wallerian degeneration. Biochem Biophys Res Commun, 2006, 350(3): 742-747.
3.	Boivin A, Pineau I, Barrette B, et al. Toll-like receptor signaling is critical for Wallerian degeneration and functional recovery after peripheral nerve injury. J Neurosci, 2007, 27(46): 12565-12576.
4.	Stoll G, Jander S, Myers RR. Degeneration and regeneration of the peripheral nervous system: from Augustus Waller’s observations to neuroinflammation. J Peripher Nerv Syst, 2002, 7(1): 13-27.
5.	Yang DP, Zhang DP, Mak KS, et al. Schwann cell proliferation during Wallerian degeneration is not necessary for regeneration and remyelination of the peripheral nerves: axon-dependent removal of newly generated Schwann cells by apoptosis. Mol Cell Neurosci, 2008, 38(1): 80-88.
6.	Dahlin LB. Prevention of macrophage invasion impairs regeneration in nerve grafts. Brain Res, 1995, 679(2): 274-280.
7.	Barrette B, Hébert MA, Filali M, et al. Requirement of myeloid cells for axon regeneration. J Neurosci, 2008, 28(38): 9363-9376.
8.	Gensel JC, Nakamura S, Guan Z, et al. Macrophages promote axon regeneration with concurrent neurotoxicity. J Neurosci, 2009, 29(12): 3956-3968.
9.	Ide C. Peripheral nerve regeneration. Neurosci Res, 1996, 25(2): 101-121.
10.	Frostick SP, Yin Q, Kemp GJ. Schwann cells, neurotrophic factors, and peripheral nerve regeneration. Microsurgery, 1998, 18(7): 397-405.
11.	Keilhoff G, Fansa H, Schneider W, et al. In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits. J Neurosci Methods, 1999, 89(1): 17-24.
12.	Kraus A, Täger J, Kohler K, et al. Efficacy of various durations of in vitro predegeneration on the cell count and purity of rat Schwann-cell cultures. J Neurotrauma, 2010, 27(1): 197-203.
13.	Lindholm D, Heumann R, Meyer M, et al. Interleukin-1 regulates synthesis of nerve growth factor in non-neuronal cells of rat sciatic nerve. Nature, 1987, 330(6149): 658-659.
14.	Lindholm D, Heumann R, Hengerer B, et al. Interleukin 1 increases stability and transcription of mRNA encoding nerve growth factor in cultured rat fibroblasts. J Biol Chem, 1988, 263(31): 16348-16351.
15.	Santala P, Heino J. Regulation of integrin-type cell adhesion receptors by cytokines. J Biol Chem, 1991, 266(34): 23505-23509.
16.	Taskinen HS, Heino J, Röyttä M. The dynamics of beta 1 integrin expression during peripheral nerve regeneration. Acta Neuropathol, 1995, 89(2): 144-151.
17.	Ridley AJ, Davis JB, Stroobant P, et al. Transforming growth factors-beta 1 and beta 2 are mitogens for rat Schwann cells. J Cell Biol, 1989, 109(6 Pt 2): 3419-3424.
18.	Stoll G, Griffin JW, Li CY, et al. Wallerian degeneration in the peripheral nervous system: participation of both Schwann cells and macrophages in myelin degradation. J Neurocytol, 1989, 18(5): 671-683.
19.	Miranda CO, Teixeira CA, Liz MA, et al. Systemic delivery of bone marrow-derived mesenchymal stromal cells diminishes neuropathology in a mouse model of Krabbe’s disease. Stem Cells, 2011, 29(11): 1738-1751.
20.	Zuo J, Hernandez YJ, Muir D. Chondroitin sulfate proteoglycan with neurite-inhibiting activity is up-regulated following peripheral nerve injury. J Neurobiol, 1998, 34(1): 41-54.
21.	Zuo J, Ferguson TA, Hernandez YJ, et al. Neuronal matrix metalloproteinase-2 degrades and inactivates a neurite-inhibiting chondroitin sulfate proteoglycan. J Neurosci, 1998, 18(14): 5203-5211.
22.	Ferguson TA, Muir D. MMP-2 and MMP-9 increase the neurite-promoting potential of schwann cell basal laminae and are upregulated in degenerated nerve. Mol Cell Neurosci, 2000, 16(2): 157-167.
23.	Yamada T, Miyazaki K, Koshikawa N, et al. Selective localization of gelatinase A, an enzyme degrading beta-amyloid protein, in white matter microglia and in Schwann cells. Acta Neuropathol, 1995, 89(3): 199-203.
24.	Krekoski CA, Neubauer D, Graham JB, et al. Metalloproteinase-dependent predegeneration in vitro enhances axonal regeneration within acellular peripheral nerve grafts. J Neurosci, 2002, 22(23): 10408-10415.
25.	Siebert H, Dippel N, Mäder M, et al. Matrix metalloproteinase expression and inhibition after sciatic nerve axotomy. J Neuropathol Exp Neurol, 2001, 60(1): 85-93.
26.	Shubayev VI, Myers RR. Upregulation and interaction of TNFalpha and gelatinases A and B in painful peripheral nerve injury. Brain Res, 2000, 855(1): 83-89.

