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Cartilage Derived Morphogenetic Protein-2 Induces Cell Migration and Its Chondrogenic Potential in C28/I2 Cells

Frank Y. Zhou, Ai-Qun Wei, PhD, Bojiang Shen, PhD, Lisa Williams, PhD, Ashish D. Diwan, FRACS, PhD

Department of Orthopaedic Research, Orthopaedic Research Institute, St George Hospital Clinical School, University of New South Wales, Sydney, Australia

Abstract

Background

Intervertebral disc degeneration is a major cause of low back pain. Previous researches have demonstrated local administration of signalling molecules as potential biological therapies for disc regeneration. Our laboratory has published encouraging results for effectiveness of injection of the cartilage derived morphogenetic protein-2 (CDMP-2) into ovine discs following annular injury. To elucidate the mechanisms underpinning these in vivo effects, this project aimed to investigate the potential of CDMP-2 on cellular migration, proliferation and extracellular matrix production in a human chondrocytic cell line.

Methods

To evaluate cell motility, cells were seeded into Boyden chambers and CDMP-2 as a chemo-attractant or a stimulant was placed into either the bottom or top chambers respectively. Cells that had completed migration through the porous membrane were visualized by immunocytochemical staining and analysed using Image J. The effect of CDMP-2 on cell proliferation, proteoglycan and collagen production, as well as chondrogenic gene expression in human chondrocytic cell line C28/I2 was also examined.

Results

The results revealed that cells migrated significantly under the influence of CDMP-2 (200 ng/ml) stimulation compared to control (3-fold increase, p=0.033) and demonstrated a significant chemotactic movement towards a solution of 200ng/ml CDMP-2 (>2-fold increase, p=0.027). A 35% increase in C28/I2 proliferation was observed after CDMP-2 stimulation (p<0.0001) compared to control, and in the presence of 100ng/ml CDMP-2, proteoglycan synthesis had an 8-fold increase (p=0.048). Similarly, gene expression analysis demonstrated increased expression of aggrecan, collagen types II, X and XXVII, BMPR-1A and BMPR-2 when cells were treated with CDMP-2.

Conclusion

The study shows that C28/I2 cells can migrate under the influence of CDMP-2 as a chemoattractant or migration stimulator, suggestive of an effect on chondrocytic cells in the intervertebral disc. Further, CDMP-2 can stimulate C28/I2 cells to proliferate and synthesize key extracellular matrix proteins. 

keywords: 
intervertebral disc, degenerative disc disease, extracellular matrix, CDMP-2, cell migration, chemoattractant
Volume 9 Article 52
doi: 
10.14444/2052