Influence of Protein Adsorption Onto Titanium Surfaces on Osteoblasts Cell Activity

Protein adsorption occurs immediately following implantation of a biomaterial, and it is widely assumed that this will have an influence on the subsequent cellular response.¹ Despite numerous studies, there are still questions on the degree of influence that this process has on initial cellular events, particularly when the surface exhibits topography.² To investigate this question, we have compared the osteoblastic cell growth onto polished and nanoporous titanium and glass as control by modulating the exposure to serum proteins during the initial phase of cell culture. 

Substrates consisted of: 1) commercial grade 2 titanium disks polished to a mirror finish, 2) polished disks nanotextured by treatment with H₂SO₄/H₂O₂for 2h, and 3) glass coverslips as control, (n=33 for each). In the pre-adsorption phase, substrates were treated for 1h with alpha Minimum Essential Medium (αMEM) alone (MnoS) or with αMEM supplemented with 10% foetal bovine serum (MS). Mouse calvaria-derived osteoblastic cells (MC3T3-EI) were seeded on these pre-treated substrates and cultured for 3h in MnoS and MS. Subsequently, cells were maintained in culture with MS for 72h. Cell number was evaluated using Alamar blue and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assays. Osteogenic cell activity was determined by immunolabeling for Osteopontin (OPN) and staining with alkaline phosphatase (ALP).

Using both Alamar Blue and MTT assays, we were unable to discern any difference in cell number within each condition. However, with the MTT, there was a tendency for fewer cells on glass when serum was absent in the pre-adsorption phase and/or during culture. Staining with ALP and immunolabeling for OPN showed that the osteogenic activity was ongoing at 72h, irrespective of the substrate. When serum was omitted during the pre-adsorption phase or from the culture medium, expression of ALP and OPN decreased on all substrates. In all cases, glass surfaces showed the least ALP and OPN positive cells as compared to titanium surfaces.

Proteins adsorption from normal tissue fluids, which are complex and multi-factorial, appears to have no significant effect on the short-term behaviour of MC3T3-E1 cells. As such, initial proteins adsorption may not be critical for the early interaction of cells with biomaterials. The fact that under all conditions cells fared well, and in particular in the total absence of serum during the first 3h, is intriguing and suggests that physico-chemical interactions without mediation by proteins are sufficient to sustain this initial phase and guide osteogenic cells toward differentiation. In this context, the challenge may actually be avoiding adsorption of molecules that may negatively impact on the cueing that the cells receive from the surface. As our data suggest, this may not be a problem in healthy patients, but may have an important impact in medically compromised individuals in which the composition of tissue fluids is altered.

Supported by Marco Polo programme of University of Bologna; CIHR; NSERC; RSBO. The authors wish to thank Dr.Marianne Ariganello (Université de Montréal, Québec, Canada) for her contribution in the study design.

1. Scotchford CA, Ball M, Winkelmann M, Vörös J, Csucs C, Brunette DM, Danuser G, Textor M. Chemically patterned, metal-oxide-based surfaces produced by photolithographic techniques for studying protein- and cell-interactions. II: Protein adsorption and early cell interactions. Biomaterials 2003;24(7):1147-58.
2. Variola F, Vetrone F, Richert L, Jedrzejowski P, Yi JH, Zalzal S, Clair S, Sarkissian A, Perepichka DF, Wuest JD, Rosei F, Nanci A. Improving biocompatibility of implantable metals by nanoscale modification of surfaces: an overview of strategies, fabrication methods, and challenges. Small 2009;5(9):996-1006.