*Article* **Heparin Enriched-WPI Coating on Ti6Al4V Increases Hydrophilicity and Improves Proliferation and Differentiation of Human Bone Marrow Stromal Cells**

**Davide Facchetti 1,2,\* , Ute Hempel <sup>3</sup> , Laurine Martocq <sup>1</sup> , Alan M. Smith <sup>4</sup> , Andrey Koptyug <sup>5</sup> , Roman A. Surmenev 6,7 , Maria A. Surmeneva 6,7 and Timothy E. L. Douglas 1,8,\***


**Abstract:** Titanium alloy (Ti6Al4V) is one of the most prominent biomaterials for bone contact because of its ability to bear mechanical loading and resist corrosion. The success of Ti6Al4V implants depends on bone formation on the implant surface. Hence, implant coatings which promote adhesion, proliferation and differentiation of bone-forming cells are desirable. One coating strategy is by adsorption of biomacromolecules. In this study, Ti6Al4V substrates produced by additive manufacturing (AM) were coated with whey protein isolate (WPI) fibrils, obtained at pH 2, and heparin or tinzaparin (a low molecular weight heparin LMWH) in order to improve the proliferation and differentiation of bone-forming cells. WPI fibrils proved to be an excellent support for the growth of human bone marrow stromal cells (hBMSC). Indeed, WPI fibrils were resistant to sterilization and were stable during storage. This WPI-heparin-enriched coating, especially the LMWH, enhanced the differentiation of hBMSC by increasing tissue non-specific alkaline phosphatase (TNAP) activity. Finally, the coating increased the hydrophilicity of the material. The results confirmed that WPI fibrils are an excellent biomaterial which can be used for biomedical coatings, as they are easily modifiable and resistant to heat treatments. Indeed, the already known positive effect on osteogenic integration of WPI-only coated substrates has been further enhanced by a simple adsorption procedure.

**Keywords:** WPI fibrils; Ti6Al4V; additive manufacturing; osseointegration; heparin; tinzaparin; osteoblast differentiation; coating; enriched
