Introduction: For the last several decades, metallic implants have been used as many types of medical devices, such as vascular stents, orthopedics, and dental prosthetics [1]. These metallic implants have several great properties such like the resistance to erosion, bio-inertness, high fatigue limit and so on. However, to the long term, they still have some problems like incomplete integration with surrounding tissues. Especially, metallic stents still have a possibility of danger like thrombosis due to their poor adhesion or bond deterioration.
To improve their poor adhesion, many researchers have investigated to introduce the modification on the surface of metallic implants. They had tried to treat physical modification such as oxygen plasma and chemical oxidation. However, the problems still remain unsolved.
In this study, we sequentially coated polydopamine (pDA) and fibronectin (FN) on cobalt-chromium alloys (CoCr) to successfully introduce the extracellular matrix (ECM) for the efficient adhesion of vascular endothelial cells (ECs). We evaluated whether pDA-FN coating can effectively hold ECM molecules without loss of natural structures and ECM-coated CoCr can enhance the cellular adhesion of ECs.
Materials and Methods: To prepare pDA-FN coated CoCr substrates, polished CoCr substrates were treated by dip-coating in the dopamine solution at 37 ℃ for 6 h. Those substrates were covered with 50 μg/ml fibronectin at 37 ℃.
After that, NIH3T3 cells were cultured on pDA-FN CoCr substrate for 10 days and decellularized using a mixture of 20 mM NH4OH and 0.5% Triton X-100 in PBS for 15 min at 37 ℃. Subsequently, the decellularized surfaces were treated with nuclease solution (50 mg/ml RNase A and 50 μg/ml DNase I) at 37 ℃ overnight and washed by PBS thoroughly.
To assess the expression and secretion of ECM molecules, immunostaining with anti-FN and anti-collagen was performed on NIH3T3-cultured substrate and modified substrate. The surface morphology and roughness of the modified substrate were characterized by using characterized by using FE-SEM and AFM, respectively.
Results and Discussion: We evaluated the surface properties of the substrates modified with pDA-FN coating followed by cell culture and subsequent decellularization for endothelial cell adhesion. Before the assessment of surface properties, it was observed that the expression of FN and collagen I in cells on the modified substrates has no changes between the experimental groups. However, after decellularization process, FN and collagen molecules were remained on only pDA-FN coated CoCr substrates (Fig. 1). The results from FE-SEM and AFM data showed that the structure of ECM from decellularized pDA-FN coated CoCr substrate has the fiber-like structures, which is similar to natural collagen fibers. Thus, it was demonstrated that the sequential pDA-FN coating could successfully introduce the natural ECM molecules on metal surfaces.
Conclusions: We produced the pDA-FN coated CoCr substrates for successful immolization of ECM molecules. It was showed that the only pDA-FN coated substrates could maintain the natural ECM structures on metal surfaces. Therefore, the sequential pDA-FN coating could be a useful tools to modify the metallic medical devices including stents for better clinical outcomes.
[1][2]
References:
[1] E. Regar, G. Sianos, P.W. Serruys, Br. Med. Bull., 2001, 59, 227–248.
[2] T.M. Bedair, Y. Cho, T.J. Kim, Y.D. Kim, B.J. Park, Y.K. Joung, D.K. Han, Langmuir, 2014, 30, 8020-8028.