Fibronectin adsorption on functionalized electrospun polycaprolactone scaffolds: Experimental and molecular dynamics studies Shawn Regis, 1 Sina Youssefian, 2 Manisha Jassal, 1 Matthew D. Phaneuf, 3 Nima Rahbar, 4 Sankha Bhowmick 1,5 1 Biomedical Engineering and Biotechnology Program, University of Massachusetts Dartmouth, North Dartmouth, Massachusetts 02747 2 Department of Mechanical Engineering, Worcester Polytechnic Institute, Worcester, Massachusetts 01609 3 BioSurfaces, Inc., Ashland, Massachusetts 01721 4 Department of Civil and Environmental Engineering, Worcester Polytechnic Institute, Worcester, Massachusetts 01609 5 Department of Mechanical Engineering, University of Massachusetts Dartmouth, North Dartmouth, Massachusetts 02747 Received 20 May 2013; accepted 4 June 2013 Published online 22 June 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.34843 Abstract: Designing scaffolds to modulate protein adsorption is a key to building advanced scaffolds for tissue regeneration. Protein adsorption to tissue engineering scaffolds is critical in early cell attachment, survival, and eventual proliferation. The goal of this study is to examine the effect of functionalization on fibronectin adsorption to electrospun polycaprolactone (PCL) scaffolds through experimentation using fluorescently labeled fibronectin and to couple this experimental data with analysis of interaction energies obtained through molecular dynamics (MD) simulations to develop a better understanding of the adsorption process. This study is the first to analyze and compare experimental and MD simulation results of fibronectin adsorption on functionalized electrospun PCL scaffolds. Electro- spun nanofiber PCL scaffolds were treated with either 1 N NaOH (hydrolyzed) or 46% hexamethylenediamine (HMD) (ami- nated) solution to be compared with untreated (control) scaffolds. We found that aminated PCL scaffolds experimen- tally adsorbed more fibronectin than control scaffolds, whereas hydrolyzed scaffolds showed decreased adsorption. MD simu- lations carried out with NVT ensemble at a temperature of 310 K indicated a higher work of adhesion for both functionalized scaffolds over control. Also, the simulations revealed different conformations of fibronectin on each scaffold type after adsorption, with the arginine–glycine–aspartic acid sequence appearing most accessible on the aminated scaffolds. This sug- gests that functionalization affects not only the quantity of pro- tein that will adsorb on a scaffold but how it attaches as well, which could affect subsequent cell attachment. VC 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 1697–1706, 2014. Key Words: protein adsorption, fibronectin, MD simulations, electrospun PCL, functionalization How to cite this article: Regis S, Youssefian S, Jassal M, Phaneuf MD, Rahbar N, Bhowmick S. 2014. Fibronectin adsorption on functionalized electrospun polycaprolactone scaffolds: Experimental and molecular dynamics studies. J Biomed Mater Res Part A 2014:102A:1697–1706. INTRODUCTION Quantitative characterization of protein adsorption on engi- neered scaffolds is a necessary first step in tissue regenera- tion. Careful calibration of protein adsorption and a mechanistic understanding of this process would help greatly toward the design of more efficient scaffolds. It is necessary to examine this protein–scaffold interaction using a specific crucial extracellular matrix (ECM) protein rather than just serum proteins, which are far too nonspecific. Fibronectin serves as a model ECM protein with many func- tions in cell attachment. The importance of fibronectin in cell adhesion has been known for some time and thus has been studied extensively. 1–10 Fibronectin can exist in an insoluble form in tissues or the ECM of cultured cells as well as in a soluble form in bodily fluids or media. It is the insoluble form of fibronectin that promotes adhesion of many cells types, do largely in part to the arginine–glycine– aspartic acid (RGD) peptide sequence contained in its chain. This RGD domain supports cell adhesion in many other pro- teins as well, and has therefore become a popular biomole- cule for scaffold functionalization. 11 The extent to which cells interact with fibronectin directly relates to the degree of cell attachment. 3 Additionally, cells will secrete soluble fibronectin to create the ECM that will replace the artificial scaffold as it degrades. 1 For this reason, it is important to know how well the functionalized scaffolds will adsorb this vital protein. There have been many experimental studies on the adsorption of ECM proteins, including fibronectin, to various surfaces. One such study focused on the adsorption of Correspondence to: S. Regis; e-mail: sregis@umassd.edu Contract grant sponsor: University of Massachusetts, Dartmouth, Massachusetts VC 2013 WILEY PERIODICALS, INC. 1697