1. Barrette B, Hebert MA, Filali M, et al. Requirement of myeloid cells for axon regeneration. J Neurosci, 2008, 28(38): 9363-9376.
2. Lee H, Jo EK, Choi SY, et al. Necrotic neuronal cells induce inflammatory Schwann cell activation via TLR2 and TLR3: implication in Wallerian degeneration. Biochem Biophys Res Commun, 2006, 350(3): 742-747.
3. Boivin A, Pineau I, Barrette B, et al. Toll-like receptor signaling is critical for Wallerian degeneration and functional recovery after peripheral nerve injury. J Neurosci, 2007, 27(46): 12565-12576.
4. Stoll G, Jander S, Myers RR. Degeneration and regeneration of the peripheral nervous system: from Augustus Waller’s observations to neuroinflammation. J Peripher Nerv Syst, 2002, 7(1): 13-27.
5. Yang DP, Zhang DP, Mak KS, et al. Schwann cell proliferation during Wallerian degeneration is not necessary for regeneration and remyelination of the peripheral nerves: axon-dependent removal of newly generated Schwann cells by apoptosis. Mol Cell Neurosci, 2008, 38(1): 80-88.
6. Dahlin LB. Prevention of macrophage invasion impairs regeneration in nerve grafts. Brain Res, 1995, 679(2): 274-280.
7. Barrette B, Hébert MA, Filali M, et al. Requirement of myeloid cells for axon regeneration. J Neurosci, 2008, 28(38): 9363-9376.
8. Gensel JC, Nakamura S, Guan Z, et al. Macrophages promote axon regeneration with concurrent neurotoxicity. J Neurosci, 2009, 29(12): 3956-3968.
9. Ide C. Peripheral nerve regeneration. Neurosci Res, 1996, 25(2): 101-121.
10. Frostick SP, Yin Q, Kemp GJ. Schwann cells, neurotrophic factors, and peripheral nerve regeneration. Microsurgery, 1998, 18(7): 397-405.
11. Keilhoff G, Fansa H, Schneider W, et al. In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits. J Neurosci Methods, 1999, 89(1): 17-24.
12. Kraus A, Täger J, Kohler K, et al. Efficacy of various durations of in vitro predegeneration on the cell count and purity of rat Schwann-cell cultures. J Neurotrauma, 2010, 27(1): 197-203.
13. Lindholm D, Heumann R, Meyer M, et al. Interleukin-1 regulates synthesis of nerve growth factor in non-neuronal cells of rat sciatic nerve. Nature, 1987, 330(6149): 658-659.
14. Lindholm D, Heumann R, Hengerer B, et al. Interleukin 1 increases stability and transcription of mRNA encoding nerve growth factor in cultured rat fibroblasts. J Biol Chem, 1988, 263(31): 16348-16351.
15. Santala P, Heino J. Regulation of integrin-type cell adhesion receptors by cytokines. J Biol Chem, 1991, 266(34): 23505-23509.
16. Taskinen HS, Heino J, Röyttä M. The dynamics of beta 1 integrin expression during peripheral nerve regeneration. Acta Neuropathol, 1995, 89(2): 144-151.
17. Ridley AJ, Davis JB, Stroobant P, et al. Transforming growth factors-beta 1 and beta 2 are mitogens for rat Schwann cells. J Cell Biol, 1989, 109(6 Pt 2): 3419-3424.
18. Stoll G, Griffin JW, Li CY, et al. Wallerian degeneration in the peripheral nervous system: participation of both Schwann cells and macrophages in myelin degradation. J Neurocytol, 1989, 18(5): 671-683.
19. Miranda CO, Teixeira CA, Liz MA, et al. Systemic delivery of bone marrow-derived mesenchymal stromal cells diminishes neuropathology in a mouse model of Krabbe’s disease. Stem Cells, 2011, 29(11): 1738-1751.
20. Zuo J, Hernandez YJ, Muir D. Chondroitin sulfate proteoglycan with neurite-inhibiting activity is up-regulated following peripheral nerve injury. J Neurobiol, 1998, 34(1): 41-54.
21. Zuo J, Ferguson TA, Hernandez YJ, et al. Neuronal matrix metalloproteinase-2 degrades and inactivates a neurite-inhibiting chondroitin sulfate proteoglycan. J Neurosci, 1998, 18(14): 5203-5211.
22. Ferguson TA, Muir D. MMP-2 and MMP-9 increase the neurite-promoting potential of schwann cell basal laminae and are upregulated in degenerated nerve. Mol Cell Neurosci, 2000, 16(2): 157-167.
23. Yamada T, Miyazaki K, Koshikawa N, et al. Selective localization of gelatinase A, an enzyme degrading beta-amyloid protein, in white matter microglia and in Schwann cells. Acta Neuropathol, 1995, 89(3): 199-203.
24. Krekoski CA, Neubauer D, Graham JB, et al. Metalloproteinase-dependent predegeneration in vitro enhances axonal regeneration within acellular peripheral nerve grafts. J Neurosci, 2002, 22(23): 10408-10415.
25. Siebert H, Dippel N, Mäder M, et al. Matrix metalloproteinase expression and inhibition after sciatic nerve axotomy. J Neuropathol Exp Neurol, 2001, 60(1): 85-93.
26. Shubayev VI, Myers RR. Upregulation and interaction of TNFalpha and gelatinases A and B in painful peripheral nerve injury. Brain Res, 2000, 855(1): 83-89.

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BONE MARROW DERIVED CELLS PROMOTING PRE-DEGENERATION OF SCIATIC NERVE IN VITRO /

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