Effect of Nitrogen-Rich Cell Culture Surfaces on TGF BETA and MAPK Signaling in Intervertebral Disk Cells

Introduction Low back pain, often due to intervertebral disk (IVD) degeneration, is an age-associated disorder that afflicts more than half of the population, above age 70. Discs are composite structures of the peripheral collagen-rich annulus fibrosus (AF) surrounding the proteoglycan-rich central nucleus pulposus (NP). Discs have significant extracellular matrix (ECM) and low cell density, with an absence of blood vessels, a lymphatic system and nerves in all but the most peripheral annular layers. 1 ECM and proper cell-to-cell contacts are essential for normal functioning of cells. ECM is mainly composed of collagens, which support the physical structure of the cell, and contain several growth factors, signal receptors, and adhesion molecules. 2 Disk aging and degeneration are associated with a loss of viable cells in the disc. One therapeutic strategy would be to enhance the IVD cell population to replenish matrix. It has been demonstrated that cultured autologous disk cells can be used to regenerate IVD ECM and provide a solution for the repair of degenerated disk. 3 However, it is necessary to recognize that disk cells that are grown in monolayers under normal culture conditions tend to undergo hypertrophic differentiation and express type X collagen leading to ECM degradation. We have previously reported that the elevated expression of type X collagen in mesenchymal stem cells from osteoarthritis patients is significantly reduced when these cells were cultured on nitrogen-rich surfaces. 4 Although stem cells can be used for cell transplantation, the absence of marker to specifically identify NP cells has restricted their clinical application. In the present study, we are culturing disk cells on surfaces with amine rich coatings to see their effect on expression of collagens and TGF β signaling. It has been reported that TGF β signaling is influenced by the nature of the surfaces to which cells adhere. 5 TGF β signaling is known to influence proliferation, ECM deposition, and also collagen synthesis. 6 Materials and Methods The Low-Pressure Plasma-Polymerized Ethylene Rich in Nitrogen (L-PPE: N) Coatings were deposited in a low-pressure radio-frequency glow discharge on a PET substrate. The Ultraviolet Polymerized Ethylene rich in Nitrogen (UV-PE: N) coatings were deposited in a stainless steel cross chamber, using a noncoherent Krypton lamp at 123.6 nm. Samples were polymerized for 7 hours to obtain a thickness of around 25 nm and an initial NH 2 concentration of [NH 2 ]/[C] = 9.5%. Paylene diX AM was deposited via a chemical vapor deposition process. The initial concentration of [NH 2 ]/[C] is 6.0%. NP and AF tissues from the IVDs were digested and the corresponding NP and AF primary cells were isolated as previously described. 7 These cells were cultured in complete DMEM. The cells are cultured on surfaces (L-PPE: N, UV-PE: N, Paylene diX AM and PET) and the cell extracts were prepared using lysis buffer. Protein expression was analyzed by immunoblotting using antibodies to collagen I and II (Abcam), collagen X(Sigma Aldrich). The activation of the TGF-β and BMP signaling pathways was determined by using antibodies against phospho-Smad 1/5, 2, 3, and Smad 1, 2, 3, 4, 5, and 6 (cell signaling). Expression and phosphorylation of MAPKs was assessed by using specific corresponding antibodies (cell signaling). Results MAPK activation Culturing of NP cells on Paylene diX AM caused significant activation of ERK by phosphorylation compared to the substrate PET. In AF cells activation was not as significant as in NP cells. In both NP and AF cells there is no change in JNK phosphorylation as compared to PET. TGF β and BMP signaling In NP cells there is a significant decrease of Smad 1/5 phosphorylation in cells cultured on L-PPE:N, UV-PE:N, Paylene diX AM surfaces compared to the cells cultured on PET substrate. On the other hand in AF cells, this decrease was seen only in L-PPE:N, Paylene diX AM surfaces. The phosphorylation of Smad 2 and 3 was not significant and no change was observed from control PET. Conclusion Results obtained indicate that there is an altered MAPK signaling in the IVD cells cultured on these surfaces. Decreased phosphorylation of Smad 1/5 but not Smad 2 and 3 suggests altered BMP signaling. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Roughley PJ. Biology of intervertebral disk aging and degeneration: involvement of the extracellular matrix. Spine 2004;29:2691–2699 Kim SH, Turnbull J, Guimond S. Extracellular matrix and cell signalling: the dynamic cooperation of integrin, proteoglycan and growth factor receptor. Journal of Endocrinology 2011;209(2):139–151 Hohaus C, Ganey TM, Minkus Y, Meisel HJ. Cell transplantation in lumbar spine disk degeneration disease. European Spine Journal 2008;17(4):492–503 Rampersad S, Ruiz JC, Petit A, Lerouge S, Antoniou J, Wertheimer MR, Mwale F. Stem cells, nitrogen-rich plasma-polymerized culture surfaces, and type X collagen suppression. Tissue Engeneering Part A 2011;17(19–20):2551–2560 Li L, Klim JR, Derda R, Courtney AH, Kiessling LL. Spatial control of cell fate using synthetic surfaces to potentiate TGF-beta signaling. Proceedings of National Academy of Science USA 2011;108(29): 11745–11750 Murata H, Zhou L, Ochoa S, Hasan A, Badiavas E, Falanga V. TGF-beta3 stimulates and regulates collagen synthesis through TGF-beta1-dependent and independent mechanisms. Journal of Investigative Dermatology 1997;108(3): 258–262 Mwale F, Demers CN, Petit A, Roughley P, Poole AR, Steffen T, Aebi M, Antoniou J, A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc. Journal of Cell Biochemistry 2003;88(6):1202–1213

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