In Vitro Effect of Amelogenin on Selected Cell Mediators from Human Monocytes
Paper i proceeding, 2008

Introduction: Inflammation is an integral part of the normal wound healing response. Besides clearing the wound of invading microbes and debris, inflammatory cells are believed to be crucial coordinators of the repair process, acting both as phagocytes and as a major source of growth factors and other signals [1]. In non-healing skin ulcers the repair process is stuck in the inflammation phase [2]. Excessive inflammation can reflect an imbalance in the transformation of phenotype between the classically activated, inflammatory macrophage and the alternatively activated macrophage involved in immunosuppression and tissue repair [3]. Amelogenin is a hydrophobic extracellular matrix protein that under physiological conditions will self assemble into nanospheres which in turn may form larger aggregates. Treatment with amelogenin has shown enhanced skin wound healing in an in vivo study in rabbits [4]. In addition, amelogenin has been proposed to have anti-inflammatory properties by attenuation of lipopolysaccharide (LPS)- and peptidoglucan-induced production of selected pro-inflammatory cytokines by human blood cells [5]. The present study was initiated to determine the effects of amelogenin on human monocyte secretion of factors which modulate both inflammation and tissue repair. Materials and Methods: Lyophilized amelogenin from Biora AB (Malmö, Sweden) was dissolved in 17 mM acetic acid. Human monocytes were obtained from six healthy blood donors by isolation using the separation gradient PercollTM in two steps according to Pertoft et al. [6]. The isolated monocytes were cultured for 24 h at 37ºC with 5% CO2 and 95% humidity. Thereafter the supernatants and non-adherent cells were removed. Fresh medium (RPMI, 5% foetal bovine serum, antibiotics) containing amelogenin, 0, 0.01, 0.1 and 1.0 mg/ml, and with or without addition of LPS, was added to the wells in triplicates. The plates were again incubated for 24 h. The supernatants were analyzed with commercial human ELISA assays for tumour necrosis factor- (TNF-), interleukin-10 (IL-10), macrophage inflammatory protein-1 (MIP-1), monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and insulin like growth factor-1 (IGF-1). Results: Amelogenin treatment markedly altered the expression of factors by human monocytes. Amelogenin significantly reduced LPS-induced TNF- secretion, whereas the IL-10 expression was increased. Monocyte secretion of the two inflammatory chemokines MIP-1 and MCP-1 (Figure; mean ± SEM, n=6) was also affected by amelogenin treatment. Furthermore, amelogenin significantly increased monocyte secretion of VEGF (Figure; mean ± SEM, n=6) and IGF-1, although to a lesser extent, after 24 h culture. Conclusions: The amelogenin effects correlate to protein concentration, however not in a dose dependent manner, but instead the cell responses may reflect a concentration related difference in self assembly of the amelogenin protein. The observed changes in cytokine and chemokine expression are markedly affected by simultaneous LPS-induced inflammation activation, revealing possible anti-inflammatory properties of the amelogenin protein. In addition, the several-fold increase in VEGF-levels by monocytes provides a possible mechanism for the observed pro-angiogenic effect in vivo [4]. These in vitro results indicate that the extracellular matrix protein amelogenin by virtue of its interaction with human monocytes may modulate inflammation and tissue repair. Acknowledgements: The support from the Swedish Research Council (grant K2006-73X-09495-16-3), Mölnlycke Health Care Group AB and the VINNOVA VinnVäxt Program Biomedical Development in Western Sweden, is gratefully acknowledged. References: 1. Martin, P., et al. Trends Cell Biol., 15, 599, 2005. 2. Ågren, M.S., et al. Acta Derm Venereol Suppl (Stockh). 210, 3, 2000. 3. Duffield, J.S. Clin Sci (Lond), 104, 27, 2003 4. Mirastschijski, U., et al. Wound Repair Regen., 12, 100, 2004. 5. Myhre, A.E., et al. J Periodontal Res., 41, 208, 2006. 6. Pertoft, H., et al. J Immunol Methods., 33, 221, 1980.




in vitro


Sofia Almqvist

Göteborgs universitet

Maria Werthén

Göteborgs universitet

Anna Johansson

Göteborgs universitet

Peter Thomsen

Göteborgs universitet

8th World Biomaterials Congress, Amsterdam, The Netherlands


Klinisk medicin