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Cell and Tissue Research

, Volume 367, Issue 2, pp 339–350 | Cite as

A novel culture platform for fast proliferation of human annulus fibrosus cells

  • Li Xiao
  • Mengmeng Ding
  • Osama Saadoon
  • Eric Vess
  • Andrew Fernandez
  • Ping Zhao
  • Li Jin
  • Xudong Li
Regular Article

Abstract

Tissue engineering provides a promising approach to treat degenerative disc disease, which usually requires a large quantity of seed cells. A simple and reliable in vitro culture system to expand seed cells in a timely fashion is necessary to implement the application clinically. Here, we sought to establish a cost-effective culture system for expanding human annulus fibrosus cells using extracellular matrix (ECM) proteins as culture substrates. Cells were cultured onto a plastic surface coated with various types of ECMs, including fibronectin, vitronectin, collagen type I, gelatin and cell-free matrix deposited by human nucleus pulposus cells. AF cell morphology, growth, adhesion and phenotype (anabolic and catabolic markers) were assessed by microscopy, real-time RT-PCR, western blotting, zymography, immunofluorescence staining and biochemical assays. Fibronectin, collagen and gelatin promoted cell proliferation and adhesion in a dose-dependent manner. Fibronectin elevated mRNA expression of proteoglycan and enhanced glycosaminoglycan production. Both collagen and gelatin increased protein expression of type II collagen. Consistent with increased cell adhesion, collagen and fibronectin promoted formation of focal adhesion complexes in the cell-matrix junction, suggesting enhanced binding of the actin network with both ECM substrates. On the other hand, fibronectin, collagen and gelatin decreased expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 in media. Finally, a mixture of fibronectin (1.7 μg/mL) and collagen (1.3 μg/mL) was identified as the most promising in vitro culture substrate system in promoting proliferation and maintaining anabolic-catabolic balance. Our method provides a simple and cost-effective platform for tissue engineering applications in intervertebral disc research.

Keywords

Intervertebral disc degeneration Extracellular matrix Cell proliferation In vitro culture Tissue engineering 

Notes

Acknowledgments

We appreciate the funding support from the National Institute of Arthritis and Musculoskeletal and Skin Diseases R21AR057512 and RO1AR064792.

Compliance with ethical standards

Funding

NIH NIAMS R21AR057512 and RO1AR064792.

Conflict of interest

The authors declare that they have no conflict of interest.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Li Xiao
    • 1
  • Mengmeng Ding
    • 1
    • 2
  • Osama Saadoon
    • 1
  • Eric Vess
    • 1
  • Andrew Fernandez
    • 1
  • Ping Zhao
    • 2
  • Li Jin
    • 1
  • Xudong Li
    • 1
  1. 1.Department of Orthopaedic SurgeryUniversity of VirginiaCharlottesvilleUSA
  2. 2.Department of AnesthesiologyChina Medical UniversityShenyangChina

